IOBC / WPRS Study group “ Induced resistance in plants against insects and diseases”
Proceedings of the meeting
at Wageningen (The Netherlands) 26-28 April 2001
Edited by Annegret Schmitt & Brigitte Mauch-Mani
IOBC wprs Bulletin Bulletin OILB srop
Vol. 25 (6), 2002
The IOBC/WPRS Bulletin is published by the International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palearctic Regional Section (IOBC/WPRS) Le Bulletin OILB/SROP est publié par l‘Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les Plantes Nuisibles, section Regionale Ouest Paléarctique (OILB/SROP) Copyright: IOBC/WPRS 2002 The Publication Commission of the IOBC/WPRS: Horst Bathon Federal Biological Research Center for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel +49 6151 407-225, Fax +49 6151 407-290 e-mail:
[email protected]
Luc Tirry University of Gent Laboratory of Agrozoology Department of Crop Protection Coupure Links 653 B-9000 Gent (Belgium) Tel +32-9-2646152, Fax +32-9-2646239 e-mail: luc.tirry@ rug.ac.be
Address General Secretariat: INRA – Centre de Recherches de Dijon Laboratoire de recherches sur la Flore Pathogène dans le Sol 17, Rue Sully, BV 1540 F-21034 DIJON CEDEX France ISBN 92-9067-143-0
Web: http://iobc-wprs.org
Financial support and donations
Uyttenboogaart-Eliasen Foundation Amsterdam, The Netherlands Syngenta, Switzerland Bayer, Germany BASF, Germany Calliope, France Wageningen University, The Netherlands BBA, Institute for Biological Control, Germany IOBC/wprs, Switzerland
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Preface In 1999, IOBC/wprs established a new study group on induced resistance in plants against insects and diseases. Its goal is to augment the understanding of the general and causal processes involved in induced defence reactions of plants against both, insects and plant pathogens, and to promote the use of induced resistance in practice. The newly formed study group held its first meeting from April 26 to 28, 2001 at the Wageningen International Congress Centre (WICC), Wageningen, The Netherlands. About 115 scientists from 17 countries attended the conference. It was the first time that entomologists, phytopathologists and plant physiologists convened to discuss a common subject: the defence reactions and mechanisms against herbivore arthropods and plant pathogens that can be induced in plants. The conference successfully brought researchers together working on fundamental, as well as applied aspects of induced resistance. The interchange with colleagues in other, but related, fields of research created many new ideas and laid ground for new links and co-operations. The congress was structured into three sessions: 1. Cross-talk among herbivore- and pathogen-induced signal cascades 2. Risks and benefits of induced resistance and induced tolerance 3. General aspects of induced resistance and induced tolerance One evening, a workshop was organised with the title “Practical implication of induced resistance / induced tolerance into crop protection programmes: Where are we and where can we go?” A total of 36 oral presentations and 27 posters covering these topics were given and 36 authors took the chance to present their oral or poster contributions as manuscripts in this volume of the IOBC/wprs Bulletin. The contents of the manuscripts are under sole responsibility of their authors. Large credits belong to Marcel Dicke (Wageningen University, Department of Entomology, Wageningen, The Netherlands) and his group for the smooth local organisation and furthermore to Brigitte Mauch-Mani (Université de Neuchâtel, Institut de Botanique, Neuchâtel, Switzerland), Ian Baldwin, (Max Planck Institute for Chemical Ecology, Department for Molecular Ecology, Jena, Germany) and Erkki Haukioja (University of Turku, Department of Biology, Turku, Finland) as members of the organising committee, as well as to Jürg Huber (Biological Research Centre for Agriculture and Forestry (BBA), Institute for Biological Control, Darmstadt, Germany) as IOBC/wprs liaison officer, for their support in setting up the study group and efforts in preparing the meeting. Silvia Schneider (BBA, Institute for Biological Control, Darmstadt, Germany) was of tremendous help when dealing with registration of participants and preparation of the Bulletin. The sponsors are thanked for their generous donations which made it possible to financially support the participation of students and scientists and to invite renowned scientists as key note speakers. The contributions of the keynote speakers created a solid backbone for the congress for which they are highly acknowledged. In this respect, special thanks are given to Joseph Kuc, who gave a great introductory talk and honoured us with his participation. Finally, all participants are thanked for their contributions and lively discussions in the meeting.
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The organising committee of this first IOBC/wprs study group conference and the steering committee of the “First International Symposium on Induced Resistance to Plant Diseases” which was held in Corfu in 2000 agreed to organise and hold the follow-up meeting jointly as successor of both conferences.
Annegret Schmitt Convenor of the IOBC/wprs Study Group Induced Resistance in Plants against Insects and Diseases
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Participants Abdel-Alim, Ahmed Ismail Federal Biological Research Centre for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel.: 0049 6151 4070, Fax: 0049 6151 407290 e-mail:
[email protected]
Boller, Thomas University of Basel Botanisches Institut Hebelstr. 1 CH-4056 Basel (Switzerland) Tel.: 0041 612672320, Fax: 0041 612672330 e-mail:
[email protected]
Anderson, Anne Utah State University Dept. Biology Logan, Utah (USA) Tel.: 001 843225305 e-mail:
[email protected]
Bostock, Richard M. University of California Dept. of Plant Pathology One Shields Ave. Davis, CA 95616 (USA) Tel.: 001 5307524269, Fax: 001 5307525674 e-mail:
[email protected]
Baldwin, Ian Thomas Max Planck Institute for Chemical Ecology Carl Zeiss Promenade 10 D-07745 Jena (Germany) Tel.: 0049 3642643659, Fax: 0049 3642643653 e-mail:
[email protected]
Buchholz, Günther Institute of Viticulture, Freiburg Merzhauserstr. 19 D-79100 Freiburg (Germany) Tel.: 0761 4016511 e-mail:
[email protected]
Balkema-Boomstra, Anneke Plant Research International P.O.B. 16 NL-6700 AA Wageningen (The Netherlands) Tel.: 0031 317477275 e-mail:
[email protected]
Burketova, Lenka Institute of Experimental Botany CAS Na Karlovce 1a CZ-16000 Prague (Czech Republic) Tel.: 0042 224310109, Fax: 0042 224310113 e-mail:
[email protected]
Bell, Doris Cognis Deutschland GmbH Henkelstr. 67 D-40551 Düsseldorf (Germany) Tel.: 0049 21179406781, Fax: 0049 2117982228 e-mail:
[email protected]
Buschmann, Holger University of Hohenheim Dept. Agroecology of the Tropics and Subtropics Fruwirthstrasse 12, Zi. 046 D-70593 Stuttgart (Germany) Tel.: 0049 7114593624, Fax: 0049 7114593843 e-mail:
[email protected]
Bezemer, T. Martyn Netherlands Institute of Ecology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791111, Fax: 0031 264723227 e-mail:
[email protected] Biere, Arjen Netherlands Institute of Ecology Dept. Plant Population Biology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791212, Fax: 0031 264723227 e-mail:
[email protected]
Chen, Ying Leuven University Lab. Phytopathology and Plant Protection Willem De Croylaan 42 B-3001 Leuven (Belgium) Tel.: 0032 16322373, Fax: 0032 16322976 e-mail:
[email protected] Conrath, Uwe University of Kaiserslautern P.O. Box 3049 D-67653 Kaiserslautern (Germany) Tel.: 049 6312053631, Fax: 0049 6312052600 e-mail:
[email protected]
viii De Boer, Jetske Wageningen University Laboratory of Entomology Postbox 8031 NL-6700 BH Wageningen (The Netherlands) Tel.: 0031 317484647, Fax: 0031 317484821 e-mail:
[email protected]
Draper, John University of Wales Institute of Biological Science Edward Lloyd Building GB-ST23 3DA Aberystwyth (Great Britain) Tel.: 0044 1970622981, Fax: 0044 1970622380 e-mail:
[email protected]
De Maagd, Ruud Plant Research International P.O. Box 16 NL-6700 AA Wageningen (The Netherlands) Tel.: 0031 317477128, Fax: 0031 317410094 e-mail:
[email protected]
Ergon, Ashild Norwegian Crop Research Institute Plant Protection Centre Hogskolevn 7 N-1432 As (Norway) Tel.: 0047 64949274, Fax: 0047 64949226 e-mail:
[email protected]
Del Campo, Francisca University of Madrid Facultad de Ciencias,Dept. de Biologia Cantoblanco E-28049 Madrid (Spain) Tel.: 0034 913978189, Fax: 0034 913978344 e-mail:
[email protected] Desmyter, S. Leuven University Lab. Phytopathology and Plant Protection Willem de Croylaan 42 B-3001 Leuven (Belgium) Tel.: 0032 16322372, Fax: 0032 16322976 e-mail:
[email protected] Devey, Jean-Paul Eden Bioscience Europe SARL 15 rue de Geispitzen F-68440 Schlierbach (France) Tel.: 0033 389268741 e-mail:
[email protected] Dicke, Marcel Wageningen Agricultural University Dept. of Entomology P.O. Box 8031 NL-6700 EH Wageningen (The Netherlands) Tel.: 0031 317484311, Fax: 0031 317484821 e-mail:
[email protected]
Ernst, Annegret Gebr. Schaette KG Stahlstr. 5 D-88339 Bad Waldsee (Germany) Tel.: 0049 752440150, Fax: 0049 7524401540 e-mail:
[email protected] Farmer, Edward University of Lausanne Dept. Plant Biology CH-1015 Lausanne (Switzerland) Tel.: 0041 216924190, Fax: 0041 216924195 e-mail:
[email protected] Felton, Gary Dept. of Entomology Penn. State University 501 ASI Building PA 16802 University Park (USA) e-mail:
[email protected] Fritzsche Hoballah, Maria Elena University of Neuchâtel Institute de Zoologie (L.E.A.E.) Case Postale 2 CH-2007 Neuchâtel (Switzerland) Tel.: 0041 327183164, Fax: 0041 327183001 e-mail:
[email protected]
Dietrich, Robert A. Syngenta Research Triangle Park, NC 27709 (USA) Tel.: 001 9195418664, Fax: 001 9195418585 e-mail:
[email protected]
Gabriels, Suzan Plant Research International, Wageningen Droevendaalsesteeg 1 NL-6708 PB Wageningen (The Netherlands) Tel.: 0031 317477333, Fax: 0031 317418094 e-mail:
[email protected]
Dorn, Silvia ETH Zürich Institute of Plant Sciences ETH Zentrum /NW D76.1, Clausiusstr. 25 CH-8092 Zürich (Switzerland) Tel.: 0041 16323921, Fax: 0041 16321171 e-mail:
[email protected]
Geldermann, Utal-Heinz Justus-Liebig-University Institute Phytopathologie und Angwew. Zoologie Heinrich- Buff-Ring 26-32 D-35392 Gießen (Germany) Tel.: 0049 6419937490, Fax: 0049 6419937499 e-mail:
[email protected]
vii Geraats, Bart Utrecht University Phytopathology P.O. Box 800.84 NL-3508 TB Utrecht (The Netherlands) Tel.: 0031 302537438, Fax: 0031 302518366 e-mail:
[email protected]
Heyens, Kathleen Limburgs Universitair Centrum Diepenbeek Dept. SBG Universitaire Campus B-3590 Diepenbeek (Belgium) Tel.: 0032 11268378, Fax: 0032 11268301 e-mail:
[email protected]
Guillon, M.R. Calliope/N.P.P. Route D'Artix, B.P. 80 F-64150 Nogueres (France) Tel.: 0033 559609292, Fax: 0033 559609219
Hoang, Thang Dole Asia Ltd. Stanfilco Division Dona Socorro Street Lanang/Davao City 8000 (Philippines) Tel.: 0063 822340241, Fax: 0063 822331512 e-mail:
[email protected]
Haring, M.A. University of Amsterdam Institute of Life Sciences Plant Physiology Kruislaan 318 NL-1098 SM Amsterdam (The Netherlands) Tel.: 0031 205257663, Fax: 0031 205257934 e-mail:
[email protected] Harvey, Jeff A. Netherlands Institute of Ecology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791111, Fax: 0031 264723227 e-mail:
[email protected] Haukiioja, Erkki University of Turku Dept. of Biology SF-20 500 Turku (Finland) Tel.: 00358 23335778, Fax: 00358 23336550 e-mail:
[email protected] Heil, Martin Biozentrum, Zoologie III Am Hubland D-97074 Würzburg (Germany) Tel.: 0049 9318884378, Fax: 0049 9318884352 e-mail:
[email protected] Heitz, Thierry CNRS Institut de Biologie Moleculaire des Plantes 12 rue du General Zimmer F-67041 Strasbourg Cedex (France) Tel.: 0033 388417271, Fax: 0033 388614442 e-mail:
[email protected] Herz, Annette Federal Biological Research Centre for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel.: 0049 6151 407236, Fax: 0049 6151407290 e-mail:
[email protected]
Howe, Gregg Michigan State University Dept. Biochemistry & Molec. Biology S-206 Plant Biology Building MI 48824 East Lansing (USA) Tel.: 001 5173555159, Fax: 001 5173539168 e-mail:
[email protected] Huber, Jürg Federal Biological Research Centre for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 64287 Darmstadt (Germany) Tel.: 0049 6151 407220, Fax: 0049 6151 407290 e-mail:
[email protected] Hyakumachi, Mitsuro Gifu University Laboratory of Plant Disease Science Faculty of Agriculture 501 1193 Gifu (Japan) Tel.: 0081 582932847, Fax: 0081 582932847 e-mail:
[email protected] Kang, Jin Ho Max Planck Institute for Chemical Ecology Carl Zeiss Promenade 10 D-0745 Jena (Germany) Tel.: 049 3641643654, Fax: 0049 3641643653 e-mail:
[email protected] Kant, Merijn University of Amsterdam Fac. Science, Dept. Population Biology Kruislaan 320 NL-1098 SM Amsterdam (The Netherlands) Tel.: 0031 205257754, Fax: 0031 205257793 e-mail:
[email protected]
x Karjalainen, Reijo University of Kuopio MTT, Plant Protection FIN-31600 Jokioinen (Finland) e-mail:
[email protected] Kessler, Andre Max Planck Institute for Chemical Ecology Carl Zeiss Promenade 10 D-07745 Jena (Germany) Tel.: 0049 3641643655, Fax: 0049 3641643653 e-mail:
[email protected] Kielkiewicz, Margaret Warsaw Agricultural University Dept. Applied Entomology Nowoursynowska 166 PL-02 787 Warsaw (Poland) Tel.: 0048 228434942, Fax: 0048 228434942 e-mail:
[email protected] Kleeberg, Hubertus Trifolio-M GmbH Sonnenstr. 22 D-35633 Lahnau (Germany) Tel.: 0049 644163114, Fax: 0049 644164650 e-mail:
[email protected] Koike, Nobuyo Gifu University Fac. Agriculture, Lab. of Plant Disease Science 1-1 Yanagido 501 1193 Gifu (Japan) Tel.: 0081 582932847, Fax: 0081 582932847 e-mail:
[email protected] Kraemer, Marco Justus-Liebig-University Institute of Crop Science and Plant Breeding Heinrich-Buff-Ring 26-32 D-35392 Gießen (Germany) Tel.: 0049 6419937426 e-mail:
[email protected] Krens, F.A. Plant Research International P.O. Box 16 NL-6700 AA Wageningen (The Netherlands) Tel.: 0031 317477147, Fax: 0031 317418094 e-mail:
[email protected] Kuc, Joseph University of Kentucky 5502 LORNA Street CA 90503-4070 Torrance (USA) Tel.: 001 3107925496, Fax: 001 3107925496 e-mail:
[email protected]
Lamb, Chris John Innes Centre Norwich Research Park Colney NR4 7UH Norwich Norfolk (Great Britain) Tel.: 0044 1603450000, Fax: 0044 1603450045 e-mail:
[email protected] Maris, Paul Wageningen Agricultural University Dept. Plant Sciences, Lab. Virology Binnenhaven 11 NL-6709 PD Wageningen (The Netherlands) Tel.: 0031 7483440, Fax: 0031 7483440 e-mail:
[email protected] Martinez, Felix Eden Biosciences Garraf 35 E-08192 Sant Quirze Valles (Spain) Tel.: 0034 937215377 e-mail:
[email protected] Mauch-Mani, Brigitte University of Fribourg Dept. Biology/Plant Biology 3, Route Albert Gockel CH-1700 Fribourg (Switzerland) Tel.: 0041 263008820, Fax: 0041 263009740 e-mail:
[email protected] McKenzie, Duncan Syngenta Crop Protection AG CH- Basel (Switzerland) e-mail:
[email protected] Meiners, Torsten University of Berlin Angewandte Zoologie, Oekologie der Tiere Haderslebener Str. 19 D-12163 Berlin (Germany) Tel.: 0049 3083855907, Fax: 0049 3083853897 e-mail:
[email protected] Melgarejo, Paloma I.N.I.A. Madrid Dept. Proteccion Vegetal Carretera de la Coruna km 7 E-28040 Madrid (Spain) Tel.: 0034 913476846, Fax: 0034 913572293 e-mail:
[email protected] Mercke, Per Plant Research International P.O. Box 16 NL-6700 AA Wageningen (The Netherlands)
vii Metraux, Jean-Pierre University of Fribourg Dept. Biology/Plant Biology 3, Route Albert Gockel CH-1700 Fribourg (Switzerland) Tel.: 0041 263008820, Fax: 0041 263009740 e-mail:
[email protected] Mur, Luis University of Wales Institute of Biological Sciences Edward Lloyd Building GB-S423 3DA Aberystwyth (Great Britain) Tel.: 0044 1970622981, Fax: 0044 1970622350 e-mail:
[email protected] Neemann, Marcus University of Hohenheim Institute of Phytomedicine D-70593 Stuttgart (Germany) Tel.: 0049 7114592389, Fax: 0049 7114592408 e-mail:
[email protected] Parker, Jane John Innes Centre Stainsbury Laboratory Colney Lane NR4 7UH Norwich (Great Britain) Tel.: 0044 1603450403, Fax: 0044 1603450011 e-mail:
[email protected] Pascual L¢pez, Susana I.N.I.A. Madrid Plant Protection Dept. Carretera de la Coruna Km 7 E-28040 Madrid (Spain) Tel.: 0091 3476811, Fax: 0091 3572293 e-mail:
[email protected] Pieterse, Corne M.J. Utrecht University Section Plant Pathology, Fac. Biology P.O. Box 800 84 NL-3508 TB Utrecht (The Netherlands) Tel.: 0031 302536887, Fax: 0031 30251836 e-mail:
[email protected] Pospieszny, Henryk Institute of Plant Protection Poznan Miczurina 20 Str. PL-60 318 Poznan (Poland) Tel.: 004861 8649094, Fax: 004861 8676301 e-mail:
[email protected]
Roda, Amy Max Planck Institute for Chemical Ecology Carl Zeiss Promenade 10 D-07745 Jena (Germany) Tel.: 0049 3641643654, Fax: 0049 3641643653 e-mail:
[email protected] Roese, Ursula Max-Planck-Institute for Chemical Ecology Carl-Zeiss-Promenade 10 D-07745 JENA (Germany) Tel.: 0049 3641643652, Fax: 0049 3641643650 e-mail:
[email protected] Schaller, Andreas ETH Zürich Plant Sciences Universitaetsstr. 2 CH-8092 Zürich (Switzerland) Tel.: 0041 16326016, Fax: 0041 16321084 e-mail:
[email protected] Schmitt, Annegret Federal Biological Research Centre for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel.: 0049 6151407241, Fax: 0049 6151407290 e-mail:
[email protected] Schoofs, Hilde RSF-Royal Research Station of Gorsem De Brede Akker 13 B-3500 Hasselt (Belgium) Tel.: 032 11586960, Fax: 0032 11674318 e-mail:
[email protected] Schweitzer, Celine Centre de Recherche Public Gabriel Lippmann cellule CREBS 162A Avenue de la Faiencerie L-1511 Luxembourg (Luxemburg) Tel.: 00352 470261438, Fax: 00352 470262389 e-mail:
[email protected] Scutareanu, Petru University of Amsterdam IBED, Sec. Population Biology Kruislaan 320 NL-1090 GB Amsterdam (The Netherlands) Tel.: 0031 205257748, Fax: 0031 205257754 e-mail:
[email protected]
xii Seibicke, Tobias Institute of Viticulture, Freiburg Merzhauserstr. 119 D-79100 Freiburg (Germany) Tel.: 0761 4016511 e-mail:
[email protected]
Thürig, Barbara FIBL Ackerstrasse CH-5070 Frick (Switzerland) Tel.: 0041 628657272, Fax: 0041 628657273 e-mail:
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Shelley, Greg University of Wales Institute of Biological Science Edward Lloyd Building GB-ST23 3DA Aberystwyth (Great Britain) Tel.: 0044 1970622981, Fax: 0044 1970622380 e-mail:
[email protected]
Tomczyk, Anna Warsaw Agricultural University Dept. Applied Entomology Nowoursynowska 166 PL-02 787 Warsaw (Poland) Tel.: 0048 228434942, Fax: 0048 228434942 e-mail:
[email protected]
Siegrist, Jürgen University of Hohenheim Institute of Phytomedicine Otto-Sander-Str. 5 D-70593 Stuttgart (Germany) Tel.: 0049 7114592388, Fax: 0049 7114592408 e-mail:
[email protected]
Ton, Jurriaan Utrecht University Phytopathology B.P.800.84 NL-3508 TB Utrecht (The Netherlands) Tel.: 0031 302537538, Fax: 0031 30251036 e-mail:
[email protected]
Stratmann, Bernd-Gerd Eden Biosciences Am Vilser Holz 17 27305 Bruchhausen-Vilsen (Germany) Tel.: 04252 2781, Fax: 042523598 e-mail:
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Torres, Enrique Universidad Nacional de Colombia Apt. Aereo 14490 Bogota (Colombia) e-mail:
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Sütterlin, Susanne Naktuinbouw Research & Development P.O.Box 135 NL-2370 AC Roelofarendsveen (The Netherlands) Tel.: 0031 7133190122, Fax: 0031 713313030 e-mail:
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Valentini, Giuseppe Eden Bioscience Europe Via Magenta 4 I-20010 Canegrate (Italy) Tel.: 0039 331400721, Fax: 0039 331400721 e-mail:
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Tamm, Lucius FIBL Crop Protection Division Ackerstrasse CH-5070 Frick (Switzerland) Tel.: 0041 628657238, Fax: 0041 628657273 e-mail:
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Valentova, Olga Institute of Chemical Technology Dept. Biochemistry Technicka 3 CZ-16628 Prag (Czech Republic) Tel.: 00420 224355102, Fax: 00420 224355167 e-mail:
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Tanabe, Kentaro Tokyo, Japan 3-7-20 Nihombashi, Chuo-Ku 103 8233 Tokyo (Japan) Tel.: 0081 3 52037896, Fax: 0081 332818589 e-mail:
[email protected] Thomma, Bart P.H.J. CMPG Katholieke Universiteit Leuven Kasteelpark Atenberg 20 B-3001 Heverlee-Leuven (Belgium) Tel.: 0032 16329658, Fax: 0032 16321966 e-mail:
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Van Ast, Jos Eden Bioscience Postbus 16 NL-422326 Hoornaar (The Netherlands) Tel.: 0031 183588171, Fax: 0031 183588176 e-mail:
[email protected] Van Dam, Nicole Netherlands Institute of Ecology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791303 e-mail:
[email protected]
vii Van Deventer-Troost, E. Syngenta, Leiden P.O. Box 628 NL-2300 AP Leiden (The Netherlands) Tel.: 0031 715258282, Fax: 0031 715221471 e-mail:
[email protected] Van der Putten, Wim H. Netherlands Institute of Ecology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791111, Fax: 0031 264723227 e-mail:
