ГЕНЕТИКА, 2011, том 47, № 7, с. 1–5
КРАТКИЕ СООБЩЕНИЯ УДК 575.17:633.16
TRANSCRIPTIONAL INTERACTIONS DURING BARLEY SUSCEPTIBLE GENOTYPE INFECTION WITH Cochliobolus sativus © 2011 M. I. E. Arabi, A. AlDaoude, A. Shoaib, M. Jawhar Department of Molecular Biology and Microbiology, AECS, Damascus, 6091, Syria email:
[email protected] Received November 12, 2010
A systematic sequencing of expressed sequence tags (ESTs) was used to obtain a global picture of the assembly of barley genes differentially expressed during the hypersensitive reaction of a susceptible genotype in re sponse to an incompatible Cochliobolus sativus pathovar. To identify a large number of plant ESTs, which are induced at different time points, an amplified fragment length polymorphism (AFLP) display of complemen tary DNA (cDNA) was ulilized. Significant transcriptional changes in the host plant occurred already 4 h post inoculation. Four hundred and fifty six ESTs have been generated, of which 17 (c. 53% upregulated, 47% downregulated) have no previously described function. On one hand, the majority of ESTannotations showed protein synthesis, but genes related to signal transduction pathway were also identified. This study provides novel global catalogue of gene regulations involved in C. sativusbarley interaction not currently rep resented in EST databases.
that carries suitable restriction sites leads to an accurate way for understanding plant responses to pathogens [9, 10]. The cDNAAFLP approach, once established, is an efficient and economical method to display whole tran script profiles of single tissues, particular developmental stages or other inducible characters [11]. Therefore, the aim of the present research was to better understand the interaction between the fungal pathogen C. sativus and the barley susceptible genotype WI 2291, via cDNA AFLP method. The major Syrian pathotype C. sativus (C41) used in the study was the most virulent of 40 isolates as de scribed by Arabi and Jawhar [4, 12, 13]. The fungus was incubated on Petri dishes containing potato dex trose agar (PDA, DIFCO, Detroit, MI, USA) for 8 days at 20–22°C in the dark to allow mycelial growth. After an extensive screening for over ten years in the greenhouse and in the laboratory, the Australian cv. WI2291 was proved to be the most susceptible geno type to all SB isolates available so far. Therefore, it was selected for the cDNAAFLP analysis. Inoculation tests of C41 isolate was performed using the method described by Arabi and Jawhar [12]. Seeds were planted in plastic flats (60 × 40 × 8 cm) filled with sterilized peatmoss and placed in a growth chamber at temperatures 22 ± 1°C (day) and 17 ± 1°C (night) with a daylength of 12 h and a relative humidity of 80–90%. Seedlings were irrigated by Knop nutrient solu tion (1 g NaNO3; 0.25 g KNO3; 0.25 g MgSO4 ⋅ 7H2O; 0.25 g KH2PO4; and 10 mg FeCl3 per 1000 ml water). Infections were initiated by spraying each plant with 0.2 ml of conidial suspension of 2 × 104 conidia/ml in pure water. Tween 20 (polyoxyethylenesorbitan
Cochliobolus sativus (Ito & Kuribayashi) Drechs. ex Dastur (anamorph: Bipolaris sorokiniana (Sacc.) Shoem), the causal agent of spot blotch (SB), is a common foliar pathogen of barley (Hordeum vulgare L.) worldwide, a disease responsible for heavy crop losses [1, 2]. The infection process of C. sativus on leaves usually occurs through natural wounding, stomata or with the use of an appresoriumlike structure through the cell wall [2, 3]. In susceptible plants the disease can result more dead and collapsed tissue and further uncon trolled spread leading to visible necrotic spots [2]. Development of stable forms of resistance to SB depends upon identification of resistances effective against the prevalent isolates in barley growing areas [1–4]. However, even susceptible hosts are not fully accessible to C. sativus but express different degrees of background resistance that is mainly achieved by pen etration defense [5]. Few studies demonstrate differential gene expres sion in resistant SB barley genotypes during the early phase of infection, before any visible symptoms are apparent in the tissues [6, 7]. However, still, little is known about the genetic background and regulation of interaction mechanisms. In addition, understanding the basis of susceptibility would greatly facilitate the development of new control strategies and the identi fication of pathogen and host factors required for dis ease progression [8]. One useful approach to the molecular analysis of plantpathogen interactions is the determination of changes in steady state mRNA levels occurring during in fection. Such amplified fragment length polymorphism (AFLP) display of complementary DNA (cDNA) has been undertaken to reveal altered expression of any gene 1
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ARABI et al.
