proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

RESEARCH ARTICLE

Proteomic analysis of bacterial-blight defense-

responsive proteins in rice leaf blades

Tariq Mahmood1, 2, Asad Jan3, Makoto Kakishima2 and Setsuko Komatsu1, 3

1 National Institute of Crop Science, Tsukuba, Japan2 University of Tsukuba, Tsukuba, Japan3 National Institute of Agrobiological Sciences, Tsukuba, Japan

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses aspart of an integrated defense mechanism. To study the compatible and incompatible interactionsbetween rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings wereinoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv.oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and sepa-rated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response tobacterial inoculation. These proteins were categorized into classes related to energy (30%), me-tabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carbox-ylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins byT7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plantdefense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thauma-tin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway ofrice against bacterial blight.

Received: July 2, 2006Accepted: August 12, 2006

Keywords:

Bacterial blight / Jasmonic acid / Rice / Xanthomonas oryzae pv. oryzae

Proteomics 2006, 6, 6053–6065 6053

1 Introduction

Rice is one of the most widely cultivated food cropsthroughout the world; nevertheless, production is con-strained by diseases of fungal, bacterial, and viral origin [1].Among the bacterial diseases, bacterial blight of rice, caused

by Xanthomonas oryzae pv. oryzae (Xoo) is one of the mostdestructive disease, causing losses of up to 50% in severecases of infection [1]. The pathogen is a yellow, slime-pro-ducing, motile, Gram-negative rod with a polar flagellumthat enters the host through wounds or natural openings.When the organism reaches xylem tissues of the host, itmultiplies and spreads throughout the plant [2]. The inter-action between Xoo and rice is either compatible or incom-patible, with the former causing disease and the latterresulting into resistance. The use of resistant varieties is themost effective control measure against this disease, whichrequires the identification and characterization of resistance(R) genes. Twenty-one bacterial-blight resistant genes (Xa1,Xa2, Xa3, Xa4, Xa7, Xa10, Xa11, Xa12, Xa14, Xa16, Xa17,Xa18, Xa21, Xa22, and Xa27 as dominant and xa5, xa8, xa13,xa15, xa19, and xa20 as recessive) have been found in rice[3–6]. Combinations of these genes result in durable resist-ance against the disease.

Correspondence: Dr. Setsuko Komatsu, National Institute of CropScience, Kannondai 2–1-18, Tsukuba 305–8518, JapanE-mail: [email protected]: 181-29-838-8392

Abbreviations: GADPH, glyceraldehyde-3-phosphate dehydro-genase; JA, jasmonic acid; OEE, oxygen evolving enhancer pro-tein; PBZ1, probenazole-inducible protein 1; PR, pathogenesis-related; RuBisCO LSU, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit; SOD, superoxide dismutase; Xoo,Xanthomonas oryzae pv. oryzae

DOI 10.1002/pmic.200600470

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

6054 T. Mahmood et al. Proteomics 2006, 6, 6053–6065

Plant resistance to pathogens depends upon timelyrecognition of the invading pathogen and rapid activation ofa defense response via a number of signal transductionpathways [7]. To recognize effectors of the pathogens, plantshave evolved a very sophisticated recognition system throughthe R proteins, a form of gene-for-gene interaction [8]. Of themany apparent plant defense responses to invasion by var-ious pathogens, one of the most studied is the expression of agroup of host-encoded proteins referred to as pathogenesis-related (PR) proteins. PR proteins occur in a wide variety ofplant species [9] and their expression is governed by a cas-cade of signal transduction molecules involving jasmonicacid (JA), salicylic acid, and ethylene [10]. JA has been shownto be involved in the expression of PR proteins [11], thusplaying an essential role in the plant defense system throughsystemic acquired resistance [12]. Despite the major advan-ces in the field of plant disease resistance, the precise mo-lecular mechanisms of interaction between plant and patho-gen, and the detailed elucidation of R protein-mediated sig-naling pathway remain elusive.