[email protected] Van Loon, L.C. Utrecht University Fac. Biology, Sec. Phytopathology P.O. Box 800.84 NL-3508 TB Utrecht (The Netherlands) Tel.: 0031 302536862, Fax: 0031 302518366 e-mail:
[email protected] Van Poecke, R.M.P. Wageningen University Lab. for Entomology P.O. Box 9101 NL-6700 HB Wakening (The Netherlands) Tel.: 0031 317485433, Fax: 0031 317484821 e-mail:
[email protected] Van Tol, Rob Applied Plant Research Nursery Stock Research Unit P.O. Box 118 NL-2770 AC Boskoop (The Netherlands) Tel.: 0031 172236700, Fax: 0031 172236710 e-mail:
[email protected] Verhagen, Bas W.M. Utrecht University Phytopathology P.O. Box 800.84 NL-3508 TB Utrecht (The Netherlands) Tel.: 0031 302537438, Fax: 0031 30251836 e-mail:
[email protected] Vet, Louise Netherlands Institute of Ecology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791111, Fax: 0031 264723227 e-mail:
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Von Amsberg, Hans J. KHH BIOSCI, Inc. Suite 400, 920 Main Campus Dr. Centennial Campus Raleigh NC 27606 (USA) Tel.: 001 9194243737, Fax: 001 9194243738 e-mail:
[email protected] Waeckers, Felix Wageningen University Dutch Institute of Ecology P.O. Box 40 NL-6666 ZG Heteren (The Netherlands) Tel.: 0031 264791306, Fax: 0031 264723227 e-mail:
[email protected] Walling, Linda L. University of California Dept. Botany and Plant Sciences CA 92521-0124 Riverside, CA (USA) Tel.: 001 9097874687, Fax: 001 9097874437 e-mail:
[email protected] Watanabe, Shunnosuke Agro-Kanesho Cop. Ltd. Manager Research and Development Div. 7F Akasaka Shasta-East 4-2-19 Akasaka, Minato-Ku, Tokyo 107-0052 (Japan) Tel.: 0081 355704711, Fax: 0081 355704708 e-mail:
[email protected] Weiskorn, Claudia IFZ Gießen Institut für Pflanzenbau und Pflanzenzüchtung Heinrich-Buff-Ring 26-32 D-35392 Gießen (Germany) Tel.: 0049 641 9937426, Fax: 0049 641 9937429 e-mail:
[email protected] Wei, Zhongmin Eden Bioscience Corp. 11816 North Creek Parkway N. Bothwell, WA 98011 (USA) Tel.: 001 4258067300, Fax: 001 4248067400 e-mail:
[email protected] Wiese, Joachim University of Gießen Institut für Pflanzenernährung Heinrich-Buff-Ring 26-32 (IFZ) D-35392 Gießen (Germany) Tel.: 0049 6419939176, Fax: 0049 6419939169 e-mail:
[email protected]
xiv Wojtasek, Hubert University of Opole Dept. of Chemistry Oleska 48 PL-45 052 Opole (Poland) Tel.: 0048 774545841 2233, Fax: 0048 774538387 e-mail:
[email protected] Wolfram, Jana University of Berlin Günzelstr. 66 D-10717 Berlin (Germany) Tel.: 030 8548166 e-mail:
[email protected] Wydra, Kerstin K. University of Hannover Institute of Plant Diseases and Plant Protection Herrenhaeuser Str. 2 D-30419 Hannover (Germany) Tel.: 0049 5117622643, Fax: 0049 511 7623015 e-mail:
[email protected]
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Contents Cross-talk among herbivore- and pathogen-induced signal cascades Induced resistance in plants – molecular, environmental and practical implications Joseph Kuc.......................................................................................................................... 1 Rhizobacteria-mediated induced systemic resistance in Arabidopsis Corné M.J. Pieterse, Jurriaan Ton, Saskia C.M. Van Wees, Shu Hase, Karen M. Léon-Kloosterziel, Bas W.M. Verhagen, Johan A. Van Pelt, L.C. Van Loon..................................................................................................................... 9 Arabidopsis defense against pathogens: a set of superimposed shields Bart P.H.J. Thomma, Iris A.M.A. Penninckx, Willem F. Broekaert, Bruno P.A. Cammue ......................................................................................................... 17 Involvement of EDS1 and PAD4 at multiple levels of plant defense Jane E. Parker, Bart J. Feys, Lisa J. Moisan, Marie-Anne Newman, Christine Rustérucci, Danny Aviv, Jeffery L. Dangl ........................................................ 25 Activation of novel signalling pathways by phloem-feeding whiteflies Wilhelmina van de Ven, David Puthoff, Cynthia LeVesque, Thomas Perring, Linda L. Walling ............................................................................................................... 33 Novel phospholipases A2 induced during pathogen resistance responses in tobacco and with potential role in oxylipin biosynthesis Thierry Heitz, Sandrine Dhondt, Guillaume Gouzerh, Pierrette Geoffroy, Michel Legrand ................................................................................................................ 41 Genetic dissection of induced resistance in tomato Gregg A. Howe, Lei Li, Gyu In Lee, Chuanyou Li, David Shaffer................................... 47 Ethylene insensitivity in tobacco and Arabidopsis thaliana affects resistance to soilborne pathogens Bart P.J. Geraats, Peter A.H.M. Bakker, L.C. van Loon ................................................. 53 Phenolic acids in tomato plants induced by carmine spider mite (Tetranychus cinnabarinus Boisduval) feeding M. Kielkiewicz .................................................................................................................. 57 Fast assay to test compounds for their potential to induce PR-gene expression in grapevine (Vitis spec.) Tobias Seibicke, Alexander Rügner, Gunther Neuhaus, Hanns-Heinz Kassemeyer, Günther Buchholz ............................................................................................................. 63
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Changes in secondary plant metabolites in cucumber leaves induced by spider mites and plant growth promoting rhizobacteria (PGPR) Anna Tomczyk................................................................................................................... 67 Identification of genes involved in rhizobacteria-mediated induced systemic resistance in Arabidopsis Karen M. Léon-Kloosterziel, Bas W.M. Verhagen, Joost J.B. Keurentjes, L.C. Van Loon, Corné M.J. Pieterse ................................................................................ 71
Risks and benefits of induced resistance and induced tolerance Prospects and challenges for practical application of rhizobacteria-mediated induced systemic resistance L.C. van Loon, P.A.H.M. Bakker, C.M.J. Pieterse ........................................................... 75 Induced responses by plant extracts from Reynoutria sachalinensis: a case study Annegret Schmitt .............................................................................................................. 83 Costs of induced resistance – what do we know and what can they explain? Martin Heil ...................................................................................................................... 89 Benefits and costs of induced volatile production in maize plants Maria Elena Fritzsche Hoballah, Ted J. C. Turlings ....................................................... 95 Time course of induced volatile emission of mature fruits upon herbivory, and response of conspecific adult herbivores and of a natural antagonist Silvia Dorn, Alan Hern, Letizia Mattiacc......................................................................... 99 Incidence of potato late blight assessed on stems and lower hierarchic levels can distinguish heterogeneous disease intensity effected by host reaction and application of salicylic acid and benzothiadiazole Diego Fernando Meza, Enrique Torres ......................................................................... 103 Constitutive and induced variation in plants: what triggers the interaction between the polyphagous insect Helicoverpa armigera (Lep.: Noctuidae) and its antagonist, the baculovirus HaSNPV? Annette Herz, Annegret Schmitt, Jürg Huber ................................................................. 107 Variation of C/N ratio and total protein content in Cacopsylla-infested leaves of three pear cultivars: relationships with an induced phenolic compound P. Scutareanu, A. Johansson .......................................................................................... 111 General aspects of induced resistance and induced tolerance ß-aminobutyric acid as a useful tool to dissect the priming phenomenon in induced resistance Valérie Toquin, Gabor Jakab, Muriel Nirina Maeder, Brigitte Mauch-Mani ............... 117 Priming as a mechanism in plant systemic acquired resistance Uwe Conrath, Oliver Thulke, Vera Katz, Sandra Schwindling, Annegret Kohler ......... 125
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PR1a promoter activation and MAP kinase phosphorylation are involved in the stimulation of plant defenses by OxycomTM Kris Blee, Kwang-Yeol Yang, Anne Anderson................................................................ 129 Induced resistance: an enhancement of basal resistance? Jurriaan Ton, Sylke Davison, Martin De Vos, Charlotte Robben, Hans Van Pelt, L.C. Van Loon, Corné M.J. Pieterse .............................................................................. 133 Biological control of Erwinia carotovora subsp. carotovora on potatoes by fluorescent pseudomonads and Bacillus subtilis A.I. Abdel-Alim, M.S. Mikhail, F.M. Barakat, P. Laux, W. Zeller ................................. 139 Induced resistance in sunflower against Orobanche cumana Holger Buschmann, Joachim Sauerborn........................................................................ 145 Induced resistance in barley (Hordeum vulgare L.) against Rhynchosporium secalis and Barley Yellow Dwarf Virus (BYDV) Claudia Weiskorn, Marco Krämer, Frank Ordon, Wolfgang Friedt ............................. 149 Benzothiadiazole enhances phenolic compound production and resistance to powdery mildew in strawberry R.O. Karjalainen, A. Hukkanen, M. Anttonen, H. Kokko, S. Kärenlampi, K. Tiilikkala .................................................................................................................... .155 Cloning of tomato proteases by direct selection in yeast for enzymes that cleave the polypeptide wound signal systemin Jochen Strassner, Yoann Huet, Andreas Schaller .......................................................... 159 Plants’ defensive responses towards insect oviposition Torsten Meiners, Monika Hilker .................................................................................... 165 Immunohistological analysis of chemically induced proteins in sugar beet Lenka Burketova, Katerina Stillerova, Marcela Feltlova, Milada Sindelarova ............ 169 Induction of defence responses and snow mould resistance in cereals and perennial ryegrass Åshild Ergon, Ingerd S. Hofgaard, Anne Marte Tronsmo ............................................. 173 Extrafloral nectar produced by Macaranga tanarius is an induced, indirect defence against herbivores Martin Heil, Thomas Koch, Andrea Hilpert, Brigitte Fiala, Wilhelm Boland, K. Eduard Linsenmair ................................................................................................... 177 Mechanisms of induction of resistance to diseases in tomato plants by Penicillium oxalicum Antonieta De Cal, Raúl García-Lepe, Paloma Melgarejo ............................................. 183 Characterization of induced PR-1 in a late blight resistant Solanum phureja Céline Schweitzer, Danièle Evers, Jean-François Hausman ......................................... 187 Does Milsana® bioprotectant induce resistance in greenhouse as well as in field-grown plants? Hans von Amsberg, Shunnosuke Watanabe ................................................................... 193
Cross-talk among herbivore- and pathogen-induced signal cascades
Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 1-7
Induced Resistance in Plants - Molecular, Environmental and Practical Implications Joseph Kuc Professor Emeritus, University of Kentucky, 5502 Lorna St., Torrance, CA 90503, USA
Abstract: Induced Systemic Resistance (ISR) in plants to pathogens and insects has likely been with us from the time plants appeared on earth and animal immune systems have been with us since they appeared. However, neither ISR nor animal immune systems have prevented serious epidemics and serious losses continue. Nevertheless, the defense systems in plants and animals are very effective for the survival of the species, but upon occasion they are ineffective as judged by human standards. A major question is why and how do the defense mechanisms become ineffective and how can we maintain and enhance their effectiveness? Resistance is the rule and susceptibility is the exception, but why the exception? Defense systems are layered and have increased in complexity in the process of evolution of complex life forms. These innate and layered defense systems are likely not lost during evolution and they can be activated and enhanced. Is ISR evidence for such layered innate systems? Defensins and protegrins are antimicrobial peptides found in plants and animals that are part of an innate immune system which evolved before antibodies and lymphocytes. Upon such innate systems have been added other systems that differ in defense compounds, their elicitation and specificity for recognition/response and signal transduction/transport. Thus, ISR can be elicited by pathogens and insects and compounds that are as structurally unrelated as proteins and simple organics and inorganics. The compounds associated with defense also differ widely in structure and complexity. Superimposed on these observations is the fact that resistance/susceptibility can be highly specific on the species and cultivar level. Key words: specificity and nonspecificity, sensitization, application
Specificity and Nonspecificity In the abstract three major issues have been raised. The first, if resistance is the rule and susceptibility the exception, then why the exception? The exception may be the result of compounds/pathways/receptors distinct from the mechanisms for resistance and their elicitation but which regulate the expression and/or magnitude of resistance responses. If we knew how to minimize the exceptions we would have taken a big step toward disease control. Granted, environment, pathogen pressure, plant vigor and emergence of new strains/races of pathogens influence the severity of disease, but are suppressors also part of the exception? There is an almost forgotten literature documenting a role for suppressors in determining susceptibility (Doke et al., 1982; Ouchi & Oku, 1982; Kuc et al., 1984; Preisig & Kuc, 1987). The second issue concerns the lack of specificity in the agents that elicit ISR. These include living organisms (as varied as insects, nematodes, fungi and bacteria), virus, components and products of organisms, plant components, complex and structurally unrelated polymers, naturally occurring organic molecules, synthesized organics, inorganics and even gases. Coupled with this lack of specificity for elicitation is the lack of structural specificity in the putative defense compounds produced by the plant. These include phytoalexins, oxidative enzymes, hydrolytic enzymes, antimicrobial proteins, polysaccharides, lipids, reactive oxygen species and inorganics. It is difficult to separate cause and effect. Granted, not all elicitors of 1
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ISR elicit identical responses, but many of them do and others may differ quantitatively and not qualitatively in response. To understand ISR, it is extremely important to grasp the significance of the great diversity of factors which elicit ISR. Clearly, it is not what the compound is but, rather, what it does that is important for initiating ISR. It is the "does" that requires major research effort for understanding. The "does" is not likely to be identical for all agents which elicit ISR and in all plants, and the major compounds contributing to containing the specific pathogen or insect may differ. The end result, however, is ISR-protection of the plant against pathogens and insects. The third issue is how can we resolve the apparent lack of specificity in elicitation and response with the specificity encountered in many pathogen and insect interactions with plants? There is more than ISR or our concept of resistance genes that determines whether an elm bark beetle attacks elms but not oaks and Cladosporium cucumerinum causes disease in cucumber but not spinach. Some cultivars of a "host" plant may be resistant to some races of a pathogen and highly susceptible to others. In Phaseolus vulgaris, cultivars may be resistant to some race of Colletotrichum lindemuthianum and susceptible to others or they may be susceptible to all races. However, resistance has been systemically induced in all cultivars to all races, including cultivars susceptible to all races (often considered to lack genes for resistance) (Elliston et al., 1971, 1976ab, 1977). This specificity in an environment of nonspecificity for ISR needs emphasis for research. As I have repeatedly stated in earlier lectures and publications, the genes for response-dependent resistance mechanisms are likely present in both resistant and susceptible plants and the responses for ISR and resistance are often qualitatively identical. The key consideration is the expression of the genes for resistance mechanisms soon enough and in sufficient magnitude to contain the infectious agent or insect.
Sensitization (Priming) An important feature of ISR is that it has often been reported with the phenomenon of sensitization, sometimes referred to as priming. In sensitization the induction of systemic resistance, abiotically or biotically, results in the rapid and enhanced expression of defense compounds/systems after subsequent infection. The plant with ISR reacts as the resistant plant in which resistance has not been induced prior to infection. Sensitization can persist for the life of some crops, e.g., cucumber, tobacco, green bean, rice, millet, and it includes resistance components as varied as phytoalexins, lignin, chitinases, β-1,3-glucanases, phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase, and hydroxyproline-rich glycoproteins (Dean & Kuc, 1987; Hammerschmidt & Kuc, 1982; Kuc, 1984; Pan et al., 1991, Conrath et al., 2001). In recent experiments, Conrath et al. (2000) reported that pretreating Arabidopsis thaliana seedlings with BTH/Bion [(Benzo (1,2,3) thiadiazole-7-carbothioic acid methyl ester] sensitized the plants to rapidly activate PAL genes after infection with Pseudomonas syringae. Pretreatment with BTH also potentiated both PAL gene expression and callose deposition in response to wounding. The BTH-induced response to wounding was not evident in the Arabidopsis mutant non expresser of PR genes (npr-1) which is defective in the ISR signal transduction mechanisms. The authors suggest that the Arabidopsis NPR-1 protein may function as a switch that modulates cross talk between ISR and wound response pathways. The molecular basis for sensitization is not clearly defined. Since sensitization and ISR have been reported in fully expanded leaves of cucumber, tobacco and many other plants, even leaves which had not yet emerged from the growing point at the time of ISR induction, it is possible that there is a long-term effect on DNA/protein synthesis which is potentiated by a
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signal and this effect persists for extended periods. This signal is translated but may be in addition to the initial signal for ISR. Sensitization is induced without necrotization by some abiotic inducers. ISR and sensitization persist in leaves after the site of induction (factory leaf or site) has been removed. The factory site, and not the leaves above or leaves which emerge, is the source of the signal(s) for ISR and sensitization (Dean & Kuc, 1986; Kuc & Richmond, 1977). Is sensitization a form of plant memory? What is the molecular basis for memory in animals? Sensitization is a very important component of ISR, and is likely more important than the production of high levels of defense compounds systemically before infection (preformed defense elicited by ISR). Further support for the long-term effects of ISR are evident. Cucumber, watermelon and muskmelon plants induced with C. lagenarium when in the second true leaf stage were protected throughout the period of fruiting (Kuc, 1983; Dalisay & Kuc, 1995). BTH, salicylic acid (SA), acetylsalicylic acid (ASA), ethidium diamine tetra acetic acid (EDTA), calcium chloride and phosphates protected pearl millet against downy mildew through the vegetative and reproductive stages (Vasanthi, 2000). Treatment of millet seeds with EDTA, SA, ASA, BABA or calcium chloride protected the resulting seedlings from downy mildew. Metal complexes of phytocyanine applied to rice seeds protected the resulting seedlings against blast for at least one month (Averyanov et al., 2000), and treatment of rice seed or seedlings with dodecyl DL-alanine hydrochloride (DAH) protected plants against blast (Arimoto & Homma, 1991; Arimoto et al., 1976). Soaking rice seeds in DAH was reported to protect plants against blast through the third generation (Arimoto & Homma, 1991).
