Plant Molecular Biology 8:405-414 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
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Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians David B. Collinge, Dawn E. Milligan, J. Maxwell Dow, Graham Scofield and Michael J. Daniels John Innes Institute, Colney Lane, Norwich N R 4 7UH, UK Received 2 September 1986; in revised form 18 December 1986; accepted 28 January 1987 Key words: hypersensitive response, Brassica campestris, X a n t h o m o n a s campestris pv. vitians, mRNA induction, gene expression
Abstract
X a n t h o m o n a s campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting. mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A + RNA in rabbit reticulocyte lysate in the presence of 35S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these bad disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.
Introduction
Genetic engineering should facilitate transfer of genes for resistance to pathogens from resistant but agriculturally unsuitable plants, to susceptible, but otherwise useful crops. However, little is understood of the molecular basis of disease resistance, particularly at the level of pathogen recognition (1, 25). A central feature of disease resistance is the ability of the host to recognise and respond to a potential pathogen (25). In many cases the plant exhibits a hypersensitive reaction (HR, 26). Cells at, or adjacent to the site of invasion undergo characteristic physiological changes involving de novo gene expression resulting in cellular collapse and
death (37). Such physiological changes include vascular blockage, callose formation, synthesis of phenylpropanoids including lignin, melanin and phytoalexins, and the release of toxic metabolites from non-toxic precursors. The role of particular gene products in the defence response has been studied both in vivo in plant-pathogen interactions and in vitro by treating model plant cell culture systems with biotic or abiotic elicitors of host defences (1, 14, 22). There is very little information on the total pattern of gene expression in plants interacting with pathogens. The published work is limited to two fungal systems (18, 19, 29, 31, 34, 41), one viral system (7, 21) and a bacterial system (37). Worldwide, the gram-negative bacterium Xantho-
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monas campestris pv. campestris is probably the most important pathogen of cultivated species of Brassica, and Raphanus and many non-crop Crucifers (43). Previous molecular work in this laboratory has concentrated on the identification and characterisation of genes of X. c. campestris involved in its growth as a pathogen of Brassica campestris (10, 33, 39), and genes of X. c. vitians necessary for the recognition of this pathovar as a potential pathogen of Brassica (11, 35). This paper concerns a complementary study of genes of Brassica campestris ssp. campestris (turnip) induced in response to challenge by the compatible pathovar X. c. campestris, and the incompatible pathovar X.c. vitians, a pathogen of Lactuca spp. (15). The present study is the first concerning plant gene expression in response to a nonhost bacterial pathogen. The results provide the basis for molecular cloning and for more extensive molecular and physiological analyses of the genes involved in resistance. Such studies can be extended to consider the effects of non-pathogenic mutants of X. c. campestris and genetically manipulated X. c. campestris, such as those carrying avirulence determinants from other pathovars (11), and further to other bacterial, fungal and viral pathogens of Brassica.
Materials and methods
Plant material Turnip (Brassica campestris ssp. campestris) cv. 'Just Right' seeds were obtained from W. Atlee Burpee Co., Warminster, PA 18974, USA. Plants were grown in peat-sand mix (1 : 1 ratio) in a greenhouse maintained at 20°C until they were 4 - 6 weeks old, and then transferred to a growth cabinet at 25 °C with 16 h lighting (at 150/zmoles quanta m -2 s -l, 4 0 0 - 7 0 0 nm) for 5 - 7 days before use.
Bacteria Xanthomonas campestris pv. campestris strains 8004 and 8005 are respectively spontaneous rifam-
picin (rW) and streptomycin (str r) resistant mutants of N C P P B 1145 (38) and cause disease on Brassica campestris cv. 'Just Right'. X. c. vitians strain 9000 is N C P P B 1839. Strain 9001 is a spontaneous mutant of strain 9000 resistant to rifampicin (100 /zg/ml). Bacteria for inoculation were grown in NYGB (38).
Inoculation of plant tissue 6 - 7 - w e e k old turnip plants were inoculated with broth cultures (c. l0 s bacteria per ml) using 1-ml plastic syringes without needles. A loaded syringe was placed against the lower surface of the leaf, supported by the thumb of the other hand. Pressure on the plunger caused infiltration of the inoculum through the stomata into the leaf. An area greater than 1 cm 2 can be infiltrated at each site by this method, and a whole leaf may be infiltrated within ten minutes by using adjacent inoculations. Leaves were detached from the plants for R N A preparation at suitable intervals after inoculation, washed with deionised water, blotted dry, weighed, frozen in liquid nitrogen and stored at - 7 0 °C before use for R N A preparation. Bacterial populations were determined essentially by the method of C o o k and Robeson (6). A 1-cm diameter cork borer was used to cut discs from inoculated areas of leaves. These were surface sterilized by brief immersion in propan-2-ol and ground using a sterile pestle and mortar in 1 ml sterile medium. Serial dilutions were plated on NYGB plates containing the appropriate antibiotic.
Electrolyte leakage 15 leaf discs (7 m m diameter) were taken from inoculated areas of leaves at intervals after inoculation, suspended in 10 ml water and vacuum infiltrated with water for 2 min. The conductance of the liquid was determined immediately using a meter, and again after 1 h at 25 °C. Control discs were obtained from uninoculated areas of the same leaves.