Homologies of sequenced AFLP fragement to sequences at 4, 24, 48 and 72 h after inoculation of barley susceptible cv. WI 2291 by the C. sativus isolate C41 Fragment no.
4h
24 h
48 h
72 h
I
II
I
II
I
II
I
II
Accession Lenght Regulation no. (bp)
8
+
+
+
+
+
+
–
+
BAA06731 121
up
13
–
–
–
–
+
+
+
–
EAZ37001 214
down
23
+
+
–
–
+
–
–
–
EAY83985
93
down
30
–
–
–
–
–
–
–
+
AJ278817
189
up
38
–
–
–
–
–
+
–
+
AJ290421
146
up
49
+
+
+
+
–
–
+
–
EAZ10281 169
down
63
–
–
–
–
–
–
+
–
AAU9536
141
down
64
–
–
–
–
–
–
+
+
AAG16625 141
down
89 106 124 128
– – – +
– – – +
– – – –
+ – + –
– – – –
– – + +
– + – –
+ – – –
EAY72268 98 EAZ23888 71 EAY86781 152 BAC43449 74
up down up up
146 152 154 155
– – – –
– – – –
– – – –
– – + –
– + – –
+ – – –
– + – –
+ – + +
AAA63149 211 AAV25286 94 EAZ12498 108 BAB44079 209
up down up up
168
–
–
–
–
+
–
+
–
EAY73848
down
157
BlastX sco
Blast match Nicotiana tabacum NPK2, serine/Treonine protein Kinases, catalytic domin Oryza sativa hypothetical protein Oryza sativa hypothetical protein Hordeum vulgare partial mRNA for putative cystene pro tease (pCYSPORT gene) Hordeum vulgare mRNA for BAX inhibitior 1 (pBI1 gene) Oryza sativa hypothetical protein Solanum phureja osmotinlike protein Solanum cryoprotective osmolinlike protein Oryza sativa hypothetical protein Oryza sativa hupothetical protein Oryza sativa hypothetical protein Arabidopsis thaliana putative dis ease resistance profection RPP8 Myosin heavy chain homolog Oryza sativa hypothetical protein Oryza sativa hypothetical protein Oryza sativa putative NBSLRR type resistance protein Oryza sativa hypothetical protein
3e10
4e19 3e22 4e18
1e08 2e22 4e21 1e20 1e21 2e07 1e19 1e31 2e18 3e24 3e16 2e22 3e23
Notes: (+), presence and (–), absence of fragment; I (controls at the time points), II (after inoculation); (4 h, 2 h, 48 h and 72 h), where mRNA was extracted from watertreated leaves incubate under the same conditions and at the same time points.