Signals, Signal Transduction, Mediators This aspect of ISR has rapidly moved forward to implicate specific genes, characterize signals/signal transduction pathways, and elucidate mediators of the metabolic pathways for defense compounds associated with ISR. Research has also been reported on the effect of different pathways for ISR on each other and therefore on the effectiveness of ISR. Added to the complexity of ISR is that it includes insects, pathogens, wounding and chemicals as mediating agents. The mediators may elicit accumulation of the same and different defense compounds and biochemical resistances may differ for any one pathogen/insect-plant interaction. What may be effective as a resistance mechanism for one may be ineffective for another. The mediators receiving most attention include SA, jasmonic acid (JA), abscisic acid (ABA), ethylene (ETH), reactive oxygen species (ROS) and nitric oxide (NO). Activation of one pathway by a mediator may antagonize the activation or effectiveness of another, e.g., SA and JA. In other cases, two mediators may result in enhanced response. This subject has been recently reviewed as it related to plant defense against insects and pathogens (see book edited by Agrawal, Tuzun & Bent, 1999; Karban & Kuc, 2000). Many of the reports which follow in this conference will consider this aspect of ISR. It is interesting to note that BABA elicited ISR in Arabidopsis against Peronospora parasitica even when JA, ETH and SA signal pathways were blocked (Mauch-Mani, 2000). Evidently, there are mediators, signaling pathways and mechanism for ISR that remain to be discovered. All ISR does not depend upon SA. ISR, whether elicited by pathogens insects or chemicals, also protects against damage caused by a variety of stresses. Strobel and Kuc (1995) reported that pro-oxidant chemicals, including the herbicide paraquat, induced systemic resistance in cucumber and tobacco to pathogens and the herbicide. They further reported that induction of resistance with pathogens or 2,6 dichloroisonicotinic acid (INA) also induced systemic protection against damage
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caused by paraquat, cupric chloride, t-butylhydroperoxide and other oxidants. An oxidative burst appears important for induction of ISR but it also helps the plant cope with oxidative agents that would cause extensive damage. It is likely that ISR protects by inhibiting development of pathogens as well as protecting against damage (oxidative, hydrolytic) caused by pathogens and damage caused by oxidants some herbicides and heavy metals. ISR has also been reported associated with enhanced growth and yield (Tuzun et al., 1986; Vashanti, 2000). Where does this end with respect to what can be protected against with ISR and the effect on plant growth and development? The broad spectrum of protection for ISR and effects on growth must be considered when conducting research on signals, signal transduction and activation of genes and pathways. As confusing as this appears, it is also exciting to realize the potential for ISR has not yet been appreciated. A simple explanation for what ISR does is that it enables the plant to do many things better for its benefit. These include defense against pathogens and insects, environmental stresses, pollutants and herbicide damage. It may also enhance uptake and utilization of plant nutrients. A report by Smith-Becker et al. (1998) is very supportive of the presence of a mobile signal that is not SA, but which conditions ISR. In my opinion, this report has not received sufficient attention. The authors report that cucumber leaves infiltrated with Pseudomonas syringae pv. syringae produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in PAL activity in the petioles of inoculated leaves and in stems above inoculated leaves, with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. SA and 4-hydroxybenzoic acid (4HBA) began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf. This report and others in the literature raise many questions concerning the relationship of SA to ISR. Clearly it has a role(s) in ISR, but what is it? In some cases of ISR it is required in some cases not. This can be explained by the existence of multiple pathways for ISR that are effective against different pathogens or insects, e.g. JA-ETH vs SA. In some cases SA suppresses resistance. It is a chelator, antioxidant and has antimicrobial activity per se. Different concentrations of SA elicit different responses and have different effects on ISR. The consensus of opinion is that SA is part of the ISR story, but it and other metabolites are part of a large cast of actors in a very complex play.
Application of ISR The application of ISR has recently been reviewed (Karban & Kuc, 1999; Kuc, 2000, 2001). There are factors favorable and unfavorable for the development and use of ISR. Favorable factors include: 1. Problems with the resistance of pathogens to classical pesticides. 2. The necessity to remove some pesticides from the market. The increased testing and cost of testing to meet requirements of regulatory agencies and the lack of substitutes for removed compounds.
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3. Health and environmental problems, real and perceived, associated with pesticides and the increased popularity for the public of "organic crops" and “sustainable agriculture". 4. The inability of pesticides to effectively control some pathogens, e. g., virus and soilborne pathogens. 5. Classical pesticides may not be economically feasible for farmers in developing countries. In these countries the level of awareness from the safe and effective application of classical pesticides is low creating dangers to human health and the environment. 6. Resistance of the public to genetically modified plants. In ISR, foreign genes are not introduced. The genes for resistance in the plant are expressed. 7. Broad spectrum and duration of effectiveness of ISR. 8. Since many defenses are activated, ISR is less likely to develop resistance in pathogens. Unfavorable factors include: 1. Some plant pathologists still scoff at the applicability of ISR. 2. Only high profit, patented and complex inducers make the major markets. Who champions for the simple non-patented compounds? 3. Lack of sufficient information exchange and financial support for non mega-agribusinessoriented scientists and a lack of adequate information flow to farmers and the public. 4. Unlike classical pesticides which directly kill or inhibit development of a pathogen, ISR depends upon the expression of genes for resistance in the plants. Therefore, ISR is more subject to physiological and environmental influences for effectiveness. 5. Public and farmer apprehension of any new technology. 6. The persistence of an "either or" mentality for disease control. ISR should not be considered an alternative to classical pesticides and sound agronomic practices for disease control. It is an important technology to integrate into a pest management system that is more "organically" oriented under an umbrella of sustainable agriculture.
Conclusions Major objectives of future research are to understand the molecular and genetic basis of ISR and to apply ISR for disease control. For the former it will be necessary to establish the role and interaction of factors as varied as SA, JA, ETH, ABA, NO, ROS. It will be necessary to determine which of the putative defense compounds contribute to ISR. We can apply ISR without knowing the nature of the mobile signal(s) which condition ISR, but research to characterize the mobile signal needs more emphasis to gain understanding of and more effectively apply ISR. The application of ISR and the understanding of ISR require an understanding of the characteristics of the pathogen and plant under study and their interaction. A knowledge of the "biology" of the systems should not be ignored and must be present to take full advantage of the advances in molecular biology and biochemistry. A greater emphasis should be placed on the use of nonpatented, simple and safe agents for ISR. It is gratifying that some agents for ISR are on the market. More are needed, and more attention should be given to ISR eliciting genes as they relate to specific pathogen-insectplant interactions under different growing conditions and stresses. In trying to unravel the mysteries of ISR and to separate cause and effect, I am reminded of something once said by Mark Twain: "Let us be thankful for the fools. Without them the rest of us could not have succeeded."
Acknowledgement
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I acknowledge the assistance of my wife, Karola Kuc, in preparing this manuscript and for many suggestions concerning its content.
References Agrawal, A., Tuzun, S. & Bent, E. 1999: eds. In: Induced Plant Defenses Against Pathogens and Herbivors. 399 pp. Arimo, J., Homma, Y., Ohtsu, N. & Misato, T. 1976: Studies on chemically induced resistance of plants to diseases I. The effect of soaking of rice seed in dodecyl DLalanate hydrochloride on seedling infection by Pyricularia oryzae. Ann. Phytopath. Soc. Japan 42:397-400. Arimo, Y. & Homma, Y. 1991: Generational succession of DL-alanine dodecylester HClinduced resistance to blast disease in rice plants. Ann. Phytopath. Soc. Japan 57:522-525. Averyanov, A., Lapikova, V., Gaevornsky, L. & Labrun, M. 2000: Two step oxidative burst associated with induced resistance to rice blast. In: First International Symposium Induced Resistance to Plant Diseases, Corfu, Greece May 22-27 pages 125-126 (Abstract). Conrath, U., Thulke, O., Katz, V., Schwindling, S. & Kohler, A. 2001: Priming as a Mechanism in Induced Systemic Resistance of Plants. Eur. J. Plant Pathol. 107:113-119. Dalisay, R. & Kuc, J. 1995: Persistence of induced resistance and enhanced peroxidase and chitinase activities in cucumber plants. Physiol. Molec. Plant Pathol. 47:315-327. Dean, R. & Kuc, J. 1986: Induced systemic protection in cucumbers; the source of the signal. Physiol. Molec. Plant Pathol. 28:227-233. Dean, R. & Kuc, J. 1987: Rapid lignification in response to wounding and infection as a mechanism for induced systemic protection in cucumber. Physiol. Molec. Plant Pathol. 31:69-81. Doke, N., Tomyjama, N. & Furiuchi, N. 1982: Elicitation and suppression of the hypersensitive response in host-parasite specificity. In: Plant Infection. eds. Asada et al., 79-94. Elliston, J., Kuc, J. & Williams, E. 1971: Induced resistance to anthracnose at a distance from the site of the inducing interaction. Phytopathology 61, 1110-1112. Elliston, J., Kuc, J. & Williams, E. 1976 a: Protection of bean against anthracnose by Colletotrichum species nonpathogenic on bean. Phytopathol. Z. 86:117-126. Elliston, J., Kuc, J. & Williams, E 1976b: A comparative study of the development of compatible, incompatible and induced incompatible interactions between Colletotrichum species and Phaseolus vulgaris. Phytopathol. Z. 87:289-303. Elliston, J., Kuc, J., Williams, E. & Rahe, J. 1977: Relation of phytoalexins accumulation to local and systemic protection of bean against anthracnose. Phytopathol. Z. 88:114-130. Hammerschmidt, R. & Kuc, J. 1982: Lignification as a mechanism for induced resistance in cucumber. Physiol. Plant Pathol. 20:61-71. Karban, R. & Kuc, J. 1999: Induced resistance against herbivores and pathogens. In: Induced Plant Defenses against Pathogens and Herbivores. eds. Agrawal, Tuzun and Bent: 1-16. Kuc, J. 1982: Plant immunization. Bioscience 32:854-860. Kuc, J. 1984: Phytoalexins and disease resistance mechanisms from a perspective of evolution and adaptation. In: Origin and Development of Adaptation, Ciba Foundation Symposium 102, 100-118. Kuc, J. 2000: Development and future direction of induced systemic resistance in plants. Crop Protection 19: 859-861.
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Kuc, J. 2001: Concepts and direction of induced systemic resistance in plants and its application. Eur. J. Plant Pathol. 107:712. Kuc, J. & Richmond, S. 1977: Aspects of the protection of cucumber against Colletotrichum lagenarium by C. lagenarium. Phytopathology 67:533-536. Kuc, J., Tjamos, E. & Bostock, R. 1984: Metabolic regulation of terpenoid accumulation and disease resistance in potato. In: isopentenoids in Plants-Biochemistry and Function. eds. Ness, Fuller and Tsai, 103-126. Mauch-Mani, B. 2000: A novel pathway for induced resistance in Arabidopsis. In: First Symposium on Induced Resistance to Plant Diseases, Corfu, Greece, May 22-27 Page 65 (Abstract). Ouchi, S. & Oku, H. 1982: Physiological basis of susceptibility induced by pathogens. In: Plant infection. eds. Asada, et al., 117-135. Pan, S., Ye, X. & Kuc, J. 1991: Association of β-1,3-glucanse activity and isoform pattern with systemic resistance to blue mould in tobacco induced by stem infection with Peronospora tabacina or leaf inoculation with tobacco mosaic virus. Physiol. Molec. Plant Pathol. 39:25-39. Preisig, C. & Kuc, J. 1987: Phytoalexins, enhancers, suppressors and other considerations in the regulation of R-gene resistance to Phytophthora infestans in potato. In: Molecular determinants of plant disease. eds. Nishimura, Vance and Doke, 203-221. Smith-Becker, J., Marois, E., Huguet, E., Midland, S., Sims, J. & Keen, N. 1998: Accumulation of salicylc acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems. Plant Physiol. 116:231-238. Strobel, N., Kuc, J. 1995: Chemical and biological inducers of systemic resistance to pathogens protect tobacco plants from damage caused by paraquat and cupric chloride. Phytopathology 85:1306-1310. Tuzun, S., Nesmith, W., Ferris, R. & Kuc, J. 1986: Effects of stem injections with Peronospora tabacina on growth of tobacco and protection against blue mold in the fields.Phytopathology 76:938-941. Vasanthi, N. 2000: Chemical inducers of systemic acquired resistance in pearl millet against downy mildew disease. First International Symposium on Induced Resistance to Plant Diseases, Corfu, Greece, May 22-27, Page 39 (Abstract).
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Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 9-16
Rhizobacteria-mediated induced systemic resistance in Arabidopsis Corné M.J. Pieterse, Jurriaan Ton, Saskia C.M. Van Wees, Shu Hase, Karen M. Léon-Kloosterziel, Bas W.M. Verhagen, Johan A. Van Pelt, L.C. Van Loon Section of Phytopathology, Faculty of Biology, Utrecht University, P.O. Box 800.84, 3508 TB Utrecht, The Netherlands Abstract: Selected strains of rhizosphere bacteria have been shown to reduce disease by activating a resistance mechanism in the plant called rhizobacteria-mediated induced systemic resistance (ISR). ISR resembles pathogen-induced systemic acquired resistance (SAR), in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of pathogens. The spectrum of effectiveness of ISR and SAR largely overlaps but is also partly divergent. In contrast to SAR, ISR induced by Pseudomonas fluorescens WCS417r is independent of salicylic acid (SA) and PR gene activation. Instead, ISR follows a signaling pathway in which components from the jasmonic acid (JA) and ethylene (ET) response are successively engaged to trigger a defense reaction that, like SAR, is controlled by the regulatory factor NPR1. To investigate the role of JA and ET in ISR, their production was monitored in ISR-expressing plants. Neither JA nor ET production changed upon induction of ISR. From this we postulate that ISR is mediated via an increase in the plants sensitivity to JA and ET. This is supported by the potentiated expression of the JA-inducible gene AtVSP observed in challenged, ISR-expressing plants. Moreover, preliminary results indicate that the ACC oxidase activity is enhanced in ISR-expressing plants, providing a greater potential to produce ET upon challenge. In our search for ISR-related genes we identified two genes that show altered expression upon induction of ISR: the JA-inducible gene AtVSP, which shows an enhanced level of expression in challenged, ISR-expressing plants, and a root-specific, ET-inducible thaumatin-like gene, which is activated upon colonization of the roots with ISR-inducing rhizobacteria. Moreover, we identified a locus (ISR1) on chromosome 3 that controls the expression of ISR. Arabidopsis genotypes that are affected in this locus are also less sensitive to ET. Together, these data confirm the important role of JA and ET in ISR signaling. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. For instance, chemical agents that activate the SAR pathway, e.g. SA and benzothiadiazole (BTH), can affect the JA-dependent wound response, which plays a role in defense against insects. We investigated possible antagonistic interactions between the SAR pathway and the ISR pathway. Simultaneous activation of SAR and ISR in Arabidopsis resulted in an additive effect on the level of induced protection against Pseudomonas syringae pv. tomato. In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene PR-1 in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control. Key words: cross-talk, defense signaling, ethylene, ISR, jasmonic acid, salicylic acid, SAR
Introduction Plants possess several pathogen-inducible defense mechanisms that are active against microbial pathogens. A classic example of induced resistance is activated after primary infection with a necrotizing pathogen, rendering distant, uninfected plant parts more resistant towards a broad spectrum of pathogens (Kuc, 1982). This form of induced resistance is often referred to as systemic acquired resistance (SAR; Ross, 1961; Ryals et al., 1996; Sticher et al., 1997), and has been demonstrated in many plant-pathogen interactions. Another form of 9
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induced disease resistance is triggered by selected strains of non-pathogenic rhizobacteria. Rhizosphere bacteria are present in large numbers on plant root surfaces, where root exudates and lysates provide nutrients. Certain strains of rhizosphere bacteria stimulate plant growth and are, therefore, called plant growth-promoting rhizobacteria. Strains that were isolated from naturally disease-suppressive soils, mainly fluorescent Pseudomonas spp., promoted plant growth by suppressing soil-borne pathogens. This biological control activity is effective under field conditions (Zehnder et al., 2001 ) and in commercial greenhouses (Leeman et al., 1995), and can be the result of competition for nutrients, siderophore-mediated competition for iron, antibiosis or the production of lytic enzymes (Bakker et al., 1991). Some of these biological control strains are also able to reduce disease through a plant-mediated mechanism that is phenotypically similar to SAR, as the induced resistance is systemically activated and extends to above-ground plant parts. To facilitate distinguishing this type of induced resistance from pathogen-induced SAR, the term rhizobacteria-mediated induced systemic resistance (ISR) was introduced (Pieterse et al., 1996; Van Loon et al., 1998). Rhizobacteria-mediated ISR has been demonstrated in many plant species, e.g. bean, carnation, cucumber, radish, tobacco, and tomato, and has been reported to be effective against a broad spectrum of plant pathogens, including fungi, bacteria and viruses (Pieterse et al., 2001a; Van Loon et al., 1998). Previously, we developed an Arabidopsis-based model system to study the molecular basis underlying rhizobacteria-mediated ISR (Pieterse et al., 1996). In this paper we will present the current state-of-the-art of the molecular basis of rhizobacteria-mediated ISR in Arabidopsis.
Material and methods For experimental details see primary literature as cited in the text.
Results and discussion Differential activation of rhizobacteria-mediated ISR in Arabidopsis The ability to induce ISR in Arabidopsis was investigated using different ISR-inducing rhizobacterial strains and different Arabidopsis accessions. Colonization of the roots by ISRinducing P. fluorescens WCS417r bacteria protected the plants against different types of pathogens, including the bacterial leaf pathogens P. syringae pv. tomato and the fungal root pathogen Fusarium oxysporum f.sp. raphani (Pieterse et al., 1996). Protection against these pathogens was typically manifested as both a reduction in disease symptoms and inhibition of pathogen growth. Since the rhizobacteria remained localized on the roots and thereby spatially separated from the challenging pathogen, it was concluded that the mode of action of disease suppression is through the activation of ISR in the plant. Elicitation of ISR against P. syringae pv. tomato depended on the host/rhizobacterium combination. For instance, Pseudomonas putida WCS358r and P. fluorescens WCS374r performed differently on different plant species: Arabidopsis was responsive to WCS358r (Van Wees et al., 1997), which is not effective in radish and carnation. Conversely, Arabidopsis was not responsive to WCS374r, a strain, which is a good inducer of ISR in radish. Also differential induction of ISR occurred between Arabidopsis accessions. Most accessions, e.g. Columbia and Landsberg erecta, were responsive to treatment with WCS417r, whereas accessions RLD and Wassilewskija were not (Ton et al., 1999; Van Wees et al., 1997). This suggests that specific recognition between the plant and the ISR-inducing rhizobacterium is required for the induction of ISR, and that ISR is genetically determined.