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Preparation of messenger RNA Messenger RNA was prepared by modification of the techniques of Martin and Northcote (30) and Covey and Grierson (8). Precautions, including the use o f sterile solutions and heat treatment of glassware, were taken to ensure the absence of ribonucleases from all materials. Apparatus was made from sterile disposable polypropylene or was treated with 1 M N a O H followed by rinsing with sterile 2°7o Decon 90 and water. Glassware was silicon-treated. About 15 g leaf material (harvested into liquid nitrogen and stored at - 7 0 °C) was used for each RNA preparation. The tissue was cooled in liquid nitrogen, ground to a fine powder in a Moulinex model 531 coffee grinder in the presence of liquid nitrogen for about 1 min and transferred to two 50-ml polypropylene centrifuge tubes (Corning No 25330). Two to three vol. of chilled extraction buffer (50 mM Tris-HCl, pH 9.0, 150 mM LiCI, 5 mM EDTA and 5°70 (w/v) SDS) were stirred into the powdered leaf tissue for about a minute. An equal vol. of phenol-chloroform (1 : 1) was added to the slurry, the tubes were shaken for a minute and centrifuged at 3000 rpm at room temperature for 10 min. The phenol-chloroform phase was reextracted with an equal vol. of extraction buffer, and after centrifugation the aqueous phases were combined and extracted a further three to four times with phenol-chloroform, transferred to an Erlenmeyer flask, and after LiC1 had been added to a final concentration of 0.2 M, two vol. of chilled ( - 2 0 ° C ) absolute ethanol were added and the RNA precipitated at - 2 0 ° C overnight. The precipitated nucleic acid was centrifuged at 10000 g (Av.) for 10 min at 4°C. The pellet was resuspended in 0.15 M sodium acetate, two vol. of chilled ethanol were added, and the suspension recentrifuged. This step was repeated, the pellet was resuspended in 0.15 M sodium acetate, two vol. of ethanol (chilled to - 2 0 ° C ) were added and the nucleic acid was precipitated overnight at - 2 0 ° C . The precipitate was recovered by centrifugation, dessiccated, then resuspended in 1 - 2 ml 0.15 M sodium acetate to which three vol. of 4 M sodium acetate were subsequently added. The tube was left in ice
for 30 min, centrifuged at 10000 g for 10 min and the pellet (chiefly rRNA and mRNA) was rewashed by the same procedure. The pellet obtained was finally resuspended in 0.15 M sodium acetate, two volumes of chilled ethanol were added and the RNA was precipitated overnight at - 2 0 ° C . This pellet was rewashed with 0.15 M sodium acetate and two vol. ethanol, resuspended in a minimum vol. of sterile water (treated with 0.1% diethyl pyrocarbonate) and the nucleic acid concentration determined spectrophotometrically. The concentration of the RNA was adjusted to 2 mg/ml and the solution was stored at - 7 0 ° C . Poly A ÷ RNA fractionation was carried out by the method of Martin and Northcote (30).
In vitro translation Rabbit reticulocyte lysate was obtained from Amersham International PLC (nuclease treated, message dependent lysate, code N90) or was a generous gift from B. A. Morris-Krsinich (prepared according to Jackson and Hunt (22)). Translations used 1/zl RNA solution (2/zg in the case of unfractionated preparations), 10 tzCi (1 #l) 35S-methionine (New England Nuclear sp. Act c. 1000 Ci/mmol cat no. NEG-009A) and 8 #1 lysate. Mixtures were incubated at 30°C for 1 h before by the addition of the appropriate sample buffer for electrophoresis (see below). A high specific activity mixture of tritiated amino acids was used as an alternative to 35S-methionine. This comprised equal quantities (by activity) L-leucine, L-lysine monohydrochloride, L-phenylalanine, L-proline and Ltyrosine (Amersham catalogue TRK.550). 10/zCi of this mixture was used for each incubation after vacuum concentration. Incorporation of labelled amino acid into protein was determined by drying a 1-/xl sample of the translation mixture onto a small square of Whatman 3 MM filter paper, washing with 10%0 TCA and 0.5% of each of the appropriate unlabelled amino acids for 30 min at 100 °C, followed by washing for 15 rain in ethanoldiethyl ether (1:1) at 37°C. The filters were then dried and radioactivity was determined by scintillation counting. Incorporation was compared with
408 translations performed with water in place of RNA.
Polyacrylamide gel electrophoresis (PAGE)
Two dimensional electrophoresis (2D-PAGE) was carried out by the method of O'Farrell (32) with minor modifications as described by Burland et al. (3). Samples were prepared for electrophoresis according to Burland et al. (3) with the omission of deoxyribonuclease treatment and leupeptin from the sample buffer, and samples were not acetone precipitated, lie/0, 0.9 m m thick gels were used for second dimension (SDS) electrophoresis. Molecular weights of polypeptides were determined by the method of Weber and Osborn (42), by running [14C]methylated polypeptide markers (Amersham code CFA 626) along the edge of selected second dimensional gels. Gels were fixed by soaking in 30% (v/v) ethanol, 10% (v/v) acetic acid for 40 min with agitation. When non-methylated molecular weight markers were used, gels were fixed and stained in 0.05°7o (w/v) Brilliant blue R (Aldrich) in 2507o (v/v) propan-2-ol, 10070 (v/v) acetic acid at 37°C for 6 0 - 9 0 min, and were destained in several changes 15°70 (v/v) propan-2-ol, 10070 (v/v) acetic acid at r o o m temperature, before fluorography which was performed by the methods of Chamberlain (4) or Jen and Thach (23). Gels were dried for fluorography and were exposed to Fuji RX medical X-ray film at - 7 0 ° C .