The completion of the rice genome sequence by theInternational Rice Genome Sequencing Project [13] and theXoo genome sequence by Lee et al. [14] are important accom-plishments in the field of agriculture. Although genomes canchange due to movement of transposable elements or fromepigenetic changes, they are generally considered to the morestatic than the highly dynamic proteome. Therefore, the chal-lenge ahead is to elucidate the functions of proteins involvedin plant disease and resistance. The level of induced PR pro-teins is higher in an incompatible interaction than a compat-ible one. The phenomenon has been studied in a variety ofplant diseases, using a proteomic approach.

Kim et al. [15] noticed the elevated levels of PR10 andprobenazole-inducible protein 1 (PBZ1) after inoculatingrice with blast fungus, Magnaporthe grisea or treating it withJA. PR5 was induced when rice was inoculated with M. gri-sea, followed by application of nitrogen fertilizer [16]. Super-oxide dismutase (SOD), heat shock proteins, and dehydrins,which are related to stress, metabolism, and translocationwere induced by rice yellow mottle virus (RYMV) in the hostafter inoculation with the virus [17]. Tsunezuka et al. [18] an-alyzed the programmed cell death and defense signalingpathways using proteomic approaches and found variousdefense and metabolism-related proteins in the rice lesionmimic mutant, cdr2, when challenged with M. griesea. Thus,proteomics has evolved as a useful tool to understand plant–microbe interactions at the molecular level.

Until now, there has been no report of proteome analysisthat focused on the defense signaling pathway in rice againstbacteria. Getting a comprehensive insight into the interac-tion of bacteria with rice will be an important step towardunderstanding diseases and resistance mechanisms thathave evolved in plants. In this study, to investigate the role ofdefense-responsive proteins in rice against Xoo and the pos-sible involvement of JA as a signaling molecule in its defensepathway, a proteomic approach was applied.

T7174 of race 1 and Xo7435 of race 4 were used againstrice cultivar Java 14, which responds to these races inincompatible and compatible manners, respectively [19].Twenty differentially expressed proteins in rice leaf bladesafter Xoo inoculation were identified with 2-D PAGE (2-DE)followed by Edman sequencing and MS analyses. Further,the role of JA in the defense mechanism causing up-regula-tion of these proteins was investigated.

2 Materials and methods

2.1 Inoculation of plants with bacteria and treatment

with JA

For inoculation, T7174 and Xo7435 were grown on mediacontaining 1% peptone, 1% sucrose, and 1.5% agar at 307Cfor 2 days. A bacterial suspension was prepared by washingthe cells from the culture slant with sterilized distilled water.Bacteria with a concentration of 108 colony forming units(cfu)/mL were used. The rice (Oryza sativa L. cv. Java 14) wasgrown in plastic seedling cases (280 mm6160 mm690 mm) in green house for 3 weeks. The uppermost fullyopened leaves were inoculated with bacterial suspension astreatment and infiltrated with sterilized distilled water ascontrol, by double or multiple-needles pricking methods. Indouble-needle pricking method, a wooden fork with one armwrapped in the cloth and the other arm glued with a rubberpiece containing a single needle was used for pricking suchas the rice leaf on each side of the midrib was singly pricked.In multiple-needles pricking method, wooden fork with therubber containing 12 needles was used for pricking the riceleaf from top to bottom. To examine the effect of JA, riceplants were treated with 100 mM JA (Wako Chemicals,Osaka, Japan).

