Ethylene Biosynthesis-Inducing Endoxylanase Is ...

Plant Physiol. (1991) 97, 1181-1186

Received for publication April 29, 1991 Accepted June 28, 1991

0032-0889/91/97/1181/06/$01 .00/0

Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants1 Bryan A. Bailey*, Rosannah Taylor, Jeffrey F. D. Dean2, and James D. Anderson3 Plant Hormone Laboratory, Beltsville Agricultural Research Center (West), Agricultural Research Service, Department of Agriculture, Beltsville, Maryland 20705 ABSTRACT

U.S.

rified to near homogeneity from xylan-grown cultures of the fungus Trichoderma viride, induces biosynthesis of the gaseous phytohormone, ethylene, in tobacco (Nicotiana tabacum cv Xanthi) leaf tissue (1 1, 15). Elevated ethylene production, a common symptom of plant infection (19), is also induced in tobacco by an EIX4 produced by an important fungal pathogen, Fusarium oxysporum (12). The T. viride EIX elicits necrosis and other symptoms commonly associated with pathogenesis when introduced into detached tobacco leaves and intact plants (3), even under conditions that block ethylene biosynthesis and ethylene action (18). Immunoblots of proteins extracted from detached, EIX-treated leaves probed with EIX-specific antibodies demonstrated that EIX had moved into leaves from the point of application (3). In this report, the same EIX-specific antibodies have been used in conjunction with the technique of tissue printing (9) to examine the means by which EIX is transported in tobacco and how that transport leads to particular patterns of symptom development.

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus

Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattem, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.

Some fungal, bacterial (1), and viral (2) pathogens are capable of moving through plant vascular systems to induce disease symptoms in infected tissues. Some of these disease symptoms, e.g. hypersensitive necrosis, are believed to be defense responses used by the plant to limit progression of the pathogen into healthy tissues (16). When applied to the plant, numerous compounds, some of which are pathogen products and others of which are host-derived, act to elicit responses resembling those in diseased tissues (7, 16). Recognition by the plant of pathogen-generated elicitors early in the course of infection could limit further spread of the pathogen (21), but this requires movement of the elicitor or elicitor-generated signal from diseased to healthy tissue in advance of the invading pathogen. Unfortunately, the movement of defense-response elicitors through diseased plant tissues in vivo is very difficult to study because of the difficulty in distinguishing between responses to pathogen products versus host-derived materials. Previous work in this laboratory showed that an endo-,B1,4-xylanase (1,4-f3-D-xylan xylanohydrolase, EC 3.2.1.8) pu-

MATERIALS AND METHODS Chemicals and Enzymes

The chemicals used were of commercial origin. The EIX was purified as previously described ( 11) from xylan-induced Trichoderma viride cultures. Treatment of Plant Materials

Tobacco (Nicotiana tabacum L. Xanthi) plants were grown under greenhouse conditions until 25 to 30 cm tall. Whole tobacco plants were exposed in a 40.6-L glass jar to 120 ,uL/ L ethylene for 14 h or maintained in an atmosphere depleted ofethylene by the use of 25 g of the organic absorbent, Purafill IIs for 14 h. After the ethylene pretreatment, a leaf located midway up the plant stem was removed, leaving the exposed petiole. A 4-cm section of Tygon tubing was attached to the petiole, and 50 Mg of EIX in 100 ML distilled water was applied to the petiole through the Tygon tubing. Control plants were treated with 50 Mg of EIX boiled for 10 min. Some plants were treated

'This research was supported in part by U.S. Department of Agriculture Competitive Research Grant Office grant No. 88-372613680. 2 Present address: Department of Biochemistry, University ofGeorgia, Athens, GA 30605. 3 Present address: Weed Science Laboratory, Beltsville Agricultural Research Center (West), Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705.

'Abbreviation: EIX, ethylene-inducing xylanase. I Mention of trademark, proprietary product, or vendor does not constitute a guarantee of warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion ofother products or vendors that may also be suitable.

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Plant Physiol. Vol. 97, 1991

BAILEY ET AL.