Results
S y m p t o m expression in Brassica leaves
The symptoms induced 24 hours after inoculation of a Brassiea leaf with both X. campestris pv. campestris and pv. vitians at a concentration of l0 s bacteria per ml are shown in Fig. 1. The inoculation method caused initial water soaking which cleared within one to two hours. The syringe caused a conspicuous wound in the centre of the inoculated zones, but the remainder of the infiltrated area had
Fig. 1. The effects of X a n t h o m o n a s campestris pv. campestris and X. c. vitians on Brassica campestris leaves 24 h after inoculation. The left hand side of the leaf was inoculated with X. c. campestris (strain 8004) in two places and the right hand side with X. c. vitians (strain 9000) in three places with 107 cells per ml.
the same appearance as the surrounding, uninoculated tissue. Inoculation with X. c. vitians rapidly led to extensive darkening and cellular collapse over the whole inoculated region. The symptoms of cellular collapse were first visible within about 8 h of inoculation: the lower surface of leaves inoculated with X. c. vitians appeared glossy in contrast to the surrounding uninoculated tissue. Another early s y m p t o m was apparent vein blockage. Eosin red dye readily penetrated uninoculated leaf tissue and tissue inoculated with X. c. campestris, but did not penetrate areas inoculated with X. c. vitians eight hours after inoculation. In contrast, X. c. campestris caused initial yellowing of the inoculated area after 48 h which subsequently spread. This was followed by rotting and melanisation. Leaves inoculated with sterile growth medium showed initial water soaking, and wounding attributable to the direct pressure of the syringe, but otherwise had the appearance of uninoculated leaves. Loss of cellular integrity, as shown by electrolyte leakage, was much
409 vitians cells decreased rapidly in Brassica leaves following inoculation, whereas those of X. c. campestris increased (data not shown). Inoculation of mixtures of strains 8005 (X. c. campestris, str r) and 9001 (X. c. vitians, rig) in various proportions resulted in a progressive reduction in the numbers of str r bacteria which could be recovered as the proportion of X. c. vitians inoculated increased. Rapid cellular collapse, electrolyte leakage, the requirement for metabolically active bacteria and limitation of bacteria spread all imply that the plant response to X. c. vitians has the characteristics of the hypersensitive response (HR) according to the definition of Klement (27).
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Analys& o f de novo gene expression following inoculation o f Brassica leaves
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The rapid development of symptoms induced in CO
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Fig. 2. Electrolyte leakage following inoculation with X . c . c a m p e s t r i s and X . c. vitians. Electrolyte leakage is expressed as
increase in conductance of the suspending solution over one hour following vacuum infiltration. Filled triangles: X . c. campestris. Filled circles: X . c. vitians. Open symbols represent controis.
more rapid in leaves inoculated with X. c. vitians than with X. c. campestris (Fig. 2). The electrolyte leakage was first recorded four hours after visible signs of tissue collapse. Visible symptoms did not develop when the inoculum contained fewer than c. 106 cells/ml. Furthermore, symptom development required metabolically active bacterial cells. X . c . vitians treated with rifampicin (50/zg/ml) or streptomycin (100/~g/ml) for 30 min and then washed did not induce symptoms. Similarly bacteria killed by heating to 100°C for 10 min prior to inoculation did not induce symptoms. Symptom development was also inhibited by infiltration of streptomycin (100 #g/ml) into leaves for up to 4 h after inoculation with X. c. vitians. The numbers of viable X. c.
Brassica campestris leaves by X. c. vitians indicates
that any specific gene expression on the part of Brassica campestris associated with H R must occur
within a few hours of inoculation, and the outcome of the response (i.e. cell death), implies that there is little point in looking beyond 24 h or so. We have therefore concentrated our study on the comparison of changes in gene expression in response to X. c. vitians, X. c. campestris and sterile growth medium over a 24-h period following inoculation. Great care has to be taken in this type of study to ensure that environmental differences between treatments are minimised. Thus, inoculations were performed on a number of plants, and were staggered so that all sampling points and treatments within any batch of plants were randomised. The results presented in this paper are from representative translation of RNA prepared from three independent sets o f inoculation timecourses. Total X a n t h o m o n a s RNA did not stimulate the incorporation of label into TCA-precipitable material, or give detectable polypeptide translation products using rabbit reticulocyte lysate. SDS-PAGE analyses of in vitro translation products of RNA prepared over a 24-h period after inoculation were performed using the gel systems of Laemmli (28) and Chua and Bennoun (5). No
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411 m a j o r differences in the polypeptide banding patterns which could be confidently ascribed to the effects of X. c. vitians or X. c. campestris were detected in any of the SDS gel systems tried. Most changes could be seen at the same times following inoculation with all three inocula, and thus probably represent responses to inoculation p e r se (results not shown). In vitro translation products were therefore analysed by 2D-PAGE which can readily resolve up to 600 independent polypeptides under our conditions. 2D-PAGE analysis of the in vitro translation products of total R N A extracted from uninoculated leaves and from leaves four and nine hours after inoculation with X. c. vitians and sterile medium are shown in Fig. 3. The major changes seen are in response to the inoculation process rather than to the bacteria, confirming the results obtained by SDSPAGE analysis of the inoculation timecourses. C o m p a r i s o n of these results with those from 2DPAGE analysis of in vitro translation products of R N A from 30 min, 1 h, 2 h and 6 h after inoculation demonstrate that inoculation causes many transient changes in leaf gene expression which are unrelated to the H R since these were seen in inoculations with medium alone (results not presented). No changes were observed 30 min after inoculation, but by 1 h m a n y of these changes could be detected; maxim u m differences in both numbers and intensity of many of the spots representing new polypeptides were not reached until 4 h. At 6 h a reduction in both numbers and intensity of these transient polypeptides was noted. Figure 3 also shows that at both 4 h and 9 h after inoculation several polypeptides were detected in response to inoculation with X. c. vitians but not with sterile medium (or with X . c . campestris, result not shown).