2.2 Preparation of cytosolic and membrane protein

fractions

A portion (170 mg) of rice leaf blade was homogenized with1 mL of homogenization buffer containing 20 mM Tris-HCl(pH 7.5), 0.25 M sucrose, 10 mM EGTA, 1 mM DTT, and1 mM phenylmethyl sulfonyl fluoride. The homogenateswere centrifuged at 30006g for 5 min, followed by the ultra-centrifugation of the supernatant at 274 0006g for 15 min.Cytosolic fraction was obtained by collecting the supernatant.The pellet was washed with 100 mL of homogenization bufferfollowed by ultracentrifugation as stated above. It was againresuspended in 50 mL of membrane solubilizing buffer con-taining 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 1 mMEDTA, and 50 mM 2-mercaptoethanol and incubated on icefor 30 min. After centrifugation at 274 0006g for 8 min,membrane fraction was obtained by collecting the super-natant [20]. For 2-DE, lysis buffer [21] with 1:1 ratio wasadded to the cytosolic and membrane fractions before load-ing.


Proteomics 2006, 6, 6053–6065 Plant Proteomics 6055

2.3 2-D PAGE

The prepared samples were separated by 2-DE [21], in the firstdimension by IEF tube gels and in the second dimension bySDS-PAGE. An IEF tube gel of 11 cm length and 3 cm diam-eter was prepared, which consisted of 8 M urea, 3.5% acryl-amide, 2% NP-40, 2% ampholines (pH 3.5–10.0 and 5.0–8.0),ammonium persulfate and tetramethylethylenediamine.Electrophoresis was carried out at 200 V for 30 min, followedby 400 V for 14 h, and 600 V for 1 h. After IEF, SDS-PAGE wasperformed in the second dimension using 15% polyacryl-amide gels with 5% stacking gels, followed by CBB stainingbefore 2-DE image analysis. 2-DE images were captured andthe positions of individual proteins on gels were automaticallyevaluated using ImageMaster 2D Elite software (AmershamBiosciences, Piscataway, TX, USA). The pI and molecularweight of each protein were determined using 2-DE markers(BioRad, Hercules, CA, USA).

2.4 Image acquisition and data analysis

CBB stained gels from three independent experiments wereanalyzed with ImageMaster 2D Elite software (version 3.01;Amersham Biosciences). Spot detection, spot measurement,background subtraction, and spot matching were performed.Following automated spot detection, gel images were care-fully edited. One gel image was selected as a reference fol-lowed by automated spot matching among the gels. Theunmatched spots of the member gels were added to thereference gel. The amount of protein spot was expressed asthe volume of that spot which was defined as the sum of theintensities of all the pixels that made up that spot. To correctthe variability due to CBB staining, and to reflect the quanti-tative variations in intensity of protein spots, the spotvolumes were normalized as the percentage of the total vol-ume in all of the spots present in the gel. Standard error wascalculated from spots of the gels from three independentexperiments and used as error bars.

2.5 N-terminal amino acid sequence analysis and

homology search

Following separation by 2-DE, proteins were electroblottedonto a polyvinylidine difluoride membrane (Pall, PortWashington, NY, USA) using a semidry transfer blotter(Nippon Eido, Tokyo, Japan) and detected by CBB staining.The protein spots were excised from the membrane andapplied to the protein sequencer, Procise 494 (Applied Bio-systems, Foster City, CA, USA). Edman degradation wasperformed according to the standard program supplied byApplied Biosystems. The amino acid sequences obtainedwere compared with those of known proteins in the Swiss-Prot (http://us.expasy.org/sprot), PIR (http://pir.george-town.edu), GenPept (http://www.genelynx.org/cgi-bin/resource), and PBD (http://www.rcsb.org/pdb) databaseswith the Web-accessible search program FastA.