1182

with a 1% solution of the vital stain Evans blue to track the movement of the applied solution in the absence of EIX. Symptom Measurements

The percentage of necrosis of leaves was monitored over 48 h for five leaves above and below the point of EIX application and rated on a scale of 0 to 100 % necrosis. For determination of ethylene production, tobacco plants were dissected 20 min after EIX application by removing each leaf, leaving the petiole on the stem. Six 1-cm leaf discs were cut from the left and right sides of each leaf and bioassayed separately over a 4-h period for ethylene production as previously described (3, 17). Ethylene biosynthesis-inducing activity is expressed as ,L ethylene evolved per h per g fresh weight of tobacco tissue.

of tobacco stems and petioles were printed onto nitrocellulose membranes (9), and EIX was immunolocalized with polyclonal antibodies raised against the 22-kD EIX polypeptide (12). The polyclonal antibodies were exposed to tobacco protein extracts localized on nitrocellulose membrane prior to use to reduce background levels of cross-reactivity. Tissues from plants treated with Evans blue were similarly printed onto nitrocellulose. RESULTS Pattern of Necrosis Development

Tobacco plants treated with EIX protein as described above were dissected 20 min after EIX application. Cross-sections

When EIX (50 ,ug) was introduced into the intact tobacco vascular system by application to a cut petiole (leafO), necrotic lesions developed in specific leaves up and down the stem from the point of application (3). When symptom development in a treated tobacco plant was charted on a radial map by leaf position around the central axis (Fig. 1), a pattern became apparent. The arrangement of leaves on Xanthi to-

Necrosis (% leaf halfl

Ethylene (uL/gm/H)

Immunolocalization of EIX Protein

-2

2

2

-5 _-

Above Application Petiole vmr...

Right side of leaf blade Left side of leaf blade

0

Below Applicaton Petiole URtWAM/01/:/.A

Rlght sle of leaf blade Left side of leaf blade

Figure 1. Radial diagrams depicting necrosis development and ethylene biosynthesis by tobacco plants in response to EIX. Whole tobacco plants were placed under an ethylene atmosphere of 120 ,/L/L for 14 h prior to EIX treatment. A 4-cm section of Tygon tubing was attached to an exposed petiole (leaf 0) midway up the plant and 50 ,ug of purified EIX protein in distilled water was applied to the petiole through the Tygon tubing. Necrosis development and ethylene production were determined on separate plants for each leaf half for five leaves above (positive numbers outside circle) and below (negative numbers outside circle) the point of EIX application. A visual estimate of necrosis was made after 24 h using a scale from 0 to 100% necrotic. Control plants treated with boiled EIX or distilled water failed to produce necrosis. Ethylene production was measured by dissecting tobacco plants 20 min after EIX application and cutting 6 discs from each leaf half. Ethylene production by the leaf discs was quantitated by GC after 4 h of incubation. Leaves from control plants failed to produce ethylene in excess of 0.02 AL/g/h and averaged 0.009 AL/g/h.

TRANSLOCATION OF AN ENDOXYLANASE IN TOBACCO XYLEM

1183

Table I. Development of Necrosis in Air- and Ethylene-Pretreated Tobacco Plants Treated with EIX through Petiole Application Plants were pretreated for 14 h in an ethylene purged atmosphere (air) or an atmosphere of 120 ,L/ L ethylene. Necrosis development was visually estimated at 4, 8, 24, and 48 h after EIX application (50 Ag) to the cut petiole of leaf 0. Values are the averages from three plants each for air and ethylene

pretreatments. Leaf Necrosis after EIX Application Leaf Number

Ethylene

Air

4h

8h

24 h

48 h

4h

8h

24 h

48 h

0 0 13 2 0

45 0 78 58 2

45 0 79 67 5

45 0 80 67 5

0 0 0 0 0

0 0 0 0 0

6 0 30 10 0

33 0 53 45 22

2 32 58 0 63

11

65 72 0 73

13 67 72 0 75

13 67 72 0 75

0 0 0 0 0

0 0 0 0 0

0 5 1 0 2

7 33 7 1 8

+5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5

bacco plants approaches a 2/5 phyllotaxy; that is, the leaves rotate around the central axis of the plant in a steep helix with approximately 1400 between leaves. Thus, the fifth leaf above (leaf +5) and the fifth below (leaf -5) the point of EIX application were attached to the stem almost directly above and below leaf 0, respectively, and consequently displayed severe necrosis. Symptomatic leaves -3, -2, and +3 were also attached to the main stalk on the same side of the plant as leaf 0, and symptoms in leaves -3 and -2 were, in fact, limited to the half of the leaf blade closest to the EIX-treated side of the plant. The remaining leaves, attached to the stalk opposite the site of application, generally failed to develop necrotic lesions. The development of necrosis was greatly enhanced by ex-

of whole plants to ethylene prior to EIX application (Table I). Although variation existed between plants, leaves +2, +3, +5, -2, -3, and -5 normally developed necrotic areas, whereas the remaining leaves evaluated showed little symptom development. The timing of necrosis development in air-pretreated plants was delayed in comparison with ethylene-pretreated plants. posure