Although many of these polypeptides were detected at both 4 h and 9 h, others appeared at either 4 h or 9 h alone. No polypeptides were seen to be induced by X. c. vitians alone before 4 h. There was no indication of disappearance or reduction in the amounts of any polypeptides in specific response to X. c. vitians other than those seen at 4 but not 9 h. No changes were recorded in response to X. c. carnpestris which were not also responses to sterile medium within 9 h of inoculation. This is perhaps not surprising since there is no difference in the visible effects of inoculation with medium and X. c. campestris over the period covered by this analysis. We conclude that the additional polypeptides observed in response to X. c. vitians are associated with H R and are not non-specific effects caused by inoculation with bacteria. Similar results to those presented (Fig. 3) were obtained using a tritiated mixture of amino acids instead of 35S-methionine in in vitro translations, and by using a wheat germ in vitro translation system with 35S-methionine (12). No major differences could be detected in the polypeptide products obtained by in vitro translation of R N A enriched for polyadenylated sequences compared to total R N A (results not presented).
Discussion
The salient features of the hypersensitive response are its rapidity, localised nature and ability to prevent the spread of the invading bacteria (26). H R in P i s u m and Phaseolus is characterised by specific gene expression on the part of the plant (18, 19, 34, 37, 41). Further, the inhibition of de novo gene expression with blastocydin S and cycloheximide in-
Fig. 3. 2D-PAGE analysis of in vitro translation products from m R N A prepared following inoculation with sterile medium and X. c. vitians. Total m R N A was prepared and translated in rabbit reticulocyte lysate using 35S-methionine, analysed by 2D-PAGE with
fluorography according to (22). Panel 1: uninoculated tissue, 2: 4-h NYGB, 3: 4-h X . c. vitians, 4: 9-h NYGB, 5: 9-h X. c. vitians. The symbols shown on panels 3 and 5 indicate spots present following inoculation with X . c. vitians but not NYGB. Differences a m o n g panels 1, 2 and 4 which represent inoculation effects are not indicated to avoid crowding. Positions of protein standards are shown for the second dimension (SDS-PAGE), numbers represent M r × 10 -3. p H gradient increases from left to right.
412 hibits the development of H R and, in some cases, results in the proliferation of a pathogen which would otherwise be contained (24, 26). We find that, as in other host-pathogen interactions involving bacterial pathogens, the development of H R in Brassica in response to X. c. vitians is dependent on the use of live or metabolically active inoculum. H R is rapid in comparison to the development of the susceptible reaction and results in the containment and subsequent death of the incompatible bacteria within the area of initial contact. The analysis of the in vitro translation products of mRNA from Brassica leaves clearly indicates that there are several changes which are specific to inoculation with the HR-inducing strain X. c. vitians, although the major numerical changes in gene expression represent a response to the inoculation procedure. Both the changes in polypeptides attributable to inoculation stress and those which correlated with the development of HR are scattered over the major part of the gels. This implies that the polypeptides involved are heterogeneous both in size and isoelectric point. These polypeptides therefore do not fall into a single group of polypeptides such as that commonly known as pathogenesis related proteins (40). The responses to the inoculation technique are likely to reflect two independent features of the process, namely physical tissue wounding caused by the syringe and flooding within the leaf from the inoculum. Wounding results in the expression of many genes (36) some of which are also expressed in response to pathogens, so it is likely that a number of these would be expressed in H R in the absence of wounding. Transient anoxia caused by flooding within the leaf may account for both the transient disappearance and appearance of some of these polypeptides. Prolonged root flooding is known to have major effects on gene expression involving the synthesis of many 'housekeeping' enzymes and the specific induction of genes for enzymes including alcohol dehydrogenase and glycolytic enzymes (13, 17). A number of probable biochemical defence responses have been identified in several plant species which involve de novo gene expression following challenge by pathogens. These are well documented elsewhere (2, 7, 14, 16, 21, 31, 37, 40).
It is likely that some of these account for a proportion of the HR-induced gene expression recorded in this study, although the physiological basis of disease resistance in Brassica has so far received little study and so it is not possible to assign functions to any individual new polypeptides observed in this study. Comparison of cloned genes from different plant sources with cloned Brassica genes, and the use of antisera raised against specific plant polypeptides in in vitro translation studies may help the identification of these defence genes. Finally, although the major effects on gene expression recorded in this study represent a response to the inoculation procedure, similar changes may take place during field infections, since a common method of spread of X. c. campestris is considered to be via rain and storm damage, leading to water soaking of plant tissues (27, 43).
Acknowledgements We are grateful to Drs B. A. Morris-Krsinich and T. M. A. Wilson for generous gifts of rabbit reticulocyte lysate used in early stages of this study, to Dr J. W. Davies who carried out translations using wheat germ extracts for us, to C. Gough for a sample of X a n t h o m o n a s campestris RNA and to Drs C. R. Martin and S. N. Covey for advice and discussion concerning methods for extracting mRNA. We also wish to thank Prof. D. A. Hopwood FRS, Drs A. J. Slusarenko and A. W. B. Johnston for critically reading the manuscript. This work was supported by the Agricultural and Food Research Council via grant-in-aid to the John Innes Institute.
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39. Turner PJ, Barber CE, Daniels M J: Evidence for clustered pathogenicity genes in Xanthomonas campestris pv campestris. Molec Gen Genet 199:338-343 (1985). 40. Van Loon LC: Pathogenesis-related proteins. Plant Mol Biol 4:111-116 (1985). 41. Wagoner W, Loscke DC, Hadwiger LA: Two dimensional electrophoretic analysis of in vivo and in vitro synthesis of proteins in peas inoculated with compatible and incompatible Fusarium solani. Physiol Plant Pathol 2 0 : 9 9 - 1 0 7 (1982). 42. Weber K, Osborn M: The reliability of molecular weight determination by dodecyl sulphate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406-4412 (1969). 43. Williams PH: Black rot: a continuing threat to world crucifers. Plant Disease 6 4 : 7 3 6 - 7 4 2 (1980).