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A novel signaling pathway controlling induced systemic resistance in Arabidopsis The signaling pathway controlling pathogen-induced SAR has been well studied in Arabidopsis. As in many other species, SAR in Arabidopsis is dependent on SA and is tightly correlated with the activation of PR genes (Mauch-Mani and Métraux, 1998; Ryals et al., 1996). To dissect the ISR signaling pathway in Arabidopsis we tested a large set of mutants that are impaired in their response to the defense signals SA, JA or ET (for overview see Table 1 in Pieterse et al. 2001a). In contrast to SAR, WCS417r-mediated ISR in Arabidopsis appeared to function independently of SA and PR gene activation, as SA-nonaccumulating NahG plants developed normal levels of ISR against P. syringae pv. tomato after colonization of the roots by WCS417r (Pieterse et al., 1996; Van Wees et al., 1997). Similarly, the SA induction-deficient mutants sid1-1 and sid2-1 (Nawrath and Métraux, 1999) expressed WCS417r-mediated ISR (C.M.J. Pieterse, unpublished results), again demonstrating that WCS417r-mediated ISR is SA-independent. Using the JA response mutant jar1-1, the ET response mutant etr1-1, and the SAR regulatory mutant npr1-1, it was demonstrated that signal transduction leading to WCS417r-mediated ISR requires responsiveness to both JA and ET and, similar to pathogen-induced SAR, is dependent on NPR1 (Pieterse et al., 1998). Like WCS417r, methyl jasmonate (MeJA) and the ET precursor 1-aminocyclopropane-1-carboxylate (ACC) were effective in inducing resistance against P. syringae pv. tomato in NahG plants. Moreover, MeJA-induced protection was blocked in jar11, etr1-1, and npr1-1 plants, whereas ACC-induced protection was affected in etr1-1 and npr1-1 plants, but not in jar1-1 plants. Hence, it was postulated that WCS417r-mediated ISR follows a novel signaling pathway in which components from the JA and ET response are successively engaged to trigger a defense reaction that, like SAR, is regulated by NPR1 (Pieterse et al., 1998). Downstream of NPR1, PR genes are activated in the SAR pathway but not in the ISR pathway (Cao et al., 1994; Pieterse et al., 1998). Evidently, NPR1 differentially regulates ISR- and SAR-related gene expression, depending on the pathway that is activated upstream of it. Production of JA and ET during ISR Increased production of JA and ET is an early symptom of active defense in infected plants. Both signaling molecules coordinate the activation of a large set of defense responses, and when applied exogenously, can induce resistance themselves. In Arabidopsis, both JA and ET activate specific sets of defense-related genes and resistance against P. syringae pv. tomato (Van Wees et al., 1999). Recently, we monitored the expression of a set of well-characterized JA- and/or ET-responsive genes in Arabidopsis plants expressing ISR. None of the genes tested were upregulated in induced plants, neither locally in the roots, nor systemically in the leaves (Van Wees et al., 1999). This suggests that WCS417r-mediated ISR in Arabidopsis was not associated with major changes in the levels of either JA or ET. Indeed, analysis of local and systemic levels of JA and ET revealed that WCS417r-mediated ISR is not associated with changes in the production of these signal molecules (Pieterse et al., 2000). By using the LOX2 co-suppressed transgenic line S-12, we confirmed that an increase in JA production is not required for the induction or expression of ISR. Transgenic S-12 plants, that are affected in the production of JA in response to wounding (Bell et al., 1995), expressed normal levels of ISR (Pieterse et al., 2000). Together, these results suggest that the JA and ET dependency of ISR is based on enhanced sensitivity to these hormones, rather than on an increase in their production.
Potentiation of JA-responsive genes in plants expressing ISR If the JA and ET dependency of ISR is based on enhanced sensitivity to these signal molecules, ISR-expressing plants would be expected to react faster or more strongly to pathogen-induced
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JA or ET production. Therefore, the expression of the JA-responsive genes AtVSP, PDF1.2, LOX2, and PAL1, the ET-responsive genes HEL and CHI-B, and the SA-inducible genes PR-1, PR-2, and PR-5 was analyzed after challenge of control, SAR- and ISR-expressing plants (Van Wees et al., 1999). Infection with P. syringae pv. tomato induced the expression of all genes tested. In challenged, SAR-expressing plants the SA-inducible genes PR-1, PR-2, and PR-5 showed a potentiated expression compared to challenged control plants. In challenged, ISRexpressing plants, only AtVSP displayed an enhanced level of expression in comparison to challenged control plants. The expression of the other JA-responsive genes was not potentiated, suggesting that ISR is associated with the potentiation of a specific set of JA-responsive genes. ISR is associated with enhanced capacity for conversion of ACC to ET In higher plants, ET is produced from methionine (Met) via S-adenosyl-L-methionine (SAM) and ACC (Met → SAM → ACC → ET; Kende and Zeevaart, 1997). The last two steps of this biosynthetic pathway are catalyzed by ACC synthase and ACC oxydase, respectively. Pathogen infections leading to chlorotic or necrotic symptoms cause an increase in ET production with ACC synthase and ACC oxidase activity being increased sequentially (De Laat and Van Loon, 1982). Under normal conditions the conversion of SAM to ACC is the rate-limiting step, however, during infections, ACC accumulates transiently, indicating that ACC oxidase activity restricts ET production. In Arabidopsis, ET production is not increased in systemic, ISR-expressing tissues compared to non-induced plants. However, after treatment with a saturating dose of 1 mM ACC, ISR-expressing plants showed a statistically significant higher level of ET emission than ACC-treated control plants (Pieterse et al., 2000; S. Hase, unpublished results). The magnitude of the increase in ACC-converting capacity varied from 20 to 50% between experiments. Also, in the first 24 hours after inoculation with P. syringae pv. tomato, ISR-expressing plants showed a significant increase in ET emission (S. Hase, unpublished results). Evidently, the capacity to convert ACC to ET is increased in Arabidopsis plants expressing ISR, providing a greater potential for producing ET upon pathogen attack. As application of ACC has been shown to induce resistance against P. syringae pv. tomato in Arabidopsis (Pieterse et al., 1998), a faster or greater production of ET in the initial phase of infection may contribute to enhanced resistance against this pathogen. Spectrum of effectiveness of ISR and SAR In Arabidopsis, SA, JA and ET are involved to different extents in basal resistance against specific pathogens. Basal resistance against the oomycetous pathogen Peronospora parasitica and to turnip crinkle virus (TCV) seems to be controlled predominantly by a SA-dependent pathway. Only SA-nonaccumulating NahG plants exhibited enhanced disease susceptibility to these pathogens (Delaney et al., 1994; Kachroo et al., 2000), whereas mutants affected in JA or ET signaling did not (Kachroo et al., 2000; Thomma et al., 1998). In contrast, basal resistance against the fungal pathogens Alternaria brassicicola and Botrytis cinerea was reduced only in JA- and ET-insensitive mutants, and not in NahG plants (Thomma et al., 1998; 1999). Interestingly, basal resistance against the bacterial pathogens P. syringae pv. tomato and Xanthomonas campestris pv. armoraciae was found to be affected in both NahG plants and in JA- and ET-response mutants (Pieterse et al., 1998; Ton et al., 2001b), suggesting that basal resistance against these pathogens is controlled by a combined action of SA, JA and ET. To compare the effectiveness of SA-dependent SAR and JA/ET-dependent ISR, we performed standard ISR and SAR bioassays using the different Arabidopsis pathogens that, in non-induced plants, are primarily resisted through either SA-dependent defenses, i.e. P. parasitica and TCV, JA/ET-dependent defenses, i.e. A. brassicicola, or a combination of SA-, JA-, and ET defenses, i.e. P. syringae pv. tomato and X. campestris pv.
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armoraciae. Induction of SAR and ISR was equally effective against P. syringae pv. tomato and X. campestris pv. armoraciae. In addition, activation of ISR resulted in a significant level of protection against A. brassicicola, whereas SAR was ineffective against this pathogen. Conversely, activation of SAR resulted in a high level of protection against P. parasitica and TCV, whereas ISR conferred only weak and no protection against P. parasitica and TCV, respectively. These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent basal resistance responses, whereas ISR is effective against pathogens that in non-induced plants are resisted through JA/ET-dependent basal resistance responses (Ton et al., 2001b; see also Ton et al. elsewhere in this issue). ISR and SAR are additive Cross-talk between defense signaling pathways has been demonstrated: JA and ET can act in concert in activating defense responses, whereas SA can suppress JA-dependent responses (Pieterse et al., 2001b). Together with the fact that ISR and SAR share the regulatory factor NPR1, the question was raised as to what extent the JA-dependent ISR pathway and the SAdependent SAR pathway interact. To investigate possible interactions between the ISR and the SAR pathway, we induced ISR and SAR against P. syringae pv. tomato simultaneously. Interestingly, simultaneous activation of both pathways resulted in an additive effect on the level of induced protection (Van Wees et al., 2000). In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, expression of the SAR marker gene PR-1 was not altered in plants expressing both ISR and SAR compared to plants expressing SAR alone, indicating that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these signaling pathways. Search for rhizobacteria-mediated ISR-related genes The state of pathogen-induced SAR is characterized by the concomitant activation of a large set of genes (Maleck et al., 2000). Of many defense-related genes tested in Arabidopsis (e.g. the SA-inducible genes PR-1, PR-2, and PR-5, and the ET- and/or JA-inducible genes HEL, CHI-B, PDF1.2, AtVSP, LOX1, LOX2, and PAL1), none were found to be up-regulated in plants expressing ISR (Van Wees et al., 1999). Thus, in contrast to SAR, the onset of ISR is not associated with major changes in gene expression. Nevertheless, ISR-expressing plants are clearly more resistant to different types of pathogens. Therefore, plants must possess as yet undiscovered defense-related gene products that contribute to broad-spectrum resistance. In another approach to search for ISR-related genes, a large collection of Arabidopsis lines containing enhancer-trap Ds transposons and the β-glucuronidase (GUS) reporter gene were screened. One enhancer-trap line showed local GUS activity in the roots upon colonization by WCS417r (see also K.M. Léon-Kloosterziel et al. elsewhere in this issue). Interestingly, the roots of this line showed a similar expression pattern after treatment of the roots with the ET precursor ACC, indicating that this line contains a transposon insertion in the vicinity of an ET-inducible gene that is up-regulated in the roots upon colonization by WCS417r. Characterization of the gene revealed that it encodes a thaumatin-like gene. Thaumatins have repeatedly been implicated in plant defense. Currently, we are investigating the role of this gene in ISR. Identification of a novel locus (ISR1) controlling rhizobacteria-mediated ISR In a genetic approach to identify ISR-related genes, we screened 10 accessions of Arabidopsis for their potential to express ISR and SAR against P. syringae pv. tomato (Ton et al., 1999). All accessions tested developed SAR. However, of the 10 accessions tested, RLD and Wassilewskija did not develop ISR after treatment of the roots with WCS417r. The WCS417r-nonresponsive phenotype was associated with a relatively high susceptibility to P.
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syringae pv. tomato. Genetic analysis of the F1, F2, and F3 progeny of a cross between the WCS417r-responsive accession Columbia and the WCS417r-nonresponsive accession RLD, revealed that both the potential to express ISR and the relatively high level of basal resistance against P. syringae pv. tomato are monogenic, dominant traits that are genetically linked. The corresponding locus, designated ISR1, was mapped between CAPS markers B4 and GL1 on chromosome III. Neither responsiveness to WCS417r, nor the relatively high level of basal resistance was complemented in the F1 progeny of crosses between RLD and Wassilewskija, indicating that both accessions are affected in the same locus. Interestingly, mutants jar1-1 and etr1-1, that are affected in their response to JA and ET, respectively, showed the same phenotype as accessions RLD and Wassilewskija in that they were both unable to express WCS417r-mediated ISR and showed enhanced susceptibility to P. syringae pv. tomato infection (Pieterse et al., 1998). Analysis of ET-responsiveness of RLD and Wassilewskija revealed that both accessions showed a reduced sensitivity to ET, that co-segregated with the recessive alleles of the ISR1 locus (Ton et al., 2001a). Therefore, it is proposed that the Arabidopsis ISR1 locus encodes a novel component of the ET-response pathway that plays an important role in disease-resistance signaling. Concluding remarks Recent advances in research on plant defense signaling pathways have shown that plants are capable of differentially activating distinct defense pathways, depending on the type of invader encountered (Pieterse and Van Loon, 1999; Pieterse et al., 2001b). Salicylic acid is an important signaling molecule in both locally and systemically induced resistance responses. However, research on rhizobacteria-mediated ISR signaling in Arabidopsis demonstrated that JA and ET play key the roles. During the past five years, research on rhizobacteria-mediated ISR has increased our knowledge of the molecular mechanisms involved in this form of induced disease resistance. An important conclusion is that different rhizobacteria utilize different mechanisms for triggering systemic resistance: some rhizobacteria trigger a SAdependent pathway, others a JA/ET-dependent pathway (Pieterse et al., 2001a). In this respect, it is interesting to note that simultaneous activation of the SA-dependent SAR pathway and the JA/ET-dependent ISR pathway resulted in an additive effect on the level of induced resistance attained (Van Wees et al., 2000). Therefore, combining rhizobacterial strains that trigger different signaling pathways in the plant provides an attractive possibility for the improvement of disease control (see also Van Loon et al. elsewhere in this issue). In contrast to SAR, rhizobacteria-mediated ISR in Arabidopsis is not associated with major changes in gene expression. Currently, research on the molecular mechanisms underlying ISR is hampered by the lack of reliable molecular markers. Therefore, future research will be focussed on identifying such marker genes using techniques such as screening of DNA microarrays, screening of enhancer/gene-trap lines, and map-based cloning approaches. Furthermore, the mechanisms involved in potentiation of JA-responsive gene expression and the increased ACC-converting capacity in ISR-expressing plants need to be investigated. Both latter findings are examples of priming that may lead to a faster and/or enhanced activation of JA- and ET-dependent defense reactions upon attack by a challenging pathogen. If priming of defense responses plays an important role in ISR, then this could explain the absence of major changes in defense-related gene expressing prior to challenge. Investigations of these phenomena will be most challenging and will certainly provide more insight in the molecular mechanisms of induced disease resistance.
References
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Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 17-24
Arabidopsis defense against pathogens: a set of superimposed shields Bart P.H.J. Thomma, Iris A.M.A. Penninckx, Willem F. Broekaert, Bruno P.A. Cammue Centre of Microbial and Plant Genetics (formerly: F.A. Janssens Laboratory of Genetics), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee-Leuven, Belgium
Abstract: Not more than a decade ago it was generally accepted that defense mechanisms induced by micro-organisms converge into a central signalling cascade that regulates a defense response consisting of multiple components. Salicylic acid was recognized as a central regulator molecule in this signalling cascade. However, by now we know that the defense response of plants to pathogens is not regulated through a single signalling cascade, but rather through a complex network of different signalling pathways. These pathways are regulated through different signalling molecules. Since a considerable amount of cross-talk is observed it is clear that pathways are interconnected. As a model, we envisage the inducible Arabidopsis defense as a set of superimposed shields, each displaying their own efficacy against different pathogens. Key words: salicylic acid, jasmonate, ethylene, camalexin, Alternaria, Botrytis, Plectosphaerella
Introduction In contrast to most living organisms, plants cannot avoid attacking organisms by moving to a more favourable place. Therefore, plants have evolved defense mechanisms to be able to combat pathogens. Some of these defenses are continuously activated, so called preformed defenses, whilst others are induced upon pathogen attack. During the last decade Arabidopsis has become a very important model plant to unravel how defense systems can control pathogen attack. This is largely due to the availability of many mutants in different defense response pathways. Studies using these mutants have led to a reasonable insight in how different defense pathways contribute to control different pathogens. Apart from locally induced defense responses, plants can also induce defense responses in tissues distant from the initial infection site. Over the last 20 years, SA has been recognised as a, or maybe even the, central molecule in systemic defence responses (Dempsey et al., 1999). Much effort has been dedicated to the elucidation of the exact role of SA in plant defence and SA-dependent signalling has been shown to be required for resistance of Arabidopsis plants to at least the pathogens Pseudomonas syringae, Peronospora parasitica and Erysiphe orontii (Cao et al., 1994; 1997; Delaney et al., 1994; Reuber et al., 1998). Recently, however, it has become clear that salicylic acid is not the only signalling molecule involved in mounting broad-spectrum systemic disease resistance responses. It was shown that a pathogen-inducible Arabidopsis gene (PDF1.2), whose product has antimicrobial properties, is not SA-inducible but depends on the plant hormones jasmonate and ethylene for induction (Penninckx et al., 1996). This finding led to the conclusion that, in addition to an SA-dependent defence response pathway, a jasmonate/ethylene-dependent defence response pathway exists in Arabidopsis (Penninckx et al., 1996; 1998).
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Material and methods Biological material The salicylate-degrading transgenic Arabidopsis line containing the NahG gene (Delaney et al., 1994), the salicylate-insensitive mutant npr1-1 (Cao et al., 1994), the jasmonate-and ethylene-insensitive Arabidopsis mutants coi1-1 (Feys et al., 1994) and ein2-1 (Guzmán and Ecker, 1990) and the phytoalexin-deficient mutant and pad3-1 (Glazebrook and Ausubel, 1994) are all derived from the Arabidopsis thaliana Columbia (Col-0) ecotype. Arabidopsis seed was sown in Petri dishes on flower potting compost containing a macronutrient supplement (Asef, Didam, The Netherlands). Seeds were vernalized for 2 days at 4°C following sowing and subsequently transferred to a growth chamber (20°C daytime temperature and 18°C night-time temperature, with a 16-hr photoperiod. After 5 days, seedlings were transferred to pots (5x4x4 cm) containing potting compost supplemented with macronutrients and grown under the same conditions as given above. Irrigation was done with tap water via the trays carrying the pots. Because the mutation in the jasmonate-insensitive coi1-1 mutant is recessive and causes male sterility, coi1-1 mutants were identified in F2 seed from selfed COI1/coi1 hemizygous plants (Feys et al., 1994). Growth and spore harvesting of the fungi Alternaria brassicicola, Botrytis cinerea, Plectosphaerella cucumerina (provided by Dr. B. Mauch-Mani, Université de Fribourg, CH) was done as described previously (Broekaert et al., 1990). Peronospora parasitica strain Wela (Delaney et al., 1994) was maintained on living Arabidopsis plants of the Weiningen ecotype and strain Noco was maintained on living Arabidopsis plants of the ecotype Col-0. In all tests four-week-old plants were used. Inoculations with micro-organisms For inoculation of Arabidopsis plants with Alternaria brassicicola or Plectosphaerella cucumerina three 5 µl drops of a suspension of 5 x 105 conidial spores/ml in water were placed on each leaf with the aid of an automated pipettor. Control plants were mockinoculated with water. For the inoculations with B. cinerea, two needle-prick wounds were applied to the leaves and the fresh wounds were covered with 5 µl drops of a suspension of 5x105 conidial spores/ml in 12 g/l potato dextrose broth (Difco, Detroit, USA) with the aid of an automated pipettor. Both wounding and the use of a rich growth medium are required by B. cinerea in order to be able to colonise Arabidopsis leaf tissue. The wounds of control plants were covered with 5 µl drops of 12 g/l potato dextrose broth. All inoculated plants were incubated at 100% relative humidity in propagator flats covered with clear polystyrene lids in the growth chamber for the time periods indicated. Inoculation with P. parasitica was done by spraying until droplet run-off with a suspension of 105 conidial spores/ml in water. Inoculated plants were incubated at 18°C at 100% relative humidity in propagator flats covered with a clear polystyrene lid for the time period indicated.