monolaurate) was added as a surfactant (100 μl per li ter) to the conidial suspension to facilitate dispersion of the inoculum over the leaf surface. Leaves were cov ered for one night with plastic bags to increase humid ity and plants were kept in the same greenhouse at 20°C with a 16h photoperiod. Leaf samples for RNA isolation were taken at dif ferent time points (4, 24, 48 and 72 h) post inoculation according to the developmental stages of the fungus during infection (Table). Leaves were collected at each time point from 20 individual, plants, labeled and im mediately frozen in liquid N2 before they were stored at –80°C until needed. As controls, mRNA was ex tracted from watertreated leaves incubated under the same conditions and at the same time points. mRNA was extracted from samples (100–200 mg) with the Nucleotrap mRNA mini kit (MachereyNagel, MN,
Germany) following the manufacturer’s instructions for plant tissues. The cDNAAFLP protocol was performed accord ing to the method described by Breyne et al. [14] with minor modifications which permits the visualization of one single cDNA fragment for each messenger orig inally present in the sample, thus reducing the redun dancy of sequences obtained. Briefly, doublestranded cDNA was synthesized from 1 μg mRNA using the Superscript II reverse transcription kit (Invitrogen, Paisley, UK) and a biotinylated oligodT primer (Roche, Mannheim, Germany). The cDNA was di gested with BstYI (restriction site RGATCY), and the 3' ends of the fragments were captured on streptavidin magnetic beads (Dynal). Digestion with MseI yielded fragments that were ligated to adapters for amplification (BstYIForw: 5'CTC GTA GAC TGC GTA GT3'; Bst ГЕНЕТИКА
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TRANSCRIPTIONAL INTERACTIONS DURING BARLEY 4h I II
24 h I II
48 h I II
72 h I II
EAZ10281 homologue EAZ37001 homologue
BAA06731 homologue
BAG43449 homologue
A portion of a silver stained AFLP gel using BstYI + AC/MseI + CT primer combination. I, controls, plants sprayed with water. II, treatments.
YIRev: 5'GAT CAC TAC GCA GTC TAC3'; MseI Forw: 5'GAC GAT GAG TCC TGA G3'; MseIRev: 5'TAC ATC AGG ACT CAT3'). Preamplification was performed with an MseI primer (Mse0: 5'GAT GAG TCC TGA GTA A3'), combined with a BstYI primer carrying either a T or a C at the 3' end (BstT0: 5'GAC TGC GTA GTG ATC T3'; BstC0: 5'GAC TGC GTA GTG ATC C3'). Preamplification PCR conditions were as follows: 5 min denaturation at 94°C and then 30 s denaturation at 94°C, 60 s annealing at 56°C, 60 s extension at 72°C (25 cycles), followed by 5 min at 72°C. After preamplification, the mixture was diluted 100 fold and 4 μl was used for selective ampli fication with 14 primer combinations, carried out with two selective nucleotides on the MseI primer. Touch down PCR conditions for selective amplifications were as follows: 5 min denaturation at 94°C, followed by 30 s denaturation at 94°C, 30 s annealing at 65°C, 60 s extension at 72°C (13 cycles, scale down of 0.7°C per cycle); 30 s denaturation at 94°C, 30 s annealing at 56°C, 60 s extension at 72°C (23 cycles) and 5 min at 72°C. Selective amplification products were separated on a 6% polyacrylamide gel in a SequiGen GT Se quencing Cell (38 × 50 cm) (BioRad, Hercules, CA, USA) running for 2.5 h at 105 W and 50°C, and silver stainned (Silver Sequence kit, Promega, Cat. Q4132). Selected cDNAAFLP bands were cut from the gels with a surgical blade and eluted in 100 μl of sterile distilled water. An aliquot of 5 μl was used as a tem plate for reamplification using nonlabeled primers identical to those employed for selective AFLP ampli fication. PCR products were purified with Multi Screen PCR μ96 plates (Millipore) and sequenced di rectly (ABI 130, PerkinElmer, Applied Biosystems, ГЕНЕТИКА
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Foster City, CA, USA). Prior to sequencing, PCR products were purified with QIAgene gel extraction kit according to the manufacturer's recommendations. Sequencing was carried out on a Genetic Analyzer (ABI 310, Perklinelmer, Applied Biosystems, USA). Each sequence was identified by homology search us ing the Basic Local Alignment Search Tool (BLAST) program against the GenBank nonredundant public sequence database using an Evalue (BLASTX expec tation values [E] of ×10–5) to database entries with as signed identities. Disease symptoms (presence of solid, dark ne crotic lesions) were more sever at 48 h after inocula tion. cDNAAFLP analysis was carried out on RNA samples of infected leaves at different points of time, and on watersprayed leaves (healthy control), as de scribed [11, 14]. Four different stages were chosen to cov er early barley responses to SB which leads within 48 h to a visible hypersensitive cell death on a susceptible geno type by considering the observations of Wisniewska et al. [15] with barley susceptibility to Bipolaris sorokiniana. For each of the 14 primer combinations, 28– 33 