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Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians David B. Collinge, Dawn E. Milligan, J. Maxwell Dow, Graham Scofield and Michael J. Daniels John Innes Institute, Colney Lane, Norwich N R 4 7UH, UK Received 2 September 1986; in revised form 18 December 1986; accepted 28 January 1987 Key words: hypersensitive response, Brassica campestris, X a n t h o m o n a s campestris pv. vitians, mRNA induction, gene expression
Abstract
X a n t h o m o n a s campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting. mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A + RNA in rabbit reticulocyte lysate in the presence of 35S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these bad disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.
Introduction
Genetic engineering should facilitate transfer of genes for resistance to pathogens from resistant but agriculturally unsuitable plants, to susceptible, but otherwise useful crops. However, little is understood of the molecular basis of disease resistance, particularly at the level of pathogen recognition (1, 25). A central feature of disease resistance is the ability of the host to recognise and respond to a potential pathogen (25). In many cases the plant exhibits a hypersensitive reaction (HR, 26). Cells at, or adjacent to the site of invasion undergo characteristic physiological changes involving de novo gene expression resulting in cellular collapse and
death (37). Such physiological changes include vascular blockage, callose formation, synthesis of phenylpropanoids including lignin, melanin and phytoalexins, and the release of toxic metabolites from non-toxic precursors. The role of particular gene products in the defence response has been studied both in vivo in plant-pathogen interactions and in vitro by treating model plant cell culture systems with biotic or abiotic elicitors of host defences (1, 14, 22). There is very little information on the total pattern of gene expression in plants interacting with pathogens. The published work is limited to two fungal systems (18, 19, 29, 31, 34, 41), one viral system (7, 21) and a bacterial system (37). Worldwide, the gram-negative bacterium Xantho-
406
monas campestris pv. campestris is probably the most important pathogen of cultivated species of Brassica, and Raphanus and many non-crop Crucifers (43). Previous molecular work in this laboratory has concentrated on the identification and characterisation of genes of X. c. campestris involved in its growth as a pathogen of Brassica campestris (10, 33, 39), and genes of X. c. vitians necessary for the recognition of this pathovar as a potential pathogen of Brassica (11, 35). This paper concerns a complementary study of genes of Brassica campestris ssp. campestris (turnip) induced in response to challenge by the compatible pathovar X. c. campestris, and the incompatible pathovar X.c. vitians, a pathogen of Lactuca spp. (15). The present study is the first concerning plant gene expression in response to a nonhost bacterial pathogen. The results provide the basis for molecular cloning and for more extensive molecular and physiological analyses of the genes involved in resistance. Such studies can be extended to consider the effects of non-pathogenic mutants of X. c. campestris and genetically manipulated X. c. campestris, such as those carrying avirulence determinants from other pathovars (11), and further to other bacterial, fungal and viral pathogens of Brassica.
Materials and methods
Plant material Turnip (Brassica campestris ssp. campestris) cv. 'Just Right' seeds were obtained from W. Atlee Burpee Co., Warminster, PA 18974, USA. Plants were grown in peat-sand mix (1 : 1 ratio) in a greenhouse maintained at 20°C until they were 4 - 6 weeks old, and then transferred to a growth cabinet at 25 °C with 16 h lighting (at 150/zmoles quanta m -2 s -l, 4 0 0 - 7 0 0 nm) for 5 - 7 days before use.
Bacteria Xanthomonas campestris pv. campestris strains 8004 and 8005 are respectively spontaneous rifam-
picin (rW) and streptomycin (str r) resistant mutants of N C P P B 1145 (38) and cause disease on Brassica campestris cv. 'Just Right'. X. c. vitians strain 9000 is N C P P B 1839. Strain 9001 is a spontaneous mutant of strain 9000 resistant to rifampicin (100 /zg/ml). Bacteria for inoculation were grown in NYGB (38).
Inoculation of plant tissue 6 - 7 - w e e k old turnip plants were inoculated with broth cultures (c. l0 s bacteria per ml) using 1-ml plastic syringes without needles. A loaded syringe was placed against the lower surface of the leaf, supported by the thumb of the other hand. Pressure on the plunger caused infiltration of the inoculum through the stomata into the leaf. An area greater than 1 cm 2 can be infiltrated at each site by this method, and a whole leaf may be infiltrated within ten minutes by using adjacent inoculations. Leaves were detached from the plants for R N A preparation at suitable intervals after inoculation, washed with deionised water, blotted dry, weighed, frozen in liquid nitrogen and stored at - 7 0 °C before use for R N A preparation. Bacterial populations were determined essentially by the method of C o o k and Robeson (6). A 1-cm diameter cork borer was used to cut discs from inoculated areas of leaves. These were surface sterilized by brief immersion in propan-2-ol and ground using a sterile pestle and mortar in 1 ml sterile medium. Serial dilutions were plated on NYGB plates containing the appropriate antibiotic.
Electrolyte leakage 15 leaf discs (7 m m diameter) were taken from inoculated areas of leaves at intervals after inoculation, suspended in 10 ml water and vacuum infiltrated with water for 2 min. The conductance of the liquid was determined immediately using a meter, and again after 1 h at 25 °C. Control discs were obtained from uninoculated areas of the same leaves.