Results and discussion Differentially acting disease response pathways Resistance against the fungi Alternaria brassicicola and Botrytis cinerea is not mediated by SAdependent signalling. Using a jasmonate- and an ethylene-insensitive Arabidopsis mutant (coi1-1 and ein2-1, respectively), it was demonstrated that both jasmonate and ethylene-dependent
19
signalling are required for resistance against Botrytis cinerea, whereas jasmonate-dependent but not ethylene-dependent signalling is required for resistance against Alternaria brassicicola (Thomma et al., 1998; 1999a). In contrast, SA-dependent signalling, yet neither jasmonatedependent signalling nor ethylene-dependent signalling is required for resistance against Peronospora parasitica. In addition, jasmonate-dependent signalling is also effective against the fungus Plectosphaerella cucumerina, although in this case SA-dependent signalling contributes as well to effective control (Thomma et al., 2000). Apart from the importance of jasmonate- and ethylene-dependent signalling for plant defense, these findings indicate that in Arabidopsis different defence response pathways that depend on different signalling molecules can be activated, and that these pathways differ in efficacy for controlling distinct (groups of) pathogens.
pathogen detection
??? salicylic acid
ethylene
jasmonate
camalexin
Botrytis Alternaria
Peronospora Plectosphaerella
Figure 1. Disease signaling pathways in Arabidopsis. This model distinguishes several inducible signaling pathways in Arabidopsis and the pathogens these pathways are effective against (Thomma et al., 2001a). A jasmonate/ethylene-dependent pathway has been shown to be effective against Botrytis cinerea (Thomma et al., 1998; 1999a), whereas an SA-dependent pathway is required for defense against Peronospora parasitica (Delaney et al., 1994; Thomma et al., 1998). A jasmonatedependent but ethylene-independent pathway in parallel with a pathway leading to production
of camalexin provides resistance against Alternaria brassicicola (Thomma et al., 1998; 1999a; 1999b). Plectosphaerella cucumerina is controlled by SA-dependent as well as by JA-dependent defense responses (Thomma et al., 2000). Cross-talk events between the signaling molecules presented in this scheme are not taken into account. Question marks indicate unknown signalling events between pathogen perception and the components mentioned in the figure.
Yet another defense response pathway leads to the production of the phytoalexin camalexin. Production of camalexin does not depend directly on either of the signalling molecules SA, jasmonate or ethylene (Thomma et al., 1999b). Production of camalexin is required for full resistance against Alternaria brassicicola but is not effective against Botrytis cinerea. Previously, camalexin has been shown not to be required for defence against Pseudomonas
20
syringae, Peronospora parasitica or Erysiphe orontii (Glazebrook and Ausubel, 1994; Glazebrook et al., 1997; Reuber et al., 1998). These results support the observation that different defence response pathways in Arabidopsis are effective against different groups of pathogens (Thomma et al., 1998; 2001a). Figure 1 depicts a simple model summarizing our findings. However, the situation in reality is likely to be more complicated, since probably additional pathways exist. Nevertheless, this figure nicely demonstrates the concept that for most pathogens the concerted activation of multiple pathways probably is responsible for resistance, rather than the effect of a single pathway, at least in Arabidopsis (Thomma et al., 2001a). Effector molecules in jasmonate-dependent signalling The Arabidopsis genes encoding PR-1, PR-2 and PR-5 are induced via an SA-dependent signalling pathway (Uknes et al., 1992), in contrast with the Arabidopsis plant defensin gene PDF1.2, which is induced via a pathway involving both jasmonate and ethylene (Penninckx et al., 1996; 1998). Also PR-3 and PR-4 genes are activated in the jasmonate-/ethylene-dependent but SA-independent pathway (Figure 2; Thomma et al., 1998; 1999a). These genes are believed to contribute at least in part to resistance against Botrytis cinerea, since both ethylene and jasmonate, two activators of these genes, confer enhanced resistance to this pathogen (Thomma et al., 1998; 1999a).
Figure 2. Jasmonate and ethylene induce a specific set of Arabidopsis PR-genes. Four-week-old soil-grown wild-type (Col-0) plants were treated with water, 5mM SA, 50 µM MeJA or 50 ppm ethylene and harvested 48 hours after treatment. RNA blots were hybridised with the probes indicated on the left of the figure.
However, also evidence is presented for a jasmonate-dependent, yet ethylene-independent disease response pathway in Arabidopsis. For Alternaria brassicicola it is shown that, additional to camalexin, a jasmonate-inducible but not ethylene-inducible component is required for resistance, since the jasmonate-insensitive mutant coi1-1 shows increased susceptibility to Alternaria brassicicola whereas the ethylene-insensitive mutant ein2-1 does not show an increased susceptibility (Thomma et al., 1998; 1999a). This implies that PDF1.2, PR-3 and PR-4 induction is not required for resistance against this pathogen. Several observations corroborate the existence of such a jasmonate-dependent but not ethylene-dependent determinant of resistance. First, it is possible to boost the resistance of Arabidopsis to Alternaria brassicicola by pre-exposure to methyljasmonate, but not to ethylene (Thomma et al., 1998; 1999a). Secondly, comparative studies have shown that the mutant coi1-1 is considerably more susceptible to Botrytis cinerea than the mutant ein2-1 (unpublished results). Thirdly, resistance to Botrytis cinerea and Plectosphaerella cucumerina can be enhanced in the mutant ein2-1 by pretreatment with methyljasmonate (Thomma et al., 1999a; 2000). Till now, there are no known candidates
21
for the jasmonate-dependent/ethylene-independent effector molecule(s). For the present time we refer to these unknown effector molecules as phytojasmonatin(s), a term which reflects our observation that jasmonate is required for their pathogen-induced production. Jasmonate-/ethylene-dependent signalling might be required for resistance to necrotrophs An obvious question is whether the separate disease response pathways, as discriminated in figure 1, control specific kinds of pathogens. The current data tend to indicate that necrotrophic pathogens, those that first macerate plant tissue by secretion of toxins or enzymes before absorbing nutrients, are among those that are effectively contained by jasmonate/ethylene-controlled effector molecules. On the other hand biotrophic pathogens, those that feed on living plant tissue, are more efficiently countered by salicylate-controlled effector events. Arabidopsis plants compromised in SA-dependent signalling display increased susceptibility to Peronospora parasitica (Delaney et al., 1994; Cao et al., 1997), to Pseudomonas syringae (Cao et al., 1994; Delaney et al., 1994) and to Erysiphe orontii (Reuber et al., 1998), all three biotrophic pathogens, while loss of jasmonate-or ethylenedependent signalling does not seem to affect host susceptibility to Peronospora parasitica (Thomma et al., 1998; 1999a) and jasmonate-insensitivity does not lead to enhanced susceptibility to Erysiphe orontii (Reuber et al., 1998). On the other hand, Arabidopsis plants compromised in jasmonate- or ethylene-dependent signalling display increased susceptibility to Botrytis cinerea, Alternaria brassicicola, Plectosphaerella cucumerina (Thomma et al., 1998; 1999a; 2000), Erwinia carotovora (Norman-Setterblad et al., 2000) and Pythium spp. (Staswick et al., 1998; Vijayan et al., 1998), all necrotrophic pathogens, whereas SAdependent signalling has shown not to be required for resistance to at least Botrytis cinerea and Alternaria brassicicola (Thomma et al., 1998; 1999a). In reality It is obvious that the model presented in figure 1 is an oversimplification of reality and many remarks can be made. First, this model depicts independent signalling cascades, whereas several studies have shown that cross-talk can occur between SA-, jasmonate- and ethylene-dependent responses. For instance, we have shown that the level of camalexin-accumulation is reduced in the Arabidopsis genotypes NahG and ein2-1, indicating that both SA and ethylene might play some role in the induction of camalexin, though the accumulation of camalexin could not be induced by exogenous application of these signalling molecules (Thomma et al., 1999b). Although the jasmonate-insensitive Arabidopsis mutant coi1-1 does not show reduced camalexin accumulation upon Alternaria brassicicola inoculation (Thomma et al., 1999b), this mutant has been reported to show reduced camalexin accumulation upon inoculation with Pseudomonas syringae (Glazebrook, 1999), indicating that also jasmonate is somehow involved in camalexin accumulation. Thus, a considerable amount of cross talk is likely to occur between all pathways. Though the model might evoke discussion about upstream interactions, the major innovation it brings over previous models is that it takes into consideration that pathogens are not restrained by the exclusive induction of a specific pathway, but merely by the action of a cocktail of components supplied by several inducible resistance response pathways. Successful containment of any potential invader probably requires an appropriate mixture of defence responses. Plants react to pathogen infection by the induction of several resistance response pathways, although the induced pathways do not always provide a noticeable contribution to the containment of the pathogen. However, there is some selectivity in the pathways that are induced, since infection with Erwinia carotovora does induce PDF1.2 but not PR-1 and vice versa for Pseudomonas syringae (Thomma et al., 2001b).
22
In nature, plants defend themselves against pathogens by constitutive barriers and inducible defence responses. It is conceivable that at least all constitutive barriers have to be broken down by an invading microorganism in order to successfully colonise the plant and cause disease. Therefore the ability to break down constitutive barriers might determine the virulence of the pathogen. A model based on this multishield concept is depicted in figure 3. Upon recognition of a pathogen that has successfully breached the constitutive shields, a number of inducible defence shields are erected, though not all barriers are erected upon recognition of a particular pathogen (Thomma et al., 2001b). Furthermore, not all barriers that are erected are effective against the attacking pathogen. If the pathogen is avirulent or poorly virulent the pathogen gets contained somewhere along the succession of shields, either completely or in major part. The speed at which a shield is build up and its efficacy to a particular pathogen determine whether or not an invading microorganism succeeds in causing disease.
Figure 3. The multi-shield defence system in Arabidopsis plants. Plant cells possess a number of constitutive defence “shields”, and a number of barriers that can be erected upon pathogen recognition. Recognition of different pathogens might lead to a combination of different inducible defence shields, some of which are not effective against the invading pathogen, while others are. Some shields are highly effective in containing the pathogen, as shown for the hypersensitive response in case of avirulent Pseudomonas or Peronospora species while other shields are only partially effective. The reduction in thickness of the arrows representing the pathogen infections is not a quantitative measure for the contribution of the defence shield, but merely indicates which shields contribute at least in part to the control of a pathogen.
23
Acknowledgements B.P.H.J.T. and I.A.M.A.P. are postdoctoral fellows of the Fonds voor wetenschappelijk Onderzoek Vlaanderen. B.P.A.C. is project leader of the Flanders Interuniversity Institute for Biotechnology (VIB). Drs. X. Dong, J. Glazebrook, J. Ryals and J. Turner are acknowledged for kindly providing seeds of the genotypes NahG, npr1-1, coi1-1 and pad3-1, respectively. Dr. B. Mauch-Mani is acknowledged for providing the fungus Plectosphaerella cucumerina.
References Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A. & Vanderleyden, J. 1990: An automated quantitative assay for fungal growth. FEMS Microbiol.Lett. 69: 55-60. Cao, H., Bowling, S.A., Gordon, A.S. & Dong, X. 1994: Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6: 1583-1592. Cao, H., Glazebrook, J., Clarke, J.D., Volko, S., & Dong, X. 1997: The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88: 57-63. Delaney, T.P., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., Gaffney, T., Gut-Rella, M., Kessmann, H., Ward, E., et al. 1994: A central role of salicylic acid in plant disease resistance. Science 266: 1247-1250. Dempsey, D.A., Shah, J. & Klessig, D.F. 1999: Salicylic acid and disease resistance in plants. Crit. Rev. Plant. Sci. 18: 547-575. Feys, B.J.F., Benedetti, C.E., Penfold, C.N. & Turner, J.G. 1994: Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6: 751-759. Glazebrook, J. 1999: Genes controlling expression of defense responses in Arabidopsis. Curr. Opin. Plant Biol. 2: 280-286. Glazebrook, J. & Ausubel, F.M. 1994: Isolation of phytoalexin-deficient mutants of Arabidopsis thaliana and characterization of their interactions with bacterial pathogens. Proc.Natl.Acad.Sci.USA 91: 8955-8959. Glazebrook, J., Zook, M., Mert, F., Kagan, I., Rogers, E.E., Crute, I.R., Holub, E.B., Hammerschmidt, R. & Ausubel, F.M. 1997: Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics 146: 381-392. Guzmán, P. & Ecker, J.R. 1990: Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. Plant Cell 2: 513-523. Norman-Setterblad, C., Vidal, S. & Palva, E.T. 2000: Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora, Mol. Plant-Microbe Interact. 13: 430-438. Penninckx, I.A.M.A., Eggermont. K., Terras. F.R.G., Thomma, B.P.H.J., De Samblanx, G.W., Buchala, A., Métraux, J.-P., Manners, J.M. & Broekaert W.F. 1996: Pathogeninduced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8: 2309-2323. Penninckx, I.A.M.A., Thomma, B.P.H.J., Buchala, A., Métraux, J.-P. & Broekaert, W.F. 1998: Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: 2103-2114. Reuber, T.L., Plotnikova, J.M., Dewdney, J., Rogers, E.E., Wood, W. & Ausubel, F.M. 1998: Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants. Plant J. 16 : 473-485.
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Staswick, P.E., Yuen, G.Y. & Lehman, C.C. 1998: Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare. Plant J. 15: 747-754. Thomma, B.P.H.J., Eggermont, K., Broekaert, W.F. & Cammue, B.P.A. 2000: Disease development of several fungi on Arabidopsis can be reduced by treatment with methyl jasmonate. Plant Physiol. Biochem. 38: 421-427. Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A., Mauch-Mani, B., Vogelsang, R., Cammue, B.P.A. & Broekaert, W.F. 1998: Separate jasmonate-dependent and salicylatedependent defense response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA 95: 15107-15111. Thomma, B.P.H.J., Eggermont, K., Tierens K.F.M.-J. & Broekaert, W.F. 1999a: Requirement of functional EIN2 (ethylene insensitive 2) gene for efficient resistance of Arabidopsis thaliana to infection by Botrytis cinerea. Plant Physiol. 121: 1093-1101. Thomma, B.P.H.J., Nelissen, I., Eggermont, K. & Broekaert, W.F. 1999b: Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola. Plant J. 19: 163-171. Thomma, B.P.H.J., Penninckx, I.A.M.A., Broekaert, W.F. & Cammue, B.P.A. 2001a: The complexity of disease signaling in Arabidopsis. Curr. Opin. Immunol. 13: 63-68. Thomma, B.P.H.J., Tierens K.F.M.-J., Penninckx, I.A.M.A., Mauch-Mani, B., Broekaert, W.F. & Cammue, B.P.A. 2001b: Different micro-organisms differentially induce Arabidopsis disease response pathways. Plant Physiol. Biochem. 39 (in press). Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E. & Ryals, J. 1992: Acquired resistance in Arabidopsis. Plant Cell 4: 645-656. Vijayan, P., Shockey, J., Lévesque, C.A., Cook, R.J. & Browse, J. 1998: A role for jasmonate in pathogen defense of Arabidopsis. Proc.Natl.Acad.Sci.USA 95: 7209-7214.
Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 25-32
Involvement of EDS1 and PAD4 at multiple levels of plant defense Jane E. Parker1,3, Bart J. Feys1, Lisa J. Moisan1, Marie-Anne Newman1, Christine Rustérucci1, Danny Aviv2, Jeffery L. Dangl2 1 Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK 2 Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA 3 Current address: Department of Molecular Plant Pathology, Max-Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, D-50829 Cologne, Germany Abstract: We have used the model plant Arabidopsis to identify genes required for pathogen recognition (Resistance or R genes) and for activation of local plant defenses. Isolation of these genes and characterization of their functions has been undertaken by comparing mutant and wild-type plant resistance phenotypes, cloning and molecular analysis of the respective proteins. In particular, we have examined the roles of EDS1 and PAD4, two lipase-like proteins that positively regulate resistance mediated by a structural subset of R gene. They share certain motifs and interact in a yeast two-hybrid assay, suggesting that physical association may be important to their cellular functions. However, they have intrinsically different roles within the resistance pathway. Whereas EDS1 is required for elaboration of the localized hypersensitive response (HR) and its accompanying oxidative burst (producing reactive oxygen intermediates, ROI), PAD4 functions downstream or independently of these events. However, both EDS1 and PAD4 are required for accumulation of the SAR signalling molecule, salicylic acid (SA) in the R gene-mediated response. The genetic and molecular data lead us to position EDS1, in the absence of PAD4, as a regulator of early plant responses but together with PAD4 in potentiation of plant defenses. In support of this model we find that EDS1 and PAD4 are equally required for basal resistance against several virulent pathogens. Moreover, EDS1 and PAD4 are necessary for signalling leading to SAR. Genetic epistasis studies also establish that EDS1 and PAD4 are essential components of runaway cell death in lsd1 mutant plants that can triggered by pathogen inoculation or artificial provision of ROI. Thus, EDS1 and PAD4 may process an ROIdependent signal in a defense potentiation loop. We are now examining EDS1 and PAD4 molecular associations in plant cells. Key words: pathogen, plant defense, reactive oxygen intermediates, Resistance genes, salicylic acid, signalling
Introduction Analysis of genetic variation in plant responses to pathogens has identified corresponding gene pairs (Resistance or R genes in the plant and avirulence or avr genes in the pathogen) that mediate recognition and lead to induction of plant resistance (Staskawicz et al., 1995; Feys and Parker, 2000). These local plant defenses are commonly associated with a form of programmed plant cell death known as the hypersensitive response (HR). Localized necrosis can also induce a plant immune response called systemic acquired resistance (SAR) that heightens defenses in uninoculated tissues against a broad spectrum of pathogens (McDowell and Dangl, 2000). One of the earliest biochemical changes associated with the HR is an oxidative burst producing reactive oxygen intermediates (ROI), including superoxide (O2-.) which is rapidly dismutated to hydrogen peroxide (H2O2) (Bolwell, 1999). Salicylic acid (SA), a phenolic molecule, accumulates in plant tissues responding to pathogen infection and is essential for 25
26
induction of SAR as well as being required for certain R gene-mediated responses (Feys and Parker, 2000). One of the major challenges in Molecular Plant Pathology is now to unravel signal transduction processes after plant-pathogen recognition that first initiate, and then restrict, the plant HR. We and others have used genetic approaches in Arabidopsis to isolate recognition and signalling components of plant defense against pathogens. The products of R genes cloned in Arabidopsis belong to the most prevalent R protein class that contains a central nucleotide binding (NB-) domain and varying numbers of carboxy-terminal leucine-rich-repeats (LRRs) (Dangl and Jones, 2001). NB-LRR proteins have been further divided into those with a coiled-coil (“CC”) motif at their amino termini and those that have amino-terminal (“TIR”) similarity to the cytoplasmic domains of human and Drosophila Toll-like receptors. Mutational analyses in Arabidopsis have uncovered positive regulators of basal defense (Glazebrook, 1999; Feys and Parker, 2000). EDS1 is a necessary component of resistance to the oomycete pathogen, Peronospora parasitica, and the bacterial pathogen, Pseudomonas syringa, mediated by R genes encoding ‘TIR-NB-LRR’ proteins (Aarts et al., 1998). EDS1 is not, however, required for resistance conferred by any of the tested ‘CC-NB-LRR’ R genes (Aarts et al., 1998). Many, but not all, CC-NB-LRR R genes examined are dependent on NDR1(Aarts et al., 1998). EDS1 encodes a 72 kDa lipase-like protein that operates upstream of SA-mediated defenses (Falk et al., 1999) whereas NDR1 encodes a 25 kDa protein that has two putative membrane attachment domains (Century et al., 1997). Mutational screens in Arabidopsis identified other plant defense signalling genes that are components of SA signalling in plant responses against pathogens. For example, PAD4 (Glazebrook et al., 1997; Zhou et al., 1998), SID1/EDS5 and SID2 (Rogers and Ausubel, 1997; Nawrath and Métraux, 1999) function upstream of SA accumulation, whereas NPR1/NIM1 is an important regulator of responses downstream of SA (Cao et al., 1994; Delaney et al., 1995). Significantly, PAD4 encodes a lipase-like protein with catalytic motifs identical to EDS1 (Jirage et al., 1999). Other Arabidopsis mutations deregulate disease resistance responses and/or plant cell necrosis, suggesting that negative control of plant defense pathways also occurs (Morel and Dangl., 1997). Of these, some exhibit a ‘disease lesion-mimic’ phenotype that is a feature of several well characterized crop plant mutants in which necrotic lesions form spontaneously or can be induced by various biotic or abiotic stresses. Arabidopsis plants carrying the recessive null lsd1 allele produce a normal HR following infection by avirulent pathogens, but initiate runaway cell death (rcd) at the margins of infection foci (Dietrich et al., 1994). Spreading lesions in lsd1 can be induced by provision of superoxide (O2-.; Jabs et al., 1996) in uninfected tissues. This, together with observations that O2-. accumulation precedes lesion formation (Jabs et al., 1996), suggests that LSD1 gauges and responds to a superoxide-dependent signal(s) emanating from an infection site. SA possibly potentiates this pathway, since lsd1 plants are acutely responsive to treatments with SA or chemically active SA-analogues (Dietrich et al., 1994; Jabs et al., 1996). It is envisaged that wild-type LSD1 may contribute to establishing a boundary to the plant HR by either negatively regulating a pro-death pathway component or activating a repressor of plant cell death (Dietrich et al., 1994). LSD1 encodes a zinc-finger protein with homology to GATA-type transcription factors (Dietrich et al., 1997). In this article we describe genetical and molecular analysis of EDS1, PAD4 and LSD1. Our results show that EDS1 and PAD4 are recruited by the same set of R genes in plant resistance. We present further data supporting the notion that a major role of EDS1 and PAD4 is to drive an LSD1-modulated defense signal potentiation loop, possibly by direct physical association between the EDS1 and PAD4 proteins.