2.6 MS analysis and database search

The protein spots were excised from gels, washed with 25%methanol and 7% acetic acid for 12 h and destained with50 mM NH4HCO3 in 50% methanol for 1 h at 407C. Proteinswere reduced with 10 mM DTT in 100 mM NH4HCO3 for 1 hat 607C and incubated with 40 mM iodoacetamide in 100 mMNH4HCO3 for 30 min. The gel pieces were minced andallowed to dry and then rehydrated in 100 mM NH4HCO3

with 1 pM trypsin (Sigma-Aldrich, St. Louis, MO, USA) at377C overnight. The digested peptides were extracted fromthe gel pieces with 0.1% TFA in 50% ACN/water three times.The peptide solution thus obtained was dried and reconcen-trated with 30 mL of 0.1% TFA in 5% ACN/water and thendesalted with NuTip C-18 pipette tips (Glygen, Columbia,MD, USA). The above peptide solution was mixed withCHCA. MALDI-TOF MS was performed using Voyager PR(Applied Biosystems, Framingham, MA, USA). The massspectra were subjected to sequence databases search usingMASCOT software (Matrix Science, London, UK). ForMALDI-TOF analysis, four criteria were used to assign apositive match with a known protein. (1) The deviation be-tween experimental and theoretical peptide masses shouldbe less than 50 ppm. (2) At least four different predictedpeptide masses were required to be matched to the observedmasses for an identification to be considered valid. (3) Thecoverage of protein sequences by the matching peptidesmust reach a minimum of 10%. (4) The score that wasobtained from the analysis with MASCOT software indicatesthe probability of a true positive identification, and must beat least 58 using full length rice cDNA database (Version20040709) and at least 67 using GeneBank database (Version20040722).

3 Results and discussion

3.1 T7174 elicits a strong disease resistance

response, while Xo7435 causes severe disease in

Java 14

Disease occurrence or resistance is the consequence of com-patible or incompatible interactions of plants with bacteria.To study the interaction between Xoo and rice, 3-week-oldJava 14 rice seedlings were inoculated with T7174 andXo7435 by the double-needle pricking method. Symptomson the rice were observed and the population characteristicsof Xoo were determined. T7174 elicited a clear resistance re-sponse in rice (Fig. 1A) since the appearance of symptomsstarted gradually. Three days after inoculation, there werewhite spots on the leaf blade that turned into light brownafter 6 days and dark brown after 9 days of inoculation. Thepopulation of T7174 was 330, 7, and 0 cfu/mL at 3, 6, and9 days after inoculation, respectively (Fig. 1B). In contrast,discoloration of leaves to brownish yellow occurred 6 daysafter inoculation with Xo7435. The discoloration persisted



Figure 1. The effect of Xooinoculation on rice seedlings.Leaf blades of 3-week-old riceseedlings were inoculated withT7174 (A and B) and Xo7435 (Cand D) of Xoo at a concentrationof 108 cfu/mL by double-needlepricking method. Photographswere taken at 0, 3, 6, 9, and12 days after inoculation (A andC). The populations of T7174and Xo7435 at 3, 6, 9, and12 days after inoculation weredetermined and displayed asgrowth curves (B and D). Valuesare the mean 6 SE of bacterialpopulations from three leafblades.

and became more severe at 9 and 12 days, and spread over amajor part of the leaf blade (Fig. 1C). The population ofXo7435 vigorously increased with an initial count of 13 cfu/mL at 3 days after inoculation, reaching to 11 000, 34 000, and46 000 cfu/mL at 6, 9, and 12 days after inoculation, respec-tively (Fig. 1D). These results indicated that Java 14 exhibiteda hypersensitive response against T7174 that restricted bac-terial growth and finally caused death of the bacteria 9 daysafter inoculation. Overall, growth rate of the rice plantsremained unaffected.

Kaku [19] noticed that no symptoms or tiny brownspots appeared around the point of inoculation when adultplants of cultivar Java 14 were inoculated with T7174. Thepresent study also demonstrated a symptomless resistancecharacterized by white spots for the first 3 days afterinoculation, which turned into slight brown and then darkbrown after 6 and 9 days of inoculation, respectively.Ogawa et al. [22] reported that the resistance of cultivarJava 14 against bacterial blight is controlled by two major