407
Preparation of messenger RNA Messenger RNA was prepared by modification of the techniques of Martin and Northcote (30) and Covey and Grierson (8). Precautions, including the use o f sterile solutions and heat treatment of glassware, were taken to ensure the absence of ribonucleases from all materials. Apparatus was made from sterile disposable polypropylene or was treated with 1 M N a O H followed by rinsing with sterile 2°7o Decon 90 and water. Glassware was silicon-treated. About 15 g leaf material (harvested into liquid nitrogen and stored at - 7 0 °C) was used for each RNA preparation. The tissue was cooled in liquid nitrogen, ground to a fine powder in a Moulinex model 531 coffee grinder in the presence of liquid nitrogen for about 1 min and transferred to two 50-ml polypropylene centrifuge tubes (Corning No 25330). Two to three vol. of chilled extraction buffer (50 mM Tris-HCl, pH 9.0, 150 mM LiCI, 5 mM EDTA and 5°70 (w/v) SDS) were stirred into the powdered leaf tissue for about a minute. An equal vol. of phenol-chloroform (1 : 1) was added to the slurry, the tubes were shaken for a minute and centrifuged at 3000 rpm at room temperature for 10 min. The phenol-chloroform phase was reextracted with an equal vol. of extraction buffer, and after centrifugation the aqueous phases were combined and extracted a further three to four times with phenol-chloroform, transferred to an Erlenmeyer flask, and after LiC1 had been added to a final concentration of 0.2 M, two vol. of chilled ( - 2 0 ° C ) absolute ethanol were added and the RNA precipitated at - 2 0 ° C overnight. The precipitated nucleic acid was centrifuged at 10000 g (Av.) for 10 min at 4°C. The pellet was resuspended in 0.15 M sodium acetate, two vol. of chilled ethanol were added, and the suspension recentrifuged. This step was repeated, the pellet was resuspended in 0.15 M sodium acetate, two vol. of ethanol (chilled to - 2 0 ° C ) were added and the nucleic acid was precipitated overnight at - 2 0 ° C . The precipitate was recovered by centrifugation, dessiccated, then resuspended in 1 - 2 ml 0.15 M sodium acetate to which three vol. of 4 M sodium acetate were subsequently added. The tube was left in ice
for 30 min, centrifuged at 10000 g for 10 min and the pellet (chiefly rRNA and mRNA) was rewashed by the same procedure. The pellet obtained was finally resuspended in 0.15 M sodium acetate, two volumes of chilled ethanol were added and the RNA was precipitated overnight at - 2 0 ° C . This pellet was rewashed with 0.15 M sodium acetate and two vol. ethanol, resuspended in a minimum vol. of sterile water (treated with 0.1% diethyl pyrocarbonate) and the nucleic acid concentration determined spectrophotometrically. The concentration of the RNA was adjusted to 2 mg/ml and the solution was stored at - 7 0 ° C . Poly A ÷ RNA fractionation was carried out by the method of Martin and Northcote (30).
In vitro translation Rabbit reticulocyte lysate was obtained from Amersham International PLC (nuclease treated, message dependent lysate, code N90) or was a generous gift from B. A. Morris-Krsinich (prepared according to Jackson and Hunt (22)). Translations used 1/zl RNA solution (2/zg in the case of unfractionated preparations), 10 tzCi (1 #l) 35S-methionine (New England Nuclear sp. Act c. 1000 Ci/mmol cat no. NEG-009A) and 8 #1 lysate. Mixtures were incubated at 30°C for 1 h before by the addition of the appropriate sample buffer for electrophoresis (see below). A high specific activity mixture of tritiated amino acids was used as an alternative to 35S-methionine. This comprised equal quantities (by activity) L-leucine, L-lysine monohydrochloride, L-phenylalanine, L-proline and Ltyrosine (Amersham catalogue TRK.550). 10/zCi of this mixture was used for each incubation after vacuum concentration. Incorporation of labelled amino acid into protein was determined by drying a 1-/xl sample of the translation mixture onto a small square of Whatman 3 MM filter paper, washing with 10%0 TCA and 0.5% of each of the appropriate unlabelled amino acids for 30 min at 100 °C, followed by washing for 15 rain in ethanoldiethyl ether (1:1) at 37°C. The filters were then dried and radioactivity was determined by scintillation counting. Incorporation was compared with
408 translations performed with water in place of RNA.
Polyacrylamide gel electrophoresis (PAGE)
Two dimensional electrophoresis (2D-PAGE) was carried out by the method of O'Farrell (32) with minor modifications as described by Burland et al. (3). Samples were prepared for electrophoresis according to Burland et al. (3) with the omission of deoxyribonuclease treatment and leupeptin from the sample buffer, and samples were not acetone precipitated, lie/0, 0.9 m m thick gels were used for second dimension (SDS) electrophoresis. Molecular weights of polypeptides were determined by the method of Weber and Osborn (42), by running [14C]methylated polypeptide markers (Amersham code CFA 626) along the edge of selected second dimensional gels. Gels were fixed by soaking in 30% (v/v) ethanol, 10% (v/v) acetic acid for 40 min with agitation. When non-methylated molecular weight markers were used, gels were fixed and stained in 0.05°7o (w/v) Brilliant blue R (Aldrich) in 2507o (v/v) propan-2-ol, 10070 (v/v) acetic acid at 37°C for 6 0 - 9 0 min, and were destained in several changes 15°70 (v/v) propan-2-ol, 10070 (v/v) acetic acid at r o o m temperature, before fluorography which was performed by the methods of Chamberlain (4) or Jen and Thach (23). Gels were dried for fluorography and were exposed to Fuji RX medical X-ray film at - 7 0 ° C .