27
Material and methods Plant materials, pathogen isolates and pathology experiments Pathogen isolates Cala2, Emoy2, Emwa1, Emco5 and Hiks1 were kindly provided by Eric Holub (Horticultural Research International, Wellesbourne, UK). Plant wild-type and mutant lines and other materials used in these analyses are described elsewhere (Feys et al., 2001; Rustérucci et al., 2001). Inoculations of plants with P. parasitica and phenotypic analysis were performed as described previously (Parker et al., 1996). Analysis of SA levels in plant tissues was done according to Newman et al. (2001). Yeast two-hybrid Assays The LexA two-hybrid system (kindly provided by Roger Brent, Massachusetts General Hospital, Boston; Gyuris et al., 1993) was used in conjunction with an Arabidopsis cDNA expression library, as described by Feys et al. (2001).
Results and discussion EDS1 and PAD4 are required by the same spectrum of R genes Null eds1 and pad4 mutant lines were identified in two Arabidopsis landraces, Landsbergerecta (Ler; eds1-2 and pad4-2) and Wassileskija (Ws-0; eds1-1 and pad4-5). This allowed us to compare the effects of each mutation on R gene-mediated resistance in the same genetic background. Results of this analysis are shown in Table 1. Table 1. Suppression of RPP gene-mediated resistance to P. parasitica in leaves of eds1 and pad4 in accessions Ler and Ws-0 Plant R gene (P. parasitica isolate) Plant Line
RPP5 (Noco2)
RPP8 (Emco5)
RPP4/8 (Emwa1)
RPP21 (Maks9)
RPP7 (Hiks1)
(Cala2)
Ler
R
R
R
R
R
S
eds1-2
S*
R
R
S*
R
S*
pad4-2
(S)
R
R
(S)
R
S*
Plant Line
RPP1A,B,C (Noco2)
RPP1A,B (Emoy2)
RPP1A (Cala2)
(Emwa1)
Ws-0
R
R
R
S
eds1-1
S*
S*
S*
S-S*
pad4-5
(S)
(S)
(S)
S-S*
Seedlings were scored after inoculation with different P. parasitica isolates that are recognized by particular RPP (Resistance to P. parasitica) genes, as indicated. Ler is genetically susceptible to P. parasitica isolate Cala2 and Ws-0 is susceptible to isolate Emwa1. Phenotypes were assigned as R (fully resistant, wild-type HR); S (susceptibility of genetically compatible lines); S* (hypersusceptible, permitting more abundant sporulation
28
than genetically susceptible line); (S) (partially susceptible, mycelium development accompanied by trailing plant cell necrosis and occasional sporophores). Data were taken from Feys et al., 2001. These experiments revealed that RPP genes with a strong requirement for EDS1 were also dependent on PAD4. While loss of EDS1 function caused a complete suppression of R gene-mediated resistance, mutations in PAD4 resulted in a partial suppression of resistance. Further phenotypic analysis showed that in these responses EDS1 operates upstream of the HR and its associated oxidative burst whereas PAD4 activity is required downstream or independently of the HR and ROI production (Rusterucci et al., 2001). A similar trend was observed after inoculation of wild-type and eds1 or pad4 mutant plants with P. syringae pv. tomato expressing different avr genes. Thus, while eds1 and pad4 are necessary components of RPS4-mediated resistance to Ps. tomato expressing avrRps4, they are dispensible for RPM1-mediated resistance to Ps. tomato expressing avrRpm1 (Feys et al., 2001; Rusterucci et al., 2001). We concluded from these analyses that EDS1 and PAD4 are likely to operate within the same plant defense pathway, although they fulfil different roles.
SA (µg/g FW)
DC3000/avrRPS4 14 12 10 8 6 4 2 0
Ler eds1 pad4
0
24
48
Time (hr)
SA (µg/g FW)
DC3000/avrRpm1 14 12 10 8 6 4 2 0
Ler eds1 pad4
0
24
48
Time (hr)
Figure 1. Accumulation of total SA in wild-type and mutant plants after bacterial inoculation. (From Feys et al., 2001)
EDS1 and PAD4 regulate SA accumulation in RPS4-mediated defense Earlier studies showed that PAD4 is an essential regulatory component of SA accumulation after inoculation with virulent P. syringae (Zhou et al., 1998). EDS1 was also placed upstream of SA-dependent defenses in resistance conditioned by RPS4 (Falk et al., 1999). We therefore compared SA accumulation profiles in wild-type, eds1 and pad4 null mutant plants after
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bacterial inoculation. As shown in Figure 1, SA accumulated in tissues of wild-type plants after inoculation with either Ps. tomato expressing avrRps4 (recognized by an EDS1- and PAD4-dependent R gene, RPS4) or avrRpm1 (recognition conferred by an EDS1- and PAD4independent R gene, RPM1). SA accumulation was abolished in eds1 and severely reduced in pad4 in RPS4-triggered responses (Figure 1). However, SA levels were not depleted in eds1 or pad4 in RPM1-conditioned resistance. We deduced from these tests that EDS1 and PAD4 are important regulators of SA generation only in R gene-triggered responses that recruit them. Since EDS1 and PAD4 are dispensable for SA generation in RPM1 resistance, we conclude that the EDS1 and PAD4 proteins are not part of the SA biosynthetic machinery itself. EDS1 and PAD4 interact specifically in a yeast two-hybrid assay The EDS1 protein was used as a bait in a yeast two-hybrid assay to identify interactors that may be part of a complex in the plant (Feys et al., 2001). One major interactor in this assay was PAD4. Association between EDS1 and PAD4 was maintained when PAD4 was inserted as the bait, as shown in Figure 2. EDS1 was also found to interact with itself. However, PAD4 was not able to dimerize in yeast. Control assays were performed in glucose medium (+GLU) to show that expression of the β-galacotsidase reporter gene (producing blue colonies) was dependent on galactose-inducible (+GAL) expression of the prey construct (Figure 2). Other constructs using Bicoid protein as bait or association between p53 and SV40-T protein served as negative and positive controls, respectively.
Figure 2. Association of PAD4 and EDS1 in a yeast two-hybrid assay. The genetic relationship of EDS1 and PAD4 (described above), coupled with their physical interaction in yeast shown here, suggests that direct EDS1-PAD4 association may be important for their defense signalling functions. Experiments using epitope-tagged forms of EDS1 and PAD4 are now in progress to investigate protein expression and molecular interactions in plant cells. Based on the phenotypic and molecular data, we envisage that EDS1 fulfils an early signalling role in R gene-mediated resistance that is upstream of the plant oxidative burst and HR and is independent of PAD4. We speculate that EDS1 has a second activity that, together with PAD4, is required for full expression of resistance. The second function may be needed to potentiate plant defenses in tissues around initial infection sites. This idea is supported by the fact that both EDS1 and PAD4 are necessary for SA accumulation (Figure 1). Recent analyses show that SA, in combination with ROI, serves to amplify plant resistance responses (Shirasu et al. 1997; Klessig et al., 2000). Exogenous applications of SA also enhance EDS1 and PAD4 expression (Falk et al., 1999; Jirage et al., 1999) implying that EDS1 and PAD4 drive an SA-associated positive feedback loop.
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EDS1 and PAD4 are essential for initiation of runaway cell death (rcd) in lsd1 mutant plants Double mutant lines between the eds1 or pad4 and lsd1 were constructed and their effects on lsd1-induced rcd assessed after pathogen infection, treatment with BTH, (benzothiodiazole, a functional SA mimic) or applications of a superoxide generator, rose bengal (Rustérucci et al., 2001). We found that both EDS1 and PAD4 are necessary for lsd1conditioned rcd initiated by each tested stimulus. Importantly, the requirement for EDS1 and PAD4 in lsd1 rcd is separable from events associated with the local HR and disease resistance. These data reinforce the notion that EDS1 and PAD4 operate at the level of defense signal potentiation. As shown in the model in Figure 3, EDS1, but not PAD4, functions upstream of localized HR and ROI production in resistance conditioned by TIR-NB-LRR type R genes. In contrast, resistance conditioned by CC-NB-LRR type R genes operates independently of EDS1 and PAD4. Irrespective of the different requirements for EDS1 and PAD4 at initial infection foci, both components are essential for signal relay leading to rcd in lsd1 in tissues adjacent to infection sites. Since EDS1 and PAD4 are also required for lsd1 rcd in response to artificial provision of ROI or BTH, we propose that EDS1 and PAD4 positively regulate an ROI/SAdependent defense signal amplification loop that is negatively regulated by LSD1.
Figure 3. Model describing two functions of EDS1 and the relationship between EDS1, PAD4 and LSD1 in defense potentiation. (From Rustérucci et al., 2001) Other analyses have revealed a requirement for EDS1 and PAD4 in constitutive SAdependent resistance pathways induced by the cpr1 and cpr6 mutations (Clarke et al., 2001; Jirage et al., 2001) that is also consistent with resistance-potentiating roles for these components. Furthermore, EDS1 and PAD4 are necessary for limiting growth of a number of virulent pathogens (Parker et al., 1996; Glazebrook et al., 1997; Aarts et al., 1998). We therefore speculate that the primary activities of EDS1 and PAD4 may be in plant defense potentiation or priming and that this mechanism is in some way engaged by a subset of R proteins to elicit rapid cellular reprogramming upon recognition of an avirulent pathogen.
Acknowledgements We thank the Gatsby Charitable Foundation and The British Biotechnology and Biological Research Council for funding. We are also grateful to Eric Holub for providing P. parasitica isolates.
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References Aarts, N., Metz, M., Holub, E., Staskawicz, B.J., Daniels, M.J. and Parker, J.E. (1998). Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signalling pathways in Arabidopsis. Proc. Natl. Acad. Sci. USA 95, 10306-10311. Bolwell, G.P. (1999). Role of active oxygen species and NO in plant defense responses. Curr. Op. Plant Biol. 2, 287-294. Cao, H., Bowling, S.A., Gordon, S. and Dong, X. (1994). Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6, 1583-1592. Century, K.S., Shapiro, A.D., Repetti, P.P., Dahlbeck, D., Holub, E. and Staskawicz, B.J. (1997). NDR1, a pathogen-induced component required for Arabidopsis disease resistance. Science 278, 1963-1965. Clarke, J.D., Aarts, N., Feys, B.J., Dong, X. and Parker, J.E. (2001). Constitutive disease resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is partially EDS1dependent in cpr5. Plant J. 26, 409-420. Dangl, J.L. and Jones, J.D.G. (2001). Plant pathogens and integrated defense responses to infection. Nature 411, 826-833. Delaney, T.P., Friedrich, L. and Ryals, J.A. (1995). Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proc.Natl.Acad.Sci.USA 92, 6602-6606. Dietrich, R.A., Delaney, T.P., Uknes, S.J., Ward, E.R., Ryals, J.A. and Dangl, J.L. (1994). Arabidopsis mutants simulating disease resistance response. Cell 77, 565-577. Dietrich, R.A., Richberg, M.H., Schmidt, R., Dean, C. and Dangl, J.L. (1997). A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death. Cell 88, 685-694. Falk, A., Feys, B.J., Frost, L.N., Jones, J.D.G., Daniels, M.J. and Parker, J.E. (1999). EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases. Proc. Natl. Acad. Sci. USA 96, 3292-3297. Feys, B.J. and Parker, J.E. (2000). Interplay of signalling pathways in plant disease resistance. Trends Genet. 16, 449-455. Feys, B.J., Moisan, L.J., Newman, M.A. and Parker, J.E. (2001). Direct interaction between the Arabidopsis disease resistance signalling proteins, EDS1 and PAD4. EMBO J., (under review). Glazebrook, J. (1999). Genes controlling expression of defense responses in Arabidopsis. Curr. Op. Plant Biol. 2, 280-286. Glazebrook, J., Zook, M., Mert, F., Kagan, I., Rogers, E.E., Crute, I.R., Holub, E.B., Hammerschmidt, R. and Ausubel, F.M. (1997). Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics 146, 381-392. Gyuris, J., Golemis, E., Chertkov, H. and Brent, R. (1993). Cdi1, a Human G1-Phase and SPhase Protein Phosphatase That Associates with Cdk2. Cell, 75, 791-803. Jabs, T., Dietrich, R.A. and Dangl, J.L. (1996). Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273, 1853-1856. Jirage, D., Tootle, T.L., Reuber, T.L., Frost, L.N., Feys, B.J., Parker, J.E., Ausubel, F.M. and Glazebrook, J. (1999). Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signalling. Proc. Natl. Acad. Sci. USA 96, 13583-13588.
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Jirage, D., Zhou, N., Cooper, B., Clarke, J.D., Dong, X. and Glazebrook, J. (2001). Constitutive salicylic acid-dependent signalling in cpr1 and cpr6 mutant requires PAD4. Plant J. 26, 395-407. Klessig, D.F., Durner, J., Noad, R., Navarre, D.A., Wendehenne, D., Kumar, D., Zhou, J.M., Shah, J., Zhang, S., Kachroo, P., Trifa, Y., Pontier, D., Lam, E., Silva, H. (2000). Nitric oxide and salicylic acid signalling in plant defense. Proc. Natl. Acad. Sci. USA 97, 88498855. McDowell, J.M. and Dangl, J.L. (2000). Signal transduction in the plant immune response. Trends Biochem.Sci. 25, 79-82. Morel, J.B. and Dangl, J.L. (1997). The hypersensitive response and the induction of cell death in plants. Cell Death and Differentiation 4, 671-683. Nawrath, C. and Metraux, J.-P. (1999). Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11, 1393-1404. Newman, M.A., von Roepenack-Lahaye, E., Parr, A., Daniels, M.J. and Dow, J.M. (2001) Induction of hydroxycinnamoyl-tyramine conjugates in pepper by Xanthomonas campestris: a plant defense response activated by hrp gene-dependent and -independent mechanisms. Mol. Plant Microbe Interact., in press. Parker, J.E., Holub, E.B., Frost, L.N., Falk, A., Gunn, N.D. and Daniels, M.J. (1996). Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 8, 2033-2046. Rogers, E.E. and Ausubel, F.M. (1997). Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression. Plant Cell 9, 305-316. Rustérucci, C., Aviv, D.H., Holt III, B.F., Dangl, J.L. and Parker, J.E. (2001). The disease resistance signalling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis. Plant Cell, in press. Shirasu, K., Nakajima, H., Rajasekhar, V.K., Dixon, R.A. and Lamb, C. (1997). Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms. Plant Cell 9, 261-270. Staskawicz, B.J., Ausubel, F.M., Baker, B.J., Ellis, J.G. and Jones, J.D.G. (1995). Molecular genetics of plant disease resistance. Science 268, 661-667. Zhou, N., Tootle, T.L., Tsui, F., Klessig, D.F. and Glazebrook, J. (1998). PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis. Plant Cell 10, 1021-1030.
40Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 33-40
Activation of novel signalling pathways by phloem-feeding whiteflies Wilhelmina van de Ven1, David Puthoff1, Cynthia LeVesque2, Thomas Perring2, Linda L. Walling1 1 Department of Botany and Plant Sciences and 2Department of Entomology, University of California, Riverside, CA 92521-012, USA.
Abstract: To investigate the signaling pathways induced by phloem-feeding whiteflies, tomato wound- and defense-response gene transcripts were evaluated after silverleaf whitefly (Bemisia argentifolii) and greenhouse whitefly (Trialeurodes vaporariorum) feeding. Temporal and spatial studies indicate that whiteflies are perceived as a bacterial or fungal pathogens and induce distinct gene sets from insects that cause extensive tissue damage. To identify novel genes that were induced in tomato and squash after whitefly feeding, differential RNA display was used. Genes expressed in apical, non-infested squash leaves after silverleaf whitefly feeding were isolated. SLW1 (SLIVERLEAF WHITEFLY-INDUCED 1) and SLW3 RNAs accumulated in apical, non-infested leaves (systemically) after feeding by silverleaf whitefly nymphs but not after feeding by silverleaf whitefly adults or sweet potato whitefly (B. tabaci Type A) adults or nymphs. Differences in SLW1 and SLW3 expression were also detected in infested leaves. SLW1 RNAs were detected in flowers and fruit, while SLW3 RNAs were not detected in any organ. Whitefly infestations did not alter this developmental programming. SLW1 (M20b peptidase-like gene) and SLW3 (a β-glucosidase-like protein gene) are modulated by different signaling pathways. SLW1 RNAs accumulate in response to exogenous MeJA. In contrast, the defense signal modulating SLW3 RNA levels is not known. SLW3 RNA levels are not influenced by pathogen infection, wounding, infection, MeJA, ethylene, salicylic acid, ABA, or reactive oxygen species. Transgenic tomato plants expressing a SLW3:GUS gene will be used as bioassay plants to assess the whitefly-induced signal important in SLW3 expression. Studies of tomato wound and defense response genes indicate that changes plant gene expression in response to whitefly feeding is distinct from aphids and caterpillars. Key words: whiteflies, defense, systemic signaling, phloem-feeding, elicitors, saliva
Introduction Induced resistance is a dynamic response providing a long-lasting and systemic resistance to a broad spectrum of pathogens and pests. Induced resistance is activated by a variety of chemicals, pathogens, biocontrol bacteria, and herbivores (Kúc, 2001). It is clear that multiple mechanisms of induced resistance are active in plants. Pathogen-induced systemic acquired resistance is mediated by salicylic acid (SA), while the induced systemic resistance activated by biocontrol rhizobacteria is mediated by jasmonic acid (JA) and ethylene. Additional signaling pathways are also important in the development of induced resistance and are currently being dissected. Furthermore, an induced resistance has also been described after wounding and herbivore attack (Karban and Baldwin, 1997). The chemical nature, quantity and duration of elicitor exposure influences the activation and cross talk of the defense signaling pathways activated. While an understanding of induced resistance mediated by plant-pathogen and -rhizobacteria interactions is emerging, less is known about plant-insect interactions and when plants are challenged sequentially or simultaneously by pathogens and herbivores (for reviews, Bostock, 1999; Felton and Korth, 2000; Walling, 2000).