Pattem of Enhanced Ethylene Production

Enhanced ethylene production occurred in leaf discs cut from leaves 20 min after EIX application, suggesting that movement of EIX or a product of EIX action is rapid (Fig. 1). The pattern of enhanced ethylene production closely fol-

Table II. Ethylene Production by Leaf Discs Cut from Fresh and Ethylene-Pretreated Tobacco Plants Treated with EIX through Petiole Application Air Pretreated8

Ler +5 +4 +3 +2 +1

Control (Rb Left Right

7 5 2 2 5

8 12 3 3 5

Plant 1 (R) Left Right

2 2 2 11 1

3 1 3 11 1

Ethylene Pretreated

Plant 2 (L)

Left

24 6 40 62 28

Right

20 8

Plant 3 (L) Left

10

17

1 11

40

20

17

4

Right

Control (R) Left

nL/g tissue/h 4 15 3 5 11 6 7

8 5 9

Plant 1

Plant 2 (R)

(R)

Plant 3 (L)

Right

Left

Right

Left

Right

Left

Right

8 8 8 6 6

734 2 324 157 2

779

48 5 259 5 6

104

194

5 48 48 6

1 77

241 3 436 145 2

1 3

170 1

26 0

0 -1 1 3 1 5 11 17 6 5 6 2 2 2 2 20 90 42 -2 7 6 62 2 52 117 80 65 12 2 64 11 9 29 12 106 -3 3 3 23 147 85 52 27 119 134 84 247 6 15 23 406 240 -4 11 5 5 3 3 8 23 12 1 31 10 2 0 0 3 3 -5 5 8 79 52 68 36 158 188 10 8 18 9 123 236 204 16 a Plants were pretreated for 14 h in an ethylene purged atmosphere (air) or an atmosphere of 120 AL/L ethylene. Twenty minutes after EIX (50 gg) application to the cut petiole of leaf 0, the leaves (+5 to -5) were cut from the plant, six discs removed from both the left and right sides b R and L indicate location of each leaf and assayed separately for ethylene production. Control plants were treated with 50 Mg boiled EIX. of leaf +1 in relation to leaf 0. L = left 1400; R = right 1400.

1184

BAILEY ET AL.

lowed the pattern previously described for necrosis. The leaves located on the stem opposite the point of enzyme application showed little enhanced ethylene biosynthesis. The total ethylene produced by tobacco leaves was enhanced by ethylene pretreatment (Table II). In addition to the previously described phyllotaxy, tobacco plants also display handedness. Some plants rotate upward clockwise from leaf 0, 1400, whereas others rotate counterclockwise 140° to reach leaf +1. When this was considered, an even more subtle pattern of symptom development was recognized. For example, leaf -2 tended to produced more ethylene on the left side of the midrib than on the right side in right-handed plants, whereas the opposite was true for left-handed plants.


Immunolocalization of EIX by Tissue Printing Techniques

Cross-reactive proteins, indicating the presence of EIX, were present within the stem vascular system of plants treated with EIX and were restricted to the EIX-treated side of the plant (Fig. 2). Cross-reactive proteins were also detected in petiole cross-sections of leaves -5, -3, -2, +2, +3, and +5 (not all shown), but were not detected in the petioles of leaves taken from the opposite side of the plant. Leaves displaying enhanced ethylene production on one side of the leaf blade (leaf +3) generally contained EIX only in the xylem on that same side of the petiole as shown in petiole +3. On occasion, limited amounts of necrosis and enhanced ethylene produc-

PETIOLE

STEM +4

.~ .Figure 2. Immunolocalization of EIX protein by tissue printing techniques. Tobacco plants were dissected 20 min after EIX application. Crosssections of tobacco stems and petioles were printed onto nitrocellulose membranes (12), and EIX was immunolocalized with polyclonal antibodies raised against the 22-kD EIX polypeptide (9). Negative and positive numbers represent leaves below and above the point of EIX application, respectively. Stem cross-sections were made at the juncture of each leaf with the stem. Experiments were repeated four times and consistently showed EIX to be spread up and down the stem more than five leaves and in the petioles of leaves -2, -3, -5, +2, +3, and +5 (not all shown). Areas of cross-reactivity with the EIX antibodies are indicated by arrows. Control plants treated with distilled water showed no cross-reactivity under these conditions.

+3 ....