genes Xa1 and Xa3. The resistance reaction in the hostvaries from symptomless to small yellow lesions orbrowning, depending on which resistance gene is present[23]. The consequence of pathogen interactions with plantsdepends on genes carried by both the host and the patho-gen. The host-encoded R gene product recognizes thepresence of a pathogen-encoded avirulent (Avr) gene prod-uct and triggers an induced defense response [24]. Xa1 isnot constitutively expressed in rice; rather its expression isinduced by inoculation with T7174 or wounding [25]. Ithas been suggested that the high disease resistance of Java14 by the additive effect of Xa1 and Xa3 might be con-trolled by a gene-for-gene interaction in AvrXa1-Xa1 and/orAvrXa3-Xa3. In order to understand the mechanism takingplace during compatible and incompatible interactions be-tween rice and Xoo, a comprehensive analysis at the mo-lecular level is needed. Toward that end, a proteomicapproach was applied to analyze changes in protein levelsduring the interaction between rice and Xoo.



3.2 Twenty proteins are differentially expressed by

T7174 and Xo7435

To investigate the protein profile of rice before any resistance ordisease symptoms were obvious, plants were selected for pro-teomic analysis 3 days after bacterial inoculation. To study theinfection mechanism of Xoo and resistance response of rice atthe proteomic level, 3-week-old Java 14 rice seedlings wereinoculated with T7174 and Xo7435 for 3 days. To cover thewhole leaf area conveniently, the multiple-needles prickingmethod was used instead of the double-needles prickingmethod. Three hundred sixty-six proteins from the cytosolicand membrane fractions were detected after leaf blade proteinswere fractionated, separated by 2-DE, and stained with CBB.Three out of 366 proteins were changed between infiltrated anduninfiltrated leaves, indicating a very little change (0.8%) in theproteins profile (Fig. 2). The level of 20 proteins, however, waschanged by T7174 and Xo7435; seven (35%) proteins were up-regulated and three (15%) proteins were down-regulated byT7174, while nine (45%) proteins were up-regulated, and ten(50%) proteins were down-regulated by Xo7435 (Fig. 3).

Based on the data from three independent experiments,the differentially expressed proteins were grouped accordingto their relative expression. Using ImageMaster 2D Elitesoftware, they were classified as either up-regulated or down-regulated proteins. All 20 proteins exhibited differentialexpression in their relative volumes. T7174 clearly up-regu-lated spots 11 and 14, while spots 5, 6, 11, 12, and 19 were up-regulated by Xo7435. On the other hand, T7174 significantlydown-regulated spot 8, while spots 1, 2, and 3 were clearlydown-regulated by Xo7435 (Fig. 4). Overall, the number ofproteins whose level changed by Xo7435 was higher than thenumber of proteins changed by T7174 in Java 14. Theseresults suggest that pathways related to energy or metabo-lism are affected during a disease or resistance condition ofthe plant.

Tsunezuka et al. [18] reported the differential expressionof 37 proteins related to metabolism and defense in the rice-blast resistant mutant, cdr2. Sixty-four proteins related tometabolism, energy, and defense were differentially expres-sed when rice plants were inoculated with RYMV [17].Among these 64 proteins, 40 proteins were differentially

Figure 2. 2-DE patterns of pro-teins from infiltrated rice leafblades. Leaf blades of 3-week-old rice seedlings were infil-trated with sterilized distilledwater by multiple-needles prick-ing method, while control wasleaves without infiltration. Threedays after infiltration, cytosolicand membrane proteins werefractionated, separated by 2-DE,and stained with CBB. Circlesshow the positions of changedproteins.