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To understand the nature of induced resistance in plant-herbivore interactions, it is critical to understand the identity of the signaling pathways activated or suppressed upon challenge with herbivores. There is an emerging literature on the changes in plant gene expression that occurs after tissue-damaging herbivore attack (Reymond et al., 2000; Ryan, 2000; Stotz et al., 2000; Baldwin et al., 2001). Similar to mechanical wounding, the octadecanoid pathway is activated producing an array of oxylipins, including JA, that have a role in activating defense responses (Vollenweider et al., 2000). Many proteins and secondary metabolites that accumulate in response to JA actively deter herbivore feeding, growth and development, oviposition, or fecundity. While there is substantial overlap in the changes in plant metabolism and gene expression after wounding and tissue-damaging insect feeding, these responses are not equivalent (Reymond et al., 2000; Walling, 2000). Insect saliva may provide suppressors or activators of defense pathways (Eichenseer et al., 1999; Miles, 1999; Felton, 2001). Herbivore oral secretions influence the blend of volatiles synthesized and released, enhance JA accumulation and proteinase inhibitor RNA levels, and suppress nicotine accumulation (Korth and Dixon, 1997; Dicke et al., 1999; Páre and Tumlinson, 1999; Kahl et al., 2000). Furthermore, many Arabidopsis genes that respond to wounding are not induced or induced to a lesser extent by larval feeding (Reymond et al., 2000). There remains a large number of herbivore feeding guilds where there is a more limited knowledge about the changes in plant gene expression, including leaf miners, phloem-feeders, and xylem-feeders. It appears that herbivores that cause little tissue-damage provoke a different array of responses in plants than tissue-damaging herbivores. Studies with several herbivore-plant interactions suggest that some plants accumulate pathogenesis-related (PR) proteins and activities, which are commonly associated with plant responses to pathogens (for review, Kombrink and Somssich, 1997; Walling, 2000). More recently, Moran and Thompson (2001) have shown that green peach aphid (Myzus persicae)-infested leaves of A. thaliana accumulate the PR-1, BGL2, PAL, and PDF1.2 transcripts. To provide a comprehensive understanding of the changes in plant gene expression in response to phloem-feeding insects, four different plant-whitefly interactions have been examined. The changes in tomato defense- and wound-response gene RNAs after feeding by two different whitefly species, the silverleaf whitefly (Bemisia argentifolii) and the greenhouse whitefly (Trialeurodes vaporariorum) were investigated. In addition, to examine the specificity of plant responses to whiteflies, squash responses to two closely related whitefly species, the silverleaf and sweet potato whitefly (B. tabaci Type A) were studied. These studies indicate that whiteflies produce both shared and species-specific elicitors that activate established and novel signaling pathways.
Material and methods Plant growth and insect rearing Cucurbita pepo L. cv. Chefini, Lycopersicon esculentum L. cv. Rutgers, cv. UC82b and cv. UC82b LapA1:GUS (Chao et al., 1999) plants were grown in soil supplemented with osmocote in a growth chamber with 14-hr (24.5°C)/10-hr (21.5°C) light/dark cycle. Plants were monitored weekly to ensure plants were not contaminated with additional insects. Bemisia argentifolii Bellows and Perring (B. tabaci Type B, silverleaf whitefly), B. tabaci Gennadius Type A (sweet potato whitefly), and Trialeurodes vaporariorum Westwood (greenhouse whitefly) were reared in separate greenhouses in insect-proof cages on Phaseolus vulgaris L. The whitefly cultures were assayed periodically for isozyme variants to ensure culture purity. Two weeks prior to infestations, plants were transferred to insect cages in the greenhouse. Infestation methods and controls for squash-whitefly and tomato-whitefly studies are detailed in van de Ven et al. (2000) and Puthoff et al. (Puthoff et al., 2001), respectively.
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Wound, defense and stress treatments Treatments are described in detail in van de Ven et al. (2000). Immediately after treatments, leaves were excised and placed into liquid nitrogen. Leaves were wounded by crushing with a needle-nosed pliers as described by Pautot et al. (1991) The wounded leaves and apical, nonwounded leaves were harvested 24 hr later. Pseudomonas syringae pv syringae was infiltrated into leaves as described by Smith et al. (1991). Infected leaves and apical, non-infected leaves were harvested 10 and 19 hr later. Shoots from 3-week-old squash plants were excised just above the second true leaf for salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), and ethylene treatments. The 24-hr MeJA (10 µM) and control treatments were performed as described by Gu et al. (1996). The SA (0.5 mM), ABA (100 µM), ethylene (10 ppm) and control treatments were performed as described by Chao et al. (1999). Squash plants were subjected to water-deficit stress by withholding water for three days and control plants were watered daily. Some of the leaves were used for measuring relative water content (van de Ven et al., 2000). Treatments with glucose and glucose oxidase and sodium nitroprusside treatments are detailed in van de Ven et al. (2000). RNA blots and GUS histochemical staining Total RNA was isolated from squash tissues using the procedure described in Martienssen et al. (1989) and tomato using the procedure described in Puthoff et al. (2001). RNA gel blot hybridizations were performed with 15 µg total RNA according to Pautot et al. (1991). Probes were labeled with [α-32P]-dCTP using the Prime-a-Gene labeling system (Promega) or by nick-translation. The SLW1 and SLW3 cDNAs were previously described (van de Ven et al., 2000). The PR-1 (P6 protein), PR-4 (P2 protein), Chi3 (acidic chitinase), Chi9 (basic chitinase), GluB (basic β-1,3-glucanase), and GluAC (acidic β-1,3-glucanase) cDNA clones were described previously (van Kan et al., 1992). The proteinase inhibitor 2 (pin2, Graham et al., 1985) and leucine aminopeptidase (LapA, Pautot et al., 1993) cDNA clones were previously described. LapA1:GUS plants and histochemical staining methods were described (Chao et al., 1999; Puthoff et al., 2001).
Results and discussion Tomato-whitefly interactions Tomatoes provide a well-characterized system to study plant responses to herbivores. Tomato responses to wounding and tissue-damaging herbivores are established (Peña-Cortés et al., 1996; Wasternack et al., 1998; Ryan, 2000; Howe et al., 2001; Pautot et al., 2001). In addition, an array of JA- and SA-regulated PR genes that respond to pathogen infection are available for study (van Kan et al., 1992; Chao et al., 1999). For this reason, the changes in wound/defense response RNAs after greenhouse and silverleaf whitefly feeding were monitored. The leucine aminopeptidase (LapA) and proteinase inhibitor 2 (pin2) genes served as molecular sentinels for the JA-responsive wound-signaling pathway. Tomato leaves were encased in nylon mesh bags and were supplemented with 250 adult whiteflies. Infested leaves and apical non-infested leaves were harvested at 0, 3, 5, 7, and 9 days. Control plants remained unbagged or were encased and no insects were added. Plants were harvested at similar times. During this period, the adult whiteflies fed, mated and oviposited. Between days 3 and 5, eggs hatched and nymphs settled at their feeding sites and fed continuously for the next 4 to 6 days.
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Unlike responses to tissue-damaging herbivores (Pautot et al., 1993; Howe et al., 2001; Pautot et al., 2001), LapA and pin2 transcripts did not accumulate locally or systemically in response to greenhouse or silverleaf whitefly feeding over the 9-day infestation period (Fig.1, Puthoff et al., 2001). Furthermore, infestation of LapA:GUS transgenic plants and control plants indicated that the LapA promoter was not activated by either species of whitefly. Instead, both species of whiteflies caused increases in PR gene RNAs (Puthoff et al., 2001). PR transcripts that accumulated in response to exogenous JA and ethylene (GluB, Chi9 and PR-1) accumulated to highest levels in infested leaves. Transcripts were first detected at the time nymphs initiated feeding (day 5) and RNA levels persisted until day 9. Chi3, GluAC, and PR-4 RNAs were less abundant or undetectable (Puthoff et al., 2001). Increases in PR transcripts did not occur in response to adult feeding alone. Collectively, these data indicate that whiteflies stimulate defense responses normally associated with fungal, bacterial or viral pathogen attack and lack the responses often associated with tissue-damaging herbivores (Kombrink and Somssich, 1997; Walling, 2000).
Greenhouse and Silverleaf Whitefly Feeding
X
? Unknown signal
?
Systemin, OGA, or chitosan ABA
JA
SA
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Ethylene
Ethylene
ROS/NO SA
Ethylene Woundresponse Genes
HR
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Chi3 GluAC PR-4
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PR-1 Chi3
Induced Systemic Resistance (ISR)
Systemic Acquired Resistance (SAR)
LapA pin2
Induced Resistance (IR) Mechanical Wounding
Rhizobacteria-induced Pathways
Pathogen-induced Pathways
Figure 1. Defense signaling pathways in tomato after whitefly feeding . This is a model incorporating known patterns of defense gene expression in response to whitefly feeding. Whiteflies suppress or do not activate the systemin-regulated octadecanoid pathway in tomato (X). Whiteflies have an unknown impact (?) on JA-independent wound-responses and their impact on the ISR has not yet been tested. PR transcripts, which accumulate in response to exogenous JA or ethylene (Chao et al., 1999; Puthoff et al., 2001), accumulate to the highest levels in response to whitefly nymph feeding (grey boxes and heavy arrows). Other PR RNAs accumulate to low levels.
Squash-whitefly interactions There are also induced plant responses that are elicited specifically by a single herbivore species. For example, the volatile blend emitted by plants to attract herbivore enemies is distinct for each plant host and herbivore species interaction (De Moraes et al., 1998; Dicke, 1999; Páre and Tumlinson, 1999). A second example emerges from studies of squash interactions with two closely related species of whiteflies: the silverleaf whitefly (B. argentifolii) and the sweet potato whitefly (B. tabaci Type A) (van de Ven et al., 2000). The squash-whitefly interactions are of interest because silverleaf whitefly nymphs, but not sweet potato whitefly nymphs, cause a developmental disorder in newly developing leaves called leaf silvering (Costa et al., 1993). Therefore, we postulated that there might be differences in
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systemic gene expression if silverleaf whitefly- and sweet potato whitefly-infested leaf RNA populations were compared. Using differential RNA display, two genes (SLW1 and SLW3) that were preferentially expressed in apical non-infested leaves from silverleaf whiteflyinfested plants were identified (van de Ven et al., 2000). The temporal and spatial expression studies showed that SLW1 and SLW3 RNAs accumulated only after nymph feeding. Adult silverleaf or sweet potato whiteflies did not cause these RNAs to accumulate in either infested leaves or apical non-infested leaves (van de Ven et al., 2000). SLW1 RNAs were abundant in infested leaves and were detected systemically in apical leaves of silverleaf whitefly-infested plants. SLW1 RNAs were not detected locally or systemically after sweet potato whitefly feeding or in control non-infested plants. SLW1 RNAs did not accumulate in response to wounding or P.s. syringae infection. However, SLW1 RNAs accumulated in response to exogenous MeJA (van de Ven et al., 2000). SLW1 RNAs were also abundant in flowers and fruit (van de Ven et al., 2000); this is similar to the developmental programming of many JA-responsive defense genes (Chao et al., 1999; Hause et al., 2000). SLW1 encodes a M20b peptidase-like protein. Its role in defense against herbivores is not understood but analyses of transgenic plants over-expressing SLW1 and a SLW1:GUS transgene are underway. After silverleaf whitefly nymphs initiated feeding, SLW3 RNAs accumulated to high levels locally and systemically (van de Ven et al., 2000). Neither sweet potato whitefly nymphs nor adults caused SLW3 RNAs to accumulate systemically. However, SLW3 RNAs were detected in sweet potato whitefly-infested leaves. These data suggest that there is a difference in the local and systemic signaling for SLW3. Unlike SLW1, SLW3 transcripts were not detected in any other organs and whitefly infestations did not alter this developmental programming. To date, the defense signal that modulates SLW3 expression is not known (van de Ven et al., 2000). SLW3 is a novel whitefly-specific defense-response gene since its RNAs do not accumulate after pathogen infection or wounding. Furthermore, SLW3 RNA levels are not influenced by MeJA, ethylene, SA, ABA, or reactive oxygen species, such as H2O2 or nitric oxide. Interesting, water-deficit stress increases the levels of both SLW1 and SLW3 RNAs. The relationship between water-deficit stress and whitefly feeding is being investigated. It is not clear if similar signals are generated by both water-deficit stress and whitefly feeding or if both SLW1 and SLW3 are regulated by multiple signaling pathways. SLW3 encodes a βglucosidase-like protein. Transgenic tomato plants expressing a SLW3:GUS gene will be used as bioassay plants to identify the whitefly-induced signal important in SLW3 gene activation. Sources of whitefly elicitors Collectively these data indicate that there are both shared and species-specific signals that modulate tomato and squash responses to whitefly feeding. Since the temporal and spatial changes in tomato gene expression were similar in response to both the greenhouse and silverleaf whiteflies, these diverse species of whiteflies must generate similar mechanical or chemical signals. Whiteflies stimulate defense responses normally associated with pathogen attack and do not induce wound-responses activated by many tissue-damaging herbivores (Kombrink and Somssich, 1997; Walling, 2000). The lack of LapA and pin2 induction is consistent with fact that whiteflies rarely puncture mesophyll or epidermal cells in their search for a feeding site (a minor vein of the phloem) (Cohen et al., 1998). In a similar manner, tomato plants do not perceive the passage of the whitefly stylet (mouthparts) between epidermal and mesophyll cells, puncturing of the phloem-sieve element, and consumption of phloem as a wound. However these mechanical forces could generate hydraulic or electrical signals to activate gene expression; both types of mechanical forces have been implicated in wound signaling (Wildon et al., 1992; Davies et al., 1997; Herde et al., 1998). It should be
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noted that mechanical forces are unlikely to account for the differences in responses to adults and nymphs seen in both tomato and squash or the differences in SLW1 and SLW3 expression after silverleaf and sweet potato whitefly feeding, since the mechanics of feeding are similar. It is more likely that the shared and species-specific signals are located in the rapidly gelling saliva that surrounds the stylet and/or the watery saliva that is egested upon whitefly probing and feeding. Unlike the salivas of aphids, which are rich sources of chemicals and proteins (Miles, 1999), little is known about the composition of whitefly saliva (Funk, 2001). It is possible that the salivas of greenhouse and silverleaf whitefly nymphs contain an elicitor to activate tomato PR genes and lack elicitors that stimulate the wound-induced octadecanoid pathway. Alternatively, the sheath and watery salivas might provide suppressors of this wound-signaling pathway, similar to that described for Helicoverpa zea (Felton, 2001). Similarly, the composition of silverleaf nymph salivas may differ from adults and from sweet potato whitefly nymph/adult salivas. Differences in elicitor quantity or potency could explain the species-specific differences in gene activation by the silverleaf and sweet potato whiteflies. Finally, the whitefly salivary elicitors may be insect derived or be synthesized by a whitefly endosymbiont. There are differences in the endosymbionts associated with the silverleaf and sweet potato whiteflies (Costa et al., 1995). Alternatively, these elicitors could be created by the concerted biochemical efforts of both the plant and insect, similar to what has been observed for the synthesis of volicitin a potent insect elicitor for volatile production (Páre et al., 1998). Studying the diversity and nature of the elicitors and novel signaling pathways activated by phloem-feeding insects is a rich avenue for future research.
Acknowledgements We thank Frances Holzer, Arthur Cooper, Nhung Nuygen, Paula Huang, and Wanida Ruangsiriluk for technical assistance. This research was supported by a University of California Biotechnology Grant, USDA Grants 95-37301-2081 and 99-35301-8077.
References Baldwin, I.T., Halitschke, R., Kessler, A., & Schittko, U. 2001: Merging molecular and ecological approaches in plant-insect interactions. Curr. Opin. Plant Biol. V4: 351-358. Bostock, R.M. 1999: Signal conflicts and synergies in induced resistance to multiple attackers. Physiol Mol. Plant Path. 55: 99-109. Chao, W.S., Gu, Y.-Q., Pautot, V., Bray, E.A., & Walling, L.L. 1999: Leucine aminopeptidase mRNAs, proteins and activities increase in response to drought, salinity and the wound signals - systemin, methyl jasmonate, and abscisic acid. Plant Physiol. 120: 979992. Cohen, A.C., Chu, C.-C., & Henneberry, T.J. 1998: Feeding biology of the silverleaf whitefly (Homoptera: Aleyrodidae). Zhonghua Kunchong 18: 65-82. Costa, H.S., Ulmanh, D.E., Johnson, M.W., & Tabashnik, B.E. 1993: Squash silverleaf symptoms induced by immature but not adult Bemisia tabaci. Phytopathology 83: 763766. Costa, H.S., Westcot, D.M., Ullman, D.E., Rosell, R., Brown, J.K., & Johnson, M.W. 1995: Morphological variation in Bemisia endosymbionts. Protoplasma 189: 194-202. Davies, E., Vian, A., Vian, C., & Stankovic, B. 1997: Rapid systemic up-regulation of genes after heat-wounding and electrical stimulation. Acta Physiol. Plant. 19: 571-576. De Moraes, C.M., Lewis, W.J., Pare, P.W., Alborn, H.T., & Tumlinson, J.H. 1998: Herbivore-infested plants selectively attract parasitoids. Nature 393: 570-573.
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Dicke, M. 1999: Are herbivore-induced plant volatiles reliable indicators of herbivore identity to foraging carnivorous arthropods? Entomol. Exp. Appl. 91: 131-142. Dicke, M., Gols, R., Ludeking, D., & Posthumus, M.A. 1999: Jasmonic acid and herbivory differentially induce carnivore-attracting plant volatiles in lima bean plants. J. Chem. Ecol. 25: 1907-1922. Eichenseer, H., Mathews, M.C., Bi, J.L., Murphy, J.B., & Felton, G.W. 1999: Salivary glucose oxidase: Multifunctional roles for Helicoverpa zea? Arch. Insect Biochem. Physiol. 42: 99-109. Felton, G.W., & Korth, K.L. 2000: Trade-offs between pathogen and herbivore resistance. Curr. Opin. Plant Biol. 3: 309-314. Felton, G.W. 2001: Mixed messages in saliva: Implications for induced resistance. Int. Org. Biol. Control. Bull. (this issue). Funk, C.J. 2001: Alkaline phosphatase activity in whitefly salivary glands and saliva. Arch. Insect Biochem. Physiol. 46: 165-174. Graham, J.S., Pearce, G., Merryweather, J., Titani, K., Ericsson, L.H., & Ryan, C.A. 1985: Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II. J. Biol. Chem. 260: 6561-6564. Gu, Y.Q., Chao, W.S., & Walling, L.L. 1996: Localization and post-translational processing of the wound-induced leucine aminopeptidase proteins of tomato. J. Biol. Chem. 271: 25880-25887. Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R., Ziegler, J., & Wasternack, C. 2000: Tissue-specific oxylipin signature of tomato flowers: Allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles. Plant J. 24: 113-126. Herde, O., Peña-Cortes , H., Willmitzer, L., & Fisahn, J. 1998: Remote stimulation by heat induces characteristic membrane-potential responses in the veins of wild-type and abscisic acid-deficient tomato plants. Planta 206: 146-153. Howe, G.A., Lee, G.I., Li, L., & Li, C. 2001: Genetic dissection of induced resistance in tomato: Prospects for engineering broad spectrum resistance to herbivores. Int. Org. Biol. Control. Bull. (this issue). Kahl, J., Siemens, D.H., Aerts, R.J., Gabler, R., Kuhnemann, F., Preston, C.A., & Baldwin, I.T. 2000: Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 210: 336-342. Karban, R., & Baldwin, I.T. 1997: Induced responses to herbivory Chicago, University of Chicago Press. Kombrink, E., & Somssich, I.E. 1997: Pathogenesis-related proteins and plant defense. In: Plant Relationships, (Eds.) Carroll, C.G., & Tudzynski, P.: Berlin, Springer-Verlag, pp. 107-128. Korth, K.L., & Dixon, R.A. 1997: Evidence for chewing insect-specific molecular events distinct from a general wound response in leaves. Plant Physiol. 115: 1299-1305. Kúc, J. 2001: Induced resistance in plants - molecular, environmental and practical implications. Int. Org. Biol. Control. Bull. (this issue). Martienssen, R.A., Barkan, A., Freeling, M., & Taylor, W.C. 1989: Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator. EMBO J. 8: 1633-1640. Miles, P.W. 1999: Aphid saliva. Biol. Rev. Camb. Phil. Soc. 74: 41-85. Moran, P., & Thompson, G. 2001: Molecular responses to aphid feeding in Arabidopsis thaliana in relation to plant defense pathways. Plant Physiol. 125: 1074-1084. Páre, P.W., Alborn, H.T., & Tumlinson, J.H. 1998: Concerted biosynthesis of an insect elicitor of plant volatiles. Proc. Natl. Acad. Sci. USA 95: 13971-13975.