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Ol~~~~

*

0

d*

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-4

TRANSLOCATION OF AN ENDOXYLANASE IN TOBACCO XYLEM

tion occurred in leaves -1 and + 1 (Tables I and II), suggesting that limited amounts of EIX not detectable by our techniques or some product of EIX action may move into these leaves. The pattern demonstrated for EIX movement was mimicked when Evans blue, a vital stain, was applied to whole tobacco plants (Fig. 3). These experiments were all performed with tobacco plants preincubated in an atmosphere purged of ethylene or in an ethylene atmosphere (14 h, 120 ,gL/L). As shown previously (3), ethylene pretreatment led to more rapid and severe symptom development but had little effect upon the overall pattern or speed of EIX protein movement (data not shown).

1185

DISCUSSION This work provides evidence that EIX, a 22-kD fungal protein capable of eliciting plant defense responses, can be transported both upward and downward through the tobacco xylem. These observations may help explain other instances in which necrosis is observed in areas distant from the point of elicitor application (20). Although specific plant proteins have been shown to be localized to the plant vascular system (6), transport of these proteins within the vascular system has not been demonstrated. Given the demonstrated ability of fungal and plant cell wall polysaccharides to elicit plant de-

PETIOLE

STEM

.4

.3 Figure 3. Movement of Evans blue in tobacco plants. A 1% solution of Evans blue (100 ML) was applied through Tygon tubing to exposed petioles on whole tobacco plants. The plants were dissected 20 min after application of Evans blue and cross-sections of stems and petioles were printed onto nitrocellulose membranes. Negative and positive numbers represent leaves below and above the point of Evans blue application, respectively. Stem cross-sections were made at the juncture of each leaf with the stem. The presence of Evans blue on tissue prints is indicated by arrows.

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1186

BAILEY ET AL.

fense responses (4, 10, 13), it is expected that oligosaccharides released from plant cell walls by EIX activity would be the actual elicitor molecules traveling through the tobacco xylem. However, previous efforts to demonstrate the release of such heat-stable signals have been unsuccessful (14). The extremely localized induction of pathogenesis-related protein synthesis in tobacco mesophyll tissue injected with minute amounts of EIX also suggests that EIX must be present locally to induce defense responses (18). Thus, even if EIX generates polysaccharide fragment elicitors, these molecules must be labile or have very limited mobility in the tobacco system. At this point, it is of interest to note that the reported size of the nondenatured EIX protein is equivalent to a 9200 D globular protein (11). Such a small size would allow EIX to penetrate even the smallest cell wall pores (8) and reach the plasmalemma. The movement of EIX both upward and downward through the xylem appears to contradict the generally accepted model of water flow in response to transpirational forces. However, Evans blue dye, introduced into the vascular system via the same route used for EIX, moved in exactly the same pattern. If the xylem elements leading into any given leaf actually intersect the primary stem xylem at points below the site of leaf attachment, then a change in the water sourcesink relationships between the leaves and stalk will suffice to explain these observations. The water (+EIX) that remains in the cut petiole and xylem elements connecting it with the stem xylem becomes a passive water source after removal of the leaf. This water (+EIX) is then drawn downward into the stem by the transpirational pull of the remaining leaves and then travels upward once it reaches vessels leading to actively transpiring leaves. Because xylem elements have limited lateral connections, the radial diffusion of EIX around the tobacco stalk is limited to only one side of the plant. The ability of fungus-produced enzymes to travel through the plant vascular system raises new questions about the mechanisms involved in pathogenesis. Many types of pathogens are known to progress through the plant via the xylem (1). One of these, the fungus F. oxysporum, is responsible for numerous vascular wilt diseases and, as previously noted, is capable of synthesizing an EIX protein when cultured on xylan. Although we have not established whether EIX is produced during host-pathogen interactions, the possibility exists, and we are vigorously pursuing this avenue of research. The potential for inducing systemic resistance to plant pathogens (5, 20, 2 1) by exogenous application of EIX remains to be explored. ACKNOWLEDGMENTS We would like to thank H. David Clark for his excellent help in photographing the figures presented in this article. LITERATURE CITED 1. Agrios CN (1978) Plant Pathology. Academic Press, New York, pp 66-67, 324-340, 464-467 2. Atabekov JG, Dorokhov YL (1984) Plant virus-specific transport function and resistance of plants to viruses. Adv Virus Res 29: 3 13-364