Results
S y m p t o m expression in Brassica leaves
The symptoms induced 24 hours after inoculation of a Brassiea leaf with both X. campestris pv. campestris and pv. vitians at a concentration of l0 s bacteria per ml are shown in Fig. 1. The inoculation method caused initial water soaking which cleared within one to two hours. The syringe caused a conspicuous wound in the centre of the inoculated zones, but the remainder of the infiltrated area had
Fig. 1. The effects of X a n t h o m o n a s campestris pv. campestris and X. c. vitians on Brassica campestris leaves 24 h after inoculation. The left hand side of the leaf was inoculated with X. c. campestris (strain 8004) in two places and the right hand side with X. c. vitians (strain 9000) in three places with 107 cells per ml.
the same appearance as the surrounding, uninoculated tissue. Inoculation with X. c. vitians rapidly led to extensive darkening and cellular collapse over the whole inoculated region. The symptoms of cellular collapse were first visible within about 8 h of inoculation: the lower surface of leaves inoculated with X. c. vitians appeared glossy in contrast to the surrounding uninoculated tissue. Another early s y m p t o m was apparent vein blockage. Eosin red dye readily penetrated uninoculated leaf tissue and tissue inoculated with X. c. campestris, but did not penetrate areas inoculated with X. c. vitians eight hours after inoculation. In contrast, X. c. campestris caused initial yellowing of the inoculated area after 48 h which subsequently spread. This was followed by rotting and melanisation. Leaves inoculated with sterile growth medium showed initial water soaking, and wounding attributable to the direct pressure of the syringe, but otherwise had the appearance of uninoculated leaves. Loss of cellular integrity, as shown by electrolyte leakage, was much
409 vitians cells decreased rapidly in Brassica leaves following inoculation, whereas those of X. c. campestris increased (data not shown). Inoculation of mixtures of strains 8005 (X. c. campestris, str r) and 9001 (X. c. vitians, rig) in various proportions resulted in a progressive reduction in the numbers of str r bacteria which could be recovered as the proportion of X. c. vitians inoculated increased. Rapid cellular collapse, electrolyte leakage, the requirement for metabolically active bacteria and limitation of bacteria spread all imply that the plant response to X. c. vitians has the characteristics of the hypersensitive response (HR) according to the definition of Klement (27).
100 IJmhos
80
60
40
Analys& o f de novo gene expression following inoculation o f Brassica leaves
20
The rapid development of symptoms induced in CO
o
12
i
24
l
48
t
72 hrs
Fig. 2. Electrolyte leakage following inoculation with X . c . c a m p e s t r i s and X . c. vitians. Electrolyte leakage is expressed as
increase in conductance of the suspending solution over one hour following vacuum infiltration. Filled triangles: X . c. campestris. Filled circles: X . c. vitians. Open symbols represent controis.
more rapid in leaves inoculated with X. c. vitians than with X. c. campestris (Fig. 2). The electrolyte leakage was first recorded four hours after visible signs of tissue collapse. Visible symptoms did not develop when the inoculum contained fewer than c. 106 cells/ml. Furthermore, symptom development required metabolically active bacterial cells. X . c . vitians treated with rifampicin (50/zg/ml) or streptomycin (100/~g/ml) for 30 min and then washed did not induce symptoms. Similarly bacteria killed by heating to 100°C for 10 min prior to inoculation did not induce symptoms. Symptom development was also inhibited by infiltration of streptomycin (100 #g/ml) into leaves for up to 4 h after inoculation with X. c. vitians. The numbers of viable X. c.
Brassica campestris leaves by X. c. vitians indicates
that any specific gene expression on the part of Brassica campestris associated with H R must occur
within a few hours of inoculation, and the outcome of the response (i.e. cell death), implies that there is little point in looking beyond 24 h or so. We have therefore concentrated our study on the comparison of changes in gene expression in response to X. c. vitians, X. c. campestris and sterile growth medium over a 24-h period following inoculation. Great care has to be taken in this type of study to ensure that environmental differences between treatments are minimised. Thus, inoculations were performed on a number of plants, and were staggered so that all sampling points and treatments within any batch of plants were randomised. The results presented in this paper are from representative translation of RNA prepared from three independent sets o f inoculation timecourses. Total X a n t h o m o n a s RNA did not stimulate the incorporation of label into TCA-precipitable material, or give detectable polypeptide translation products using rabbit reticulocyte lysate. SDS-PAGE analyses of in vitro translation products of RNA prepared over a 24-h period after inoculation were performed using the gel systems of Laemmli (28) and Chua and Bennoun (5). No
410
411 m a j o r differences in the polypeptide banding patterns which could be confidently ascribed to the effects of X. c. vitians or X. c. campestris were detected in any of the SDS gel systems tried. Most changes could be seen at the same times following inoculation with all three inocula, and thus probably represent responses to inoculation p e r se (results not shown). In vitro translation products were therefore analysed by 2D-PAGE which can readily resolve up to 600 independent polypeptides under our conditions. 2D-PAGE analysis of the in vitro translation products of total R N A extracted from uninoculated leaves and from leaves four and nine hours after inoculation with X. c. vitians and sterile medium are shown in Fig. 3. The major changes seen are in response to the inoculation process rather than to the bacteria, confirming the results obtained by SDSPAGE analysis of the inoculation timecourses. C o m p a r i s o n of these results with those from 2DPAGE analysis of in vitro translation products of R N A from 30 min, 1 h, 2 h and 6 h after inoculation demonstrate that inoculation causes many transient changes in leaf gene expression which are unrelated to the H R since these were seen in inoculations with medium alone (results not presented). No changes were observed 30 min after inoculation, but by 1 h m a n y of these changes could be detected; maxim u m differences in both numbers and intensity of many of the spots representing new polypeptides were not reached until 4 h. At 6 h a reduction in both numbers and intensity of these transient polypeptides was noted. Figure 3 also shows that at both 4 h and 9 h after inoculation several polypeptides were detected in response to inoculation with X. c. vitians but not with sterile medium (or with X . c . campestris, result not shown).