Figure 3. 2-DE patterns of proteins fromrice leaf blades inoculated with Xoo.Leaf blades of 3-week-old rice seedlingswere inoculated with T7174 and Xo7435of Xoo at a concentration of 108 cfu/mLor infiltrated with sterilized distilledwater by multiple-needles prickingmethod. Three days after inoculation,cytosolic and membrane proteins werefractionated, separated by 2-DE, andstained with CBB. Upward arrows indi-cate the positions of up-regulated pro-teins and downward arrows show thepositions of down-regulated proteins,while circles represent the same pro-teins in control.

expressed in susceptible cultivars and 24 proteins were dif-ferentially expressed in partially resistant cultivars. Thesereports and the present results indicate that dynamicchanges at the proteome level do occur in response to fungal,viral, and bacterial infection. Additionally, the expression ofmore proteins was altered by Xo7435, a compatible strain,than for T7174, an incompatible strain. Among them, mostof the proteins were down-regulated by Xo7435 that could bedue to the lethal effect of Xo7435. The production regulatedby oxidative stress plays a vital role in plant defense during

incompatible interaction [26, 27]. Thus, in plants, a “qualitycontrol” phenomenon might be in operation that maintainsthe physiological activities of the plant in a way to defendagainst the attacking incompatible strain. However, in acompatible interaction, it is most probable that this “qualitycontrol” system of plant gets a severe blow due to excessivestress, resulting into the degradation of many proteins andunwanted cell death, leading to the damage of the rice seed-ling. Though the disease and resistance symptoms werevisible 6 days after inoculation, changes at proteome level



Figure 4. Relative expression ofchanged proteins in rice leafblades after inoculation withXoo. Leaf blades of 3-week-oldrice seedlings were inoculatedwith T7174 and Xo7435 of Xooat a concentration of 108 cfu/mL.The changes in protein spotswere calculated with Image-Master 2D Elite software andplotted as the relative intensitiesof 20 spots indicated in Fig. 3.Values are the means 6 SE ofprotein volumes of gels fromthree independent experiments.Black, gray, and white barsrepresent control, T7174 andXo7435, respectively.

took place much earlier than the appearance of the symp-toms. To further confirm this phenomenon, the identity ofthe changed proteins was determined by MS and protein se-quencing.

3.3 Xoo affects proteins related to energy,

metabolism, and defense

As a starting point for understanding the functions of the 20differentially expressed proteins, each protein was analyzedby protein sequencing and MALDI-TOF MS. In the begin-ning, all spots were first subjected to sequence analysis only.Protein spots that could not be analyzed by protein sequenc-ing were subjected to MALDI-TOF MS analysis. According tothe criteria of Bevan et al. [28], the proteins were categorizedas related to energy (6/20, 30%), metabolism (4/20, 20%),defense (4/20, 20%), protein synthesis (1/20, 5%), proteinwith unknown function (1/20, 5%), and undetermined pro-tein (4/20, 20%) (Table 1). The amino acid sequence of oneprotein (spot 13) was present in the rice genome but itsfunction was not known. The sequence of four proteins

(spots 1, 3, 8, and 14) could not be determined because eitherthe N-terminus of the protein was blocked or the amount ofprotein for MS was below the required level.

There were more proteins related to energy, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit(RuBisCO LSU, spots 9 and 19), ATP synthase (spots 15 and16), oxygen evolving enhancer protein 1 (OEE1, spot 18), andOEE2 (spot 18), than the number of proteins related to me-tabolism, defense, or protein synthesis (Table 1). Ribosomalprotein (50 S; spot 20) was the only protein related to proteinsynthesis that was identified in this study. Tsunezuka et al.[18] reported that, out of 37 differentially expressed proteins,27 (73%) were related to metabolism in the rice mutant cdr2,which is resistant against rice-blast fungus. These resultssuggest that metabolism-related changes in protein levelsaffected the energy-related proteins after inoculation of ricewith Xoo. The difference in the modes of interaction betweenfungal blast and bacterial blight with their hosts might haveresulted in a difference in the number of energy-related pro-teins whose accumulation was altered. In this study, theexpression of transketolase (spot 2), glyceraldehyde-3-phos-



Table 1. The changed proteins in rice leaf blade after Xoo inoculation

Spotno.

pI Mr Sequence Homologous protein (% homology) Accessionno.