3. Bailey BA, Dean JFD, Anderson JD (1990) An ethylene biosynthesis-inducing endoxylanase elicits electrolyte leakage and necrosis in Nicotiana tabacum cv Xanthi leaves. Plant Physiol 94: 1849-1854 4. Baldwin EA, Biggs KH (1988) Cell-wall lysing enzymes and products of cell-wall digestion elicit ethylene in citrus. Physiol Plant 73: 58-64 5. Biles CL, Martyn RD (1989) Local and systemic resistance induced in watermelons by Formae Speciales of Fusarium oxysporum. Phytopathology 79: 856-860 6. Biles CL, Martyn RD, Wilson HD (1989) Isozymes and general proteins from various watermelon cultivars and tissue types. Hortic Sci 24: 810-812 7. Bowles DJ (1990) Defense-related proteins in higher plants. Annu Rev Biochem 59: 873-908 8. Carpita NC (1982) Limiting diameters of pores and the surface structure of plant cell walls. Science 218: 813-814 9. Cassab GI, Varner JE (1989) Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining by tissue printing on nitrocellulose paper. J Cell Biol 105: 2581-2588 10. Chappell J, Hahlbrock K, Boller T (1984) Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase. Planta 161: 475-480 11. Dean JFD, Anderson JD (1991) The ethylene biosynthesis-inducing xylanase. II. Purification and physical characterization of the enzyme produced by Trichoderma viride. Plant Physiol 95: 316-323 12. Dean JFD, Gamble HR, Anderson JD (1989) The ethylene biosynthesis-inducing xylanase: its induction in Trichoderma viride and certain plant pathogens. Phytopathology 79: 1071-1078 13. Doares SH, Bucheli P, Albersheim P, Darvill AG (1989) Hostpathogen interactions XXXIV. A heat-labile activity secreted by a fungal phytopathogen releases fragments of plant cell wall that kill plant cells. Mol Plant-Microbe Interact 2: 346-353 14. Fuchs Y, Anderson JD (1987) Purification and characterization of ethylene inducing proteins from Cellulysin. Plant Physiol 84: 732-736 15. Fuchs Y, Saxena A, Gamble HR, Anderson JD (1989) Ethylene biosynthesis-inducing protein from Cellulysin is an endoxylanase. Plant Physiol 89: 138-143 16. Hahn GH, Bucheli P, Cervone F, Doares SH, O'Neill RA, Darvill A, Albersheim P (1989) Role of cell wall constituents in plantpathogen interactions. In T Kosuge, EW Nester, eds, PlantMicrobe Interactions, Vol 3. McGraw Hill, New York, pp 131-181 17. Lieberman M, Kunishi AT, Mapson LW, Wardale DA (1966) Stimulation of ethylene production in apple tissue slices by methionine. Plant Physiol 41: 376-382 18. Lotan T, Fluhr R (1990) Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a nonethylene pathway for induction. Plant Physiol 93: 811-817 19. Pegg GF (1976) The involvement of ethylene in plant pathogenesis. In R Heitfuss, PH Williams, eds, Encyclopedia of Plant Physiology, Vol 4. Springer-Verlag, New York, pp 582-591 20. Ricci P, Bonnet P, Huet J-C, Sallantin M, Beauvais-Cante F, Bruneteau M, Billard V, Michel G, Pernollet J-C (1989) Structure and activity of proteins from pathogenic fungi Phytophthora eliciting necrosis and acquired resistance in tobacco. Eur J Biochem 183: 555-563 21. van Loon LC (1989) Stress proteins in infected plant. In T Kosuge, EW Nester, eds, Plant-Microbe Interactions, Vol 3. McGraw Hill, New York, pp 198-239

Plant Physiol. (1991) 97, 1608 0032-0889/91/97/1 608/01/$01 .00/0

CORRECTION Vol. 97, 1181-1186, 1991 Bryan A. Bailey, Rosannah Taylor, Jeffrey F. D. Dean, and James D. Anderson. Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem ofNicotiana tabacum cv Xanthi Plants. An error occurred in the printing of color Figure 2 on page 1184. The correct figure and its legend are shown below.

PETIOLE

STEM

.4

4.*

.W

41

1

A".,

Figure 2. Immunolocalization of EIX protein by tissue printing techniques. Tobacco plants were dissected 20 min after EIX application. Crosssections of tobacco stems and petioles were printed onto nitrocellulose membranes (12), and EIX was immunolocalized with polyclonal antibodies raised against the 22-kD EIX polypeptide (9). Negative and positive numbers represent leaves below and above the point of EIX application, respectively. Stem cross-sections were made at the juncture of each leaf with the stem. Experiments were repeated four times and consistently showed EIX to be spread up and down the stem more than five leaves and in the petioles of leaves -2, -3, -5, +2, +3, and +5 (not all shown). Areas of cross-reactivity with the EIX antibodies are indicated by arrows. Control plants treated with distilled water showed no cross-reactivity under these conditions.

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