transcript derived fragments (TDFs) were visual ized as bands, 25–760 bp in size, representing approx imately 456 transcripts overall, of which 17 have no previously described function. To determine the re producibility of these profiles, the experiments were repeated using additional samples of a biological rep licate. Differentiallyexpressed transcripts were visu ally scored relative to the first sampling time point which was arbitrarily attributed a zero value. The pattern of expressed genes at the beginning of the SB inoculation test (Figur) represents the “nor mal” set of active genes in a susceptible plant after four hours of being sprayed with water. Based on the as sumption that disease infection involves the early rec ognition of the invading pathogen, the cDNAAFLP patterns of susceptible plants were screened for newly expressed fragments which occur 4, 24, 48 and 72 hours after fungal attack. BlastX score to sequences on the database were presented in this study (Table). The most striking dis covery in our investigation was that nearly 47% of the differentiallyexpressed barley genes we identified were down regulated during infection (Table), possibly reflecting the exploitation of cellular resources and/or the suppression of defense responses [16, 17]. Additionally, most of the visualized transcripts were unaffected by infection, and 53% of the differentially expressed genes were clearly upregulated confirming the absence of a general, global, repressive environ ment. Among the upregulated genes, we identified many usually considered to have “housekeeping” func tions, such as a myosin heavy chain (AAA63149) which appeared at 48 h of inoculation (Figur). However, sev eral subsequent reports supported myosin’s role in cy toskeleton rearrangement during incompatible interac tions as well as in the maintenance of compatibility [18,
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ARABI et al.
19]. If this first line of defence is overcome by the patho gen, in many cases, it is followed by hypersensitive plant cell death, which stops growth of the penetrating fungus and finally leads to its death [20]. During infection, low level defense responses can be activated in susceptible plants, as already reported in barley [21, 22]. Therefore, it is not surprising that wellestablished C. sativus infections involve the up regulation of genes encoding different enzymes in the betaoxidation pathway, such as threonine protein ki nases, catalytic domin (BAA06731) which appeared at different times of inoculation. Additionally, fragments 63 (AAU9536) and 64 (AAG16625) showed high sim ilarities (high BlastX scores) to Solanum phureja and S. cryoprotective osmotin – like protein. Narasimhan et al. [23] found that the osmotin induces intracellular signaling in the target fungus to promote apoptosis and increase cell wall permeability, the modulation of tran scripts involved in the osmotion biosynthesis of C. sa tivus pathway needs to be investigated in more detail. Genes related to protein metabolism were also prevalently repressed in our experiment. Among them were genes encoding resistance proteins, protein mod ification and degradation enzymes (e.g. putative cys tein protease; pCYSPORT gene), as well as kinases which could also be involved in intracellular and inter cellular signaling. This suggested a general repression of protein synthesis and turnover. However, gene in volved in mRNA for BAX inhibitor 1 (PBIgene) (AJ290421) was induced, in agreement with previous findings [8]. Although barley resistance genes prevents penetra tion and does not rely on epidermal HR, it is inhibited by over expression of the potential cell death suppres sor BI1 in epidermal cells. This provides further evi dence for a link between susceptibility to fungal patho gen and cell survival [24]. Support for this hypothesis comes from the fact that B. graminis f. sp. hordei–re sistant mlo mutants are extremely susceptible to the rice blast fungus Magnaporthe grisea and to toxic cul ture filtrates of Cochliobolus sativus [1, 25]. The present study provides novel gene regulations involved in C. sativusbarley interaction not currently represented in EST databases. The data show that in fection results primarily in the downregulation of barley transcripts in major functional categories, especially protein synthesis. Myosin was upregulated in infected leaves, reflecting the occurrence of important cytoskel eton modifications during SB infection, and further in dicating that assumption of constitutive expression for “housekeeping” genes must always be considered with caution in specific physiopathological conditions. However, although further quantitative validation using RealTime PCR (qPCR) is needed, these results will hopefully serve as a basis to address new questions and design new experiments to elucidate further the biology of plantascomycota interactions and the associated re programming of host metabolism.