Score PA

Energy

09 5.2 22 N-blocked (MS) RuBisCO LSU AJ295261 67 2015 5.4 60 N-blocked (MS) ATP synthase b subunit, chloroplast M31464 103 2016 5.5 59 N-blocked (MS) ATP synthase b subunit, chloroplast AB037543 113 2017 5.3 40 N-EGVPXRLTFD OEE1 (90) O4907918 5.9 30 N-AYGEAANVFG OEE2 (100) P8166819 5.4 29 N-blocked (MS) RuBisCO LSU AB125345 70 20

Metabolism

02 5.5 66 N-blocked (MS) Transketolase, chloroplast AK067452 62 2004 5.9 43 N-VAINGFG GADPH (100) S3387205 5.1 32 N-blocked (MS) Ribose-5-phosphate isomerase AK060861 92 2006 5.5 29 N-GRKFFVGGN Triosephosphate isomerase, cytosolic (100) P46226

Defense

10 5.2 19 N-ATFTITNRXS PR5 (90) P3111011 5.1 18 N-APAXVSDEHA PBZ1 (90) D3817012 5.5 20 N-ATKKAVAVLK SOD (100) S2914607 5.4 26 N-AGGVDDAPLV Peroxiredoxin, chloroplast (100) AM039889

Protein synthesis

20 4.6 25 N-STATESPKVL 50 S ribosomal protein, chloroplast (100) Q06030

Unknown protein

13 4.5 13 N-STVLDGLKYS Function unknown AC068924

Undetermined protein

01 5.4 66 N-blocked (MS) NSM03 5.7 46 N-blocked (MS) NSM08 5.4 24 N-blocked (MS) NSM14 5.7 13 N-blocked (MS) NSM

N-, N-terminal amino acid sequences determined by Edman degradation; PA, total number of peptides assigned; NSM, not significantlymatched; MS, homologous proteins determined by MALDI-TOF MS.

phate dehydrogenase (GADPH, spot 4), ribose-5-phosphateisomerase (spot 5), and triosephosphate isomerase (spot 6),all classified as metabolism-related proteins, was altered. Outof them, transketolase and GADPH were differentiallyexpressed both in the rice-blast resistant mutant, cdr2 [18]and in Java 14 by Xoo inoculation in the present study. Thesefindings suggest that GADPH and transketolase affect themetabolism of rice during fungal and bacterial infection.

Defense-related proteins, thaumatin-like protein (PR5)and PBZ1 were also identified in this study (Table 1). Over-expression of PR5 in transgenic rice plants enhanced theresistance of rice to Rhizoctonia solani, the causal organismof sheath blight of rice [29]. Takemoto et al. [30] reported thatchitinase (PR3) and osmotin (PR5) were associated with theactin cytoskeleton that is involved in cytoplasmic aggregationin the early stages of the hypersensitive response (HR) be-tween Phytophthora infestans and potato. Takahashi et al. [31]

reported a high level of PBZ1 gene expression in highlyinduced lesion mimic mutants. Thus, the expression of PRproteins might be a common response cascade during anincompatible interaction. Defense-related proteins, SOD,and peroxiredoxin were also identified in this study (Table 1).SOD was also induced in rice leaf blades under salt stress [32]as well as during the stage of multiple lesion formation inthe blast fungus-resistant cdr2 mutant of rice [18]. Peroxi-redoxin was up-regulated in the HR of poplar against Mel-ampsora larici-populina, the causal agent of poplar rust [33].The induction of SOD and peroxiredoxin reflects the pres-ence of toxic oxides generated in the interaction of rice withXoo, which need to be quenched for the protection of riceplant. This study demonstrated that defense-related proteins,which are PR5, PBZ1, SOD, and peroxiredoxin, are activatedin the rice plant after Xoo inoculation and may function in adefense mechanism to protect the plant.