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Páre, P.W., & Tumlinson, J.H. 1999: Plant volatiles as a defense against insect herbivores. Plant Physiol. 121: 325-331. Pautot, V., Holzer, F.M., & Walling, L.L. 1991: Differential expression of tomato proteinase inhibitor I and II genes during bacterial pathogen invasion and wounding. Mol. PlantMicrobe Inter. 4: 284-292. Pautot, V., Holzer, F.M., Reisch, B., & Walling, L.L. 1993: Leucine aminopeptidase: An inducible component of the defense response in Lycopersicon esculentum (tomato). Proc. Natl. Acad. Sci. USA 90: 9906-9910. Pautot, V., Holzer, F.M., Chaufaux, J., & Walling, L.L. 2001: The induction of tomato leucine aminopeptidase genes (LapA) after Pseudomonas syringae pv. tomato infection is primarily a wound response triggered by coronatine. Mol. Plant-Microbe Inter. 14: 214224. Peña-Cortés, H., Prat, S., Atzorn, R., Wasternack, C., & Willmitzer, L. 1996: Abscisic aciddeficient plants do not accumulate proteinase inhibitor II following systemin treatment. Planta 198: 447-451. Puthoff, D.P., Chao, W.S., LeVesque, C.S., Perring, T.M., & Walling, L.L. 2001: Tomato pathogenesis-related protein genes are expressed in response to greenhouse and silverleaf whitefly feeding. Plant Physiol.: In Revision. Reymond, P., Weber, H., Damond, M., & Farmer, E.E. 2000: Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12: 707719. Ryan, C.A. 2000: The systemin signaling pathway: differential activation of defensive genes. Biochim. Biophys. Acta 1477: 112-122. Smith, J.A., Hammerschmidt, R., & Fulbricht, D.W. 1991: Rapid induction of systemic resistance in cucumber by Pseudomonas syringae pv. syringae. Mol. Plant Path. 38: 223235. Stotz, H.U., Pittendrigh, B.R., Kroymann, J., Weniger, K., Fritsche, J., Bauke, A., & MitchellOlds, T. 2000: Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth. Plant Physiol. 124: 1007-1017. van de Ven, W.T.G., LeVesque, C.S., Perring, T.M., & Walling, L.L. 2000: Local and systemic changes in squash gene expression in response to silverleaf whitefly feeding. Plant Cell 12: 1409-1423. van Kan, J.A.L., Joosten, M.H.A.J., Wagemakers, C.A.M., van den Berg-Velthuis, G.C.M., & de Wit, P.J.G.M. 1992: Differential accumulation of messenger RNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum. Plant Mol. Biol. 20: 513-527. Vollenweider, S., Weber, H., Stolz, S., Chetelat, A., & Farmer, E.E. 2000: Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves. Plant J. 24: 467-476. Walling, L.L. 2000: The myriad plant responses to herbivores. J. Plant Growth Regul. 99: 195-216. Wasternack, C., Ortel, B., Miersch, O., Kramell, R., Beale, M., Greulich, F., Feussner, I., Hause, B., Krumm, T., Boland, W., & Parthier, B. 1998: Diversity in octadecanoidinduced gene expression of tomato. J. Plant Physiol. 152: 345-352. Wildon, D.C., Thain, J.F., Minchin, P.E.H., Gubb, I.R., Reilly, A.J., Skipper, Y.D., Doherty, H.M., O'Donnell, P.J., & Bowles, D.J. 1992: Electrical signaling and systemic proteinase inhibitor induction in the wounded plant. Nature 360: 62-65.
Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 41-46
Novel phospholipases A2 induced during pathogen resistance responses in tobacco and with potential role in oxylipin biosynthesis Thierry Heitz, Sandrine Dhondt, Guillaume Gouzerh, Pierrette Geoffroy, Michel Legrand Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67000 Strasbourg, France Abstract : The induction of resistance of plants to infection by some microbes or against insect attacks is dependent on the production of fatty acid derivatives called oxylipins, whose best known representative is jasmonic acid (JA). Oxylipins display signalling or direct antimicrobial properties and may also affect cell death in the host. Their biosynthesis requires the enzymatic release of unsaturated fatty acids from membrane lipids, a rate-limiting step which remains elusive. This work reports on the isolation and cloning of the first phospholipases A that are induced after pathogen attack. Enzymes responsible for a strong increase in soluble phospholipase A2 activity in tobacco reacting hypersensitively to tobacco mosaic virus (TMV) belong to the patatin-like family of acyl hydrolases and may contribute to the massive lipid breakdown occurring during responses to pathogens. Key words : jasmonate, lipid signal, oxylipin, patatin-like, pathogen defense, phospholipase A2
Introduction Induced plant resistance against microbes is triggered by the early recognition of the aggressor and relies on complex signal transduction cascades orchestrating the activation of numerous metabolic pathways and downstream responses. The early molecular events initiated upon recognition are transduced into the biosynthesis of a limited number of secondary defense signals deriving from various cellular components. For exemple, salicylic acid, deriving from the phenylpropanoid metabolism, is one of the major defense regulators by its importance in controlling the induction of a whole set of antimicrobial “Pathogenesis-Related“ (PR) genes and resistance to a variety of pathogens. The anti-pathogen defense system as established in the so-called “hypersensitive response“ (HR) generates specific signals and responses, but also integrates pathways activated by other stresses such as wounding and herbivore attacks. For instance, oxidized fatty acid derivatives (oxylipins), first described as signals restricted to the wound response, are also produced during antimicrobial defense and are critical to the induction of resistance to some microbes (Thomma et al., 2001). Oxylipins are synthesized in response to different stimuli by complex enzymatic cascades which are currently being elucidated (Blée, 1998). However, the initial release of precursor fatty acids from structural lipids, believed to be catalyzed by phospholipases A2 (PLA2) or galactolipases, is still largely unknown and is the topic of our research.
Material and methods Most methods were described in Dhondt et al (2000). The outlines of the procedures are given below.
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PLA2 purification and lipid acyl hydrolase activities PLA2 activity was assayed on Escherichia coli cells grown on medium supplemented with [14C]oleic acid which is incorporated in the sn-2 position of phospholipids. Plant extracts were incubated with autoclaved, labelled bacterial cell suspensions. Soluble radioactivity recovered in the supernatant is a measurement of PLA2 activity. This assay was used to follow PLA2 activity during the fractionation of extracts of TMV-infected leaves. Three successive chromatographic steps allowed the identification of a protein whose abundance was correlated with PLA2 activity. The purified fraction containing this protein was blotted and the sequence of the 50 N-terminal amino acids of the active protein was determined and turned out to be similar to patatin sequences. Lipid acyl hydrolase activity of recombinant proteins was also measured on 1-palmitoyl, [14C]-2-linoleoyl-phosphatididylcholine and on galactolipids, monogalactosyl-diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). In this case, the reaction mixture was extracted with chloroform/methanol (2/1; v/v) and separated on thin layer chromatography with the solvent mixture hexane/ether/acetic acid (160/40/3; v/v/v). Fatty acids were visualized with primulin, scraped from the plate and radioactivity was counted by scintillation. Unlabelled fatty acids were eluted from silica, methylated with acidified methanol and quantified by gas chromatography on a DB-WAX column. Quantification of jasmonates JA was measured in purified plant extracts in early experiments by ELISA using a monoclonal antibody kindly provided by Prof. E. Weiler (Bochum, Germany). A method was later developed by the same group to quantify simultaneously 12-oxo-phytodienoic acid and JA by gas chromatography-mass spectrometry. Cloning of patatin-like cDNAs and production of recombinant NtPAT proteins Three related patatin-like cDNAs called NtPat were amplified by RT-PCR from RNA extracted from 5-day TMV-infected leaves using primers deriving from the N-terminal sequence of the purified protein and from consensus sequences identified by alignment of cloned patatin-like genes. Full length cDNAs were isolated by screening a λ-Zap library made from the same plant material. NtPat1 and NtPat3 cDNAs were cloned in the pGEX-KG vector, for production of recombinant GST-NtPat fusion proteins in the BL21 strain of E. coli. Proteins purified according to the manufacturer’s instructions were injected into rabbits to raise anti-NtPAT1 and anti-NtPAT3 serums. Treatment of plants and study of NtPat gene expression Tobacco (Nicotiana tabacum cv Samsun NN) were grown in a greenhouse under controlled conditions. Six week-old plants were used to study the response of NtPat genes to various stresses. Plants leaves were wounded with a hemostat across the midvein. The wounded surface represented approximately 20% of the leaf surface. Methyl jasmonate (MJ) vapors (3.5 µM) were administered by pipetting MJ onto a cotton wick (with no contact with plants) and placing plants into a translucid, airtight plexiglas box. Aminocyclopropane-carboxylic acid (ACC, 2 mM) was sprayed on the abaxial surface of leaves. For this treatment as well as for combined ACC and MJ treatment, plants were also placed in airtight boxes. ß-megaspermin (50 nM) was injected with a syringe without needle into mesophyll sectors between secondary veins.
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Results and discussion Phospholipase A2 activity and jasmonates in hypersensitively reacting tobacco We became interested in fatty-acid releasing activity about 10 years ago while studying hydrolytic enzymatic activities (ß-1,3-glucanase, chitinase, esterase) induced in tobacco reacting by an HR to tobacco mosaic virus (TMV) infection. Some of these activities were believed to degrade host components and generate molecular species endowed with defense-eliciting properties. When using a PLA2 assay based on in vivo-labelled bacterial membrane phospholipid substrate, we found a dramatic increase in soluble PLA2 activity, occurring at the onset of necrotic lesion appearance (Figure 1). PLA2 activity reaches its maximum very rapidly, in contrast to the delayed appearance of enzymatic activities borne by PR proteins.
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250 Phospholipase A OPDA JA
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Figure 1. Evolution of jasmonates (cis-OPDA and 3R,7S-JA ) and soluble PLA2 activity levels in TMV-inoculated leaves. Open symbols : mock-inoculated leaves. Closed symbols : TMV-inoculated leaves.
This burst in lipid-degrading activity suggests an early role in host membrane fatty acid turnover. Recently, Penninckx et al (1996) demonstrated for the first time the accumulation of JA in a plant-microbe interaction, as well as the JA-dependence of the pathogen-induction of a defensin gene. We then addressed the question of the activation of oxylipin metabolism in the tobacco-TMV interaction and established that a sequential accumulation of 12-oxophytodienoic acid, the precursor of JA, and of JA itself occurs in this pathosystem (Dhondt et al., 2000). Interestingly, the rise in PLA2 activity clearly preceded the accumulation of jasmonates (Figure 1), opening the possibility of an involvement of this activity in the mobilization of free fatty acid precursors for oxylipin biosynthesis. Isolation of PLA2 and cloning of patatin-like genes To gain insight into the molecular nature of PLA2 enzymes, we fractionated the soluble protein extract from infected leaves and three successive chromatographic steps allowed the isolation of a low abundance protein bearing most of enzymatic activity. The N-terminal sequence of this protein was similar to patatin, the major storage protein in potato tubers, which was previously shown to display weak acyl hydrolase (phospholipase A, galactolipase, sulfolipase) activity (Andrews et al., 1992). By using the N-terminal sequence information and sequences streches conserved between known patatin-like genes, we cloned 3 tobacco patatin-like cDNAs that were called NtPat genes, one of which encoding the isolated PLA2. NtPat1 and NtPat3 cDNAs were expressed in E. coli and recombinant proteins were assayed for their ability to hydrolyse
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phospholipids and galactolipids, the two major lipid classes in plant plasma membranes and chloroplast membranes, respectively. Both NtPAT1 and NtPAT3 proteins displayed high PLA2 and galactolipase activities, suggesting that these hydrolases probably degrade different cellular membrane constituents during the HR. The description of substrate specificity of fatty-acid releasing enzymes activated upon pathogen ingress is of particular importance as the exact cellular compartment and the exact nature of lipid species providing the oxylipin precursors have not been established clearly. Expression profiles The expression profile of NtPat genes was examined by RT-PCR in time-course experiments in response to different stress conditions and defense modulator treatments. No signal was detected in healthy plants. During the HR to TMV, the rapid accumulation of the 3 NtPat mRNAs was observed concomitantly to the appearance of necrotic lesions and preceded PLA2 activity levels with a parallel profile. A similar transcriptional activation of NtPat genes occurred after infiltration of ß-megaspermin, an HR-inducing elicitin from Phytophthora megasperma. In this case, the NtPat transcripts were detected 3 hours after treatment in the infitrated zone that will undergo cell death a few hours later, whereas a more delayed induction was observed in the surrounding living cells. Two other pathogens, the soft rot-causing bacterium Erwinia carotovora and fungus Botrytis cinerea appeared also to be powerful inducers of NtPat-encoded PLA2 activity in tobacco. Wounding, which is known to induce a very rapid (within minutes) accumulation of jasmonates in plants, was found to induce only poorly and rather lately the expression of NtPat genes. These genes may thus not be involved in the near-immediate JA synthesis occurring after mechanical damage. Among several known plant defense regulators that were tested (salicylic acid, aminocyclopropane-carboxylic acid (ACC), methyl-jasmonate (MJ), coronatine, reactive oxygen species), only the ethylene precursor ACC was able to induce significantly NtPat genes and PLA2 activity when given alone and its effect was greatly enhanced when plants were treated with MJ simultaneously. However, although being strong, the induction maximized only between 48 and 72 hours, and may reflect an indirect mechanism of activation.
Conclusions We isolated and cloned novel isoforms of patatin-like proteins that appear in leaves under pathogen defense conditions. These isoforms likely contribute to the degradation of a wide array of host lipids in response to microbial attacks. The appearance of high NtPat-encoded PLA2 activity after TMV infection and fungal elicitor treatment precedes the accumulation of jasmonates and this probably also reflects the synthesis of a spectrum of other oxylipins with various biological properties. NtPat proteins constitute the first example of pathogen-induced, fatty acid-releasing enzymes. Their involvement in disease resistance is currently under investigation. The regulation of fatty acid mobilization for subsequent conversion into oxylipins may be orchestrated by different types of phospho- and galactolipases at both transcriptional and post-translational levels during plant responses to different stresses.
Acknowledgements S. D. was supported by a predoctoral fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche. G.G. was supported by a CIFRE Fellowship granted by Aventis Crop Science and the French government. We thank members of the Weiler laboratory for help in determination of jasmonates.
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References Andrews, D.L., Beames, B., Summers, M.D. & Park, W.D. (1992) Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector. Biochem. J. 252: 199-206. Blée, E. (1998) Phytooxylipins and plant defense reactions. Prog. Lipid Res. 37: 33-72 Dhondt, S., Geoffroy, P., Stelmach, S., Legrand, M. & Heitz T. (2000). Soluble phospholipase A2 activity is induced before oxylipin accumulation in tobacco mosaic virus-infected tobacco leaves and is contributed by patatin-like enzymes. Plant J. 23: 431-440. Penninckx, I.A.M. A., Eggermont, K., Terras, F.R.G., Thomma, B.P.H.J., De Samblanx, G.W., Buchala, A., Métraux, J.P., Manners, J.M. & Broekaert, W.F. (1996) Pathogeninduced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8: 2309-2323 Thomma et al. (2001). The complexity of disease signalling in Arabidopsis. Curr. Opin. Immunol. 13: 63-68.
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Induced Resistance in Plants against Insects and Diseases IOBC/wprs Bull. Vol. 25(6) 2002 pp. 47-51
Genetic dissection of induced resistance in tomato Gregg A. Howe, Lei Li, Gyu In Lee, Chuanyou Li, David Shaffer Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824 USA
Abstract: We are using a genetic approach to dissect the mechanisms of wound signaling and induced anti-herbivore defense in tomato. Various screens were carried out to identify mutants that are defective in the systemic expression of the wound-responsive proteinase inhibitor (PIN) genes. Genetic analysis indicates that the mutants define at least five genes that are required for both systemic wound responses and the action of systemin, a peptide signal of the wound response. Phenotypic characterization suggests that the mutants can be classified as being defective either in systemin perception or JA biosynthesis and/or accumulation. The MicroTom dwarf cultivar of tomato is being used to facilitate genetic screens for new classes of wound response mutants. A mutagenized population of MicroTom was screened for plants that are insensitive to jasmonic acid (JA), a key regulator of induced resistance. Here we report the preliminary characterization of one such mutant that is blocked in JA perception and, as a consequence, is completely compromised in herbivoreinduced expression of defense-related genes. Key words: Induced systemic resistance; Jasmonic acid; Systemin; Wound response; Tomato
Introduction Induced resistance to arthropod herbivores is orchestrated by signal transduction pathways that regulate the deployment of defensive phytochemicals, both at the site of wounding and in systemic undamaged tissues. Much of what is known about the signaling events that control this process has come from the study of wound-inducible proteinase inhibitors (PINs) in tomato (Lycopersicon esculentum) (Ryan, 2000). A unique component of the wound response pathway in tomato is the 18-amino acid peptide systemin and its precursor protein prosystemin. Wounding, systemin, and cell wall-derived oligogalacturonides (OGAs) are thought to activate PIN gene expression by triggering the biosynthesis of jasmonic acid (JA) via the octadecanoid pathway (Farmer and Ryan, 1992). JA-mediated PIN expression requires ethylene (O’Donnell et al., 1996), and may also involve hydrogen peroxide as a second messenger (Orozco-Cárdenas et al., 2001). Very little is known about the process by which various wound signals are communicated within and between cells for the establishment of induced resistance. We are taking a genetic approach to dissect this process, using the wellcharacterized systemic wound response of tomato as a model system. The idea is to identify mutations that affect the wound response pathway, and to work back toward identification of gene function. We anticipate that this approach will provide new insight into the basic processes that govern plant responses to pest attack.
Material and methods Isolation of jai1 Lycopersicon esculentum cv MicroTom (MT) was grown and maintained as previously described (Howe et al., 2000). Fast neutron irradiation of MT seed was performed at the 47
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International Atomic Energy Agency (Seibersdorf, Austria), using calibrated doses in the range of 12.7 and 17.9 Gy. M2 seed was collected separately from each M1 plant. Approximately 25 M2 seedlings (18 day old) from each family were placed in an enclosed lucite box in which 5 ul pure methyl jasmonate (MeJA) (Bedoukian) was distributed to several evenly spaced cotton wicks. Plants were exposed to MeJA vapor for 24 hrs, followed by an additional 24 hrs of incubation in the absence of MeJA. A single leaflet from the lower leaf of each treated plant was assayed for polyphenol oxidase (PPO) activity as previously described (Howe and Ryan, 1999). Putative PPO-deficient mutants were retested for PIN2 accumulation as described by Howe and Ryan (1999). Tobacco hornworm (Manduca sexta) eggs were obtained from Carolina Biological Supply. Larvae were reared according to the manufacturer’s instructions.
Results and discussion Wound response mutants of tomato Two genetic screens were previously performed to identify wound response mutants of tomato. One screen involved identification of plants that fail to accumulate serine proteinase inhibitors I and II (PIN1 and PIN2) in response to mechanical wounding (Lightner et al., 1993). This approach yielded two non-allelic mutants called JL5 (renamed def1) and JL1 (herein called def2) that are deficient in wound-induced PIN expression (Lightner et al., 1993; Howe et al., 1996). A second screen for wound response mutants employed a tomato line that expresses a 35S::prosystemin (35S::prosys) transgene that activates PIN and PPO expression in a constitutive, wound-independent manner (Howe and Ryan, 1999). An EMS-mutagenized population 35S::prosys plants was screened for individuals that are deficient in PPO and PIN accumulation. Eleven spr (suppressed in prosystemin-mediated responses) mutants that show a defect in wound-induced gene expression were identified. Six mutants define two novel complementation groups designated Spr1and Spr2. Two mutants appear to be allelic to def1. The three remaining mutants are self-sterile but nevertheless can be propagated as heterozygotes (G. Howe, unpublished). Given the possibility that mutations in the wound response pathway of tomato affect fertility (see below), further characterization of these lines may provide insight into mechanism of “cross-talk” between signaling pathways for defenserelated and developmental processes. The response of mutants to wounding and various signaling intermediates was studied to gain insight into the role of these genes in the wound response pathway (Table 1). Mutations in Def1, Def2, and Spr2 severely reduce both local and systemic PIN expression in response to mechanical wounding and chewing insects (Lightner et al., 1993; Howe et al., 1996; C. Li and G. Howe, unpublished results). The phenotypic similarity between def1, def2, and spr2 (Table 1) suggests that these mutations affect closely related steps in the pathway that function in JA biosynthesis or the regulation of JA levels (Howe et al., 1996; Howe et al., 2000). Genetic mapping of these loci is in progress, as a prelude to identifying the precise molecular basis of the mutations. The phenotype of spr1 differs from def1, def2, and spr2 in two significant ways. First, spr1 plants are completely insensitive to systemin and prosystemin, but yet are responsive to the polysaccharide elicitors OGA and chitosan (Table 1). Second, spr1 exhibits near wild-type levels of wound-induced PIN expression in tissues adjacent to the wound site, but very little (