ACKNOWLEDGMENTS The authors thank the Director General of AECS and the Head of Biotechnology Department for their help throughout the period of this research. REFERENCES 1. Kumar, J., Schafer, P., Huckelhoven, R., et al., Bipo laris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control, Mol. Plant Pathol., 2002, vol. 3, pp. 185–195. 2. Schäfer, P., Hückelhoven, R., Kogel, K.H., The white barley mutant albostrians shows a super susceptible but symptomless interaction phenotype with the hemi biotrophic fungus Bipolaris sorokiniana, Mol. Plant Microbe Interact., 2004, vol.17, pp. 366–373. 3. Yadav, B.S., Behavior of Cochliobolus sativus during its infection of barley and wheat leaves, Austral. J. Bot., 1981, vol. 29, pp. 71–79. 4. Arabi, M.I.E., Jawhar, M., Identification of Cochliobo lus sativus (spot blotch) isolates expressing differential virulence on barley genotypes in Syria, J. Phytopathol., 2004, vol. 152, pp. 461–464. 5. Ghazvini, H., Tekauz, A., Host pathogen interactions among barley genotypes and Bipolaris sorokiniana iso lates, Plant Dis., 2008, vol. 92, pp. 225–233. 6. Santén, K., Pathogenesisrelated proteins in barley. Localization and accumulation patterns in response to infection by Bipolaris sorokiniana, Doctoral disserta tion, ISBN, 2007, vol. 5, pp. 576–7385. 7. AlDaoude, A., Jawhar, M., Transcriptional changes in barleyCochliobolus sativus interaction, Austral. Plant Pathol., 2009, vol. 38, pp. 1–5. 8. Hückelhoven, R., Dechert, C., Trujillo, M., Kogel, K.H., Differential expression of putative cell death regulator genes in nearisogenic, resistant and susceptible barley lines during interaction with the powdery mildew fun gus, Plant Mol. Biol., 2001, vol. 47, pp. 739–748. 9. Wendy, E.D., Rowland, O., Piedras, P., et al., cDNA AFLP reveals a striking overlap in race specific resis tance and wound response gene expression profiles, Plant Cell, 2000, vol. 12, pp. 963–977. 10. Polesani, M., Desario, F., Ferrarini, A., et al., cDNA AFLP analysis of plant and pathogen genes expressed in grapevine infected with Plasmopara viticola, BMC Ge nomics, 2008, vol. 9, pp. 142–147. 11. Bachem, C.W., van der Hoeven, R.S., de Bruijn, S.M., et al., Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber devel opment, Plant J., 1996, vol. 9, pp. 745–753. 12. Arabi, M.I.E., Jawhar, M., Pathotypes of Cochliobolus sativus (spot blotch) on barley in Syria, J. Plant Pathol., 2003, vol. 85, pp. 193–196. 13. Arabi, M.I.E., Jawhar, M., Molecular and pathogenic variation identified among isolates of Cochliobolus sati vus, Austral. Plant Pathol., 2007, vol. 36, pp. 17–21. 14. Breyne, P., Dreesen, R., Vandepoele, K., Transcrip tome analysis during cell division in plants, Proc. Nat. Acad. Sci., USA, 2002, vol. 99, pp. 14825–14830. ГЕНЕТИКА