Although many of these polypeptides were detected at both 4 h and 9 h, others appeared at either 4 h or 9 h alone. No polypeptides were seen to be induced by X. c. vitians alone before 4 h. There was no indication of disappearance or reduction in the amounts of any polypeptides in specific response to X. c. vitians other than those seen at 4 but not 9 h. No changes were recorded in response to X. c. carnpestris which were not also responses to sterile medium within 9 h of inoculation. This is perhaps not surprising since there is no difference in the visible effects of inoculation with medium and X. c. campestris over the period covered by this analysis. We conclude that the additional polypeptides observed in response to X. c. vitians are associated with H R and are not non-specific effects caused by inoculation with bacteria. Similar results to those presented (Fig. 3) were obtained using a tritiated mixture of amino acids instead of 35S-methionine in in vitro translations, and by using a wheat germ in vitro translation system with 35S-methionine (12). No major differences could be detected in the polypeptide products obtained by in vitro translation of R N A enriched for polyadenylated sequences compared to total R N A (results not presented).
Discussion
The salient features of the hypersensitive response are its rapidity, localised nature and ability to prevent the spread of the invading bacteria (26). H R in P i s u m and Phaseolus is characterised by specific gene expression on the part of the plant (18, 19, 34, 37, 41). Further, the inhibition of de novo gene expression with blastocydin S and cycloheximide in-
Fig. 3. 2D-PAGE analysis of in vitro translation products from m R N A prepared following inoculation with sterile medium and X. c. vitians. Total m R N A was prepared and translated in rabbit reticulocyte lysate using 35S-methionine, analysed by 2D-PAGE with
fluorography according to (22). Panel 1: uninoculated tissue, 2: 4-h NYGB, 3: 4-h X . c. vitians, 4: 9-h NYGB, 5: 9-h X. c. vitians. The symbols shown on panels 3 and 5 indicate spots present following inoculation with X . c. vitians but not NYGB. Differences a m o n g panels 1, 2 and 4 which represent inoculation effects are not indicated to avoid crowding. Positions of protein standards are shown for the second dimension (SDS-PAGE), numbers represent M r × 10 -3. p H gradient increases from left to right.
412 hibits the development of H R and, in some cases, results in the proliferation of a pathogen which would otherwise be contained (24, 26). We find that, as in other host-pathogen interactions involving bacterial pathogens, the development of H R in Brassica in response to X. c. vitians is dependent on the use of live or metabolically active inoculum. H R is rapid in comparison to the development of the susceptible reaction and results in the containment and subsequent death of the incompatible bacteria within the area of initial contact. The analysis of the in vitro translation products of mRNA from Brassica leaves clearly indicates that there are several changes which are specific to inoculation with the HR-inducing strain X. c. vitians, although the major numerical changes in gene expression represent a response to the inoculation procedure. Both the changes in polypeptides attributable to inoculation stress and those which correlated with the development of HR are scattered over the major part of the gels. This implies that the polypeptides involved are heterogeneous both in size and isoelectric point. These polypeptides therefore do not fall into a single group of polypeptides such as that commonly known as pathogenesis related proteins (40). The responses to the inoculation technique are likely to reflect two independent features of the process, namely physical tissue wounding caused by the syringe and flooding within the leaf from the inoculum. Wounding results in the expression of many genes (36) some of which are also expressed in response to pathogens, so it is likely that a number of these would be expressed in H R in the absence of wounding. Transient anoxia caused by flooding within the leaf may account for both the transient disappearance and appearance of some of these polypeptides. Prolonged root flooding is known to have major effects on gene expression involving the synthesis of many 'housekeeping' enzymes and the specific induction of genes for enzymes including alcohol dehydrogenase and glycolytic enzymes (13, 17). A number of probable biochemical defence responses have been identified in several plant species which involve de novo gene expression following challenge by pathogens. These are well documented elsewhere (2, 7, 14, 16, 21, 31, 37, 40).
It is likely that some of these account for a proportion of the HR-induced gene expression recorded in this study, although the physiological basis of disease resistance in Brassica has so far received little study and so it is not possible to assign functions to any individual new polypeptides observed in this study. Comparison of cloned genes from different plant sources with cloned Brassica genes, and the use of antisera raised against specific plant polypeptides in in vitro translation studies may help the identification of these defence genes. Finally, although the major effects on gene expression recorded in this study represent a response to the inoculation procedure, similar changes may take place during field infections, since a common method of spread of X. c. campestris is considered to be via rain and storm damage, leading to water soaking of plant tissues (27, 43).
Acknowledgements We are grateful to Drs B. A. Morris-Krsinich and T. M. A. Wilson for generous gifts of rabbit reticulocyte lysate used in early stages of this study, to Dr J. W. Davies who carried out translations using wheat germ extracts for us, to C. Gough for a sample of X a n t h o m o n a s campestris RNA and to Drs C. R. Martin and S. N. Covey for advice and discussion concerning methods for extracting mRNA. We also wish to thank Prof. D. A. Hopwood FRS, Drs A. J. Slusarenko and A. W. B. Johnston for critically reading the manuscript. This work was supported by the Agricultural and Food Research Council via grant-in-aid to the John Innes Institute.
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