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TRANSCRIPTIONAL INTERACTIONS DURING BARLEY 15. Wisniewska, H., Wakulinski, W., Chelkowski, J., Sus ceptibility of barleys to Bipolaris sorokiniana seedling blight determined by disease scoring and electrolyte, J. Phytopathol., 1998, vol. 146, pp. 563566. 16. Kortekamp, A., Wind, R., Zyprian, E., Investigation of the interaction of Plasmopara viticola with susceptible and resistant genotypes, J. Plant Dis. Protect., 1998, vol. 105, pp. 475–488. 17. Kiefer, B., Riemann, M., Buche, C., et al., The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola, Planta, 2002, vol. 215, pp. 387–393. 18. Yokota, E., McDonald, A.R., Liu, B., et al., Localiza tion of a 170 kDa myosin heavy chain in plant cells, Protoplasma, 1995, vol. 185, pp. 178–187. 19. Schmelzer, E., Cell polarization, a crucial process in fun gal defense, Trends Plant Sci., 2002, vol. 7, pp. 411–415. 20. Veronese, P., Ruiz, M.T., Coca, M.A., et al., Defense against Pathogens. Both Plant Sentinels and Foot Sol diers Need to Know the Enemy, Plant Physiol., 2003, vol.131, pp. 1580–1590.
21. Lipka, V., Panstruga, R., Dynamic cellular responses in plantmicrobe interactions, Curr. Op. Plant Biol., 2005, vol. 8, pp. 625–631. 22. Fung, R.W., Gonzalo, M., Fekete, C., et al., Powdery mildew induces defenseoriented reprogramming of the transcriptome in a susceptible but not in a resistant grapevine, Plant Physiol., 2008, vol. 146, pp. 236–249. 23. Narasimhan, M.L., Damsz, B., Coca, M.A., et al., A plant defense response effectors induces microbial apo ptosis, Mol. Cell, 2001, vol. 8, pp. 921–930. 24. Hückelhoven, R., Dechert, C., Kogel, K.H., Over ex pression of barley BAX Inhibitor1 induces breakdown of mlomediated penetration resistance to Blumeria graminis, Proc. Natl Acad. Sci. USA, 2003, vol. 100, pp. 5555–5560. 25. Jarosch, B., Kogel, K.H., Schaffrath, U., The ambiva lence of the barley Mlo locus: Mutations conferring re sistance against powdery mildew (Blumeria graminis f. sp. hordei) enhance susceptibility to the rice blast fun gus Magnaporthe grisea, Mol. PlantMicrobe Interact., 1999, vol. 12, pp. 508–514.
TRANSCRIPTIONAL INTERACTIONS DURING BARLEY SUSCEPTIBLE GENOTYPE INFECTION WITH Cochliobolus sativus © 2011 г. M. I. E. Arabi, A. AlDaoude, A. Shoaib, M. Jawhar Department of Molecular Biology and Microbiology, AECS, Damascus, 6091, Syria email:
[email protected] Received November 12, 2010
A systematic sequencing of expressed sequence tags (ESTs) was used to obtain a global picture of the assembly of barley genes differentially expressed during the hypersensitive reaction of a susceptible genotype in re sponse to an incompatible Cochliobolus sativus pathovar. To identify a large number of plant ESTs, which are induced at different time points, an amplified fragment length polymorphism (AFLP) display of complemen tary DNA (cDNA) was ulilized. Significant transcriptional changes in the host plant occurred already 4 h post inoculation. Four hundred and fifty six ESTs have been generated, of which 17 (c. 53% upregulated, 47% downregulated) have no previously described function. On one hand, the majority of ESTannotations showed protein synthesis, but genes related to signal transduction pathway were also identified. This study provides novel global catalogue of gene regulations involved in C. sativusbarley interaction not currently rep resented in EST databases.
ГЕНЕТИКА
том 47
№7
2011
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