3.4 JA affects 5 of the 20 proteins modulated by Xoo

in rice

The signaling role of JA during biotic and abiotic stresses orthe wound response is well known. Vijayan et al. [10]demonstrated the protective role of JA against diseases whenthe JA-deficient triple mutant of Arabidopsis, fad3–2 fad7–2fad 8, which was extremely susceptible to root rot diseasecaused by the fungal pathogen Pythium mastophorum, getsubstantial protection when treated exogenously with JA.Coi1, a JA-insensitive mutant of Arabidopsis was not pro-tected even after JA treatment, providing evidence that JA is asignal molecule in the defense response [10]. Levels of JAincreased when plant cell suspension cultures of Rauvolfiacanescens and Eschscholtzia californica were treated with yeastelicitor [34]. These results indicate the signaling role of JA,which may induce the expression of defense proteins in theinteraction between rice and Xoo.

To investigate whether JA plays some signaling role inthe defense response of rice against Xoo, 3-week-old-riceseedlings were treated with 100 mM JA for 3 days. Cytosolicand membrane proteins were fractionated from leaf blades,separated by 2-DE, and stained with CBB. Out of 20 pro-teins differentially expressed during the interaction of ricewith Xoo, five proteins also changed with JA treatment. PR5(spot 10), PBZ1 (spot 11), transketolase (spot 2), and anundetermined protein (spot 1) were up-regulated, whileOEE2 (spot 18) was down-regulated by JA (Fig. 5 andTable 1).

Kim et al. [15, 35] reported an elevated expression ofPBZ1 and PR10 in the rice-blast fungal disease in incompa-tible interactions or when treating rice with JA. The syner-gistic induction of PR5 by ethylene and JA was also reportedin tobacco seedlings [9]. Rakwal and Komatsu [11] reportedthe induction of PR1 and PR5, after exogenously treating ricewith JA. Based on the results of the present study and above

Figure 5. 2-DE patterns of proteins from rice leaf blades treated with JA. Three-week-old rice seedlings were treated without or with 100 mMJA for 3 days. Cytosolic and membrane proteins from leaf blades were fractionated, separated by 2-DE, and stained with CBB. Out of 20changed proteins in Fig. 3, the proteins changed by JA were marked. Upward arrows indicate the positions of up-regulated proteins anddownward arrows show the positions of down-regulated proteins, while circles represent the same proteins in control.



observations, we predict that JA signals lead to the expres-sion of defense proteins, PR5 and PBZ1, in the interactionbetween rice and Xoo.

Zou et al. [36] reported that 94 soybean chloroplast-asso-ciated genes including OEE2 were down-regulated by Pseu-domonas syringae in a resistance response. A large number ofphotosynthesis-related genes, including components ofphotosystems 1 and 2, were down-regulated in a resistantinteraction between Xanthomonas campestris pv. vesicatoriaand tomato plants [37]. Transcripts for OEE2 were down-regulated in the interaction between herbivores and Nicoti-ana attenuate [38]. The down-regulation of OEE2 may ham-per chloroplast-associated activities that lead to a decrease inphotosynthesis after infection of rice seedlings with Xoo(Fig. 3).

Transketolase is involved in the translocation of metabo-lites from leaves to sink tissues [39]. Cluzet et al. [40] noticedan insignificant increase of transketolase when Medicagotruncatula was treated with elicitor from green algae Ulva. Inthe present study, the level of transketolase was up-regulatedby JA treatment or down-regulated by Xo7435 inoculation(Fig. 3). The induction of transketolase by JA suggests that ina JA-mediated defense response, photosynthetic activitiesmay increase allowing mobilization of resources into hostresistance. However, these activities remain unaffected andcontinue normally in a resistant response of rice againstT7174, in contrast to their severe decrease under the lethaleffect of Xo7435. These results suggest that induction ofdefense might proceed at the expense of some other forms ofmetabolism during interactions of rice with T7174. It is pos-sible that JA treatment or T7174 inoculation do not interferewith primary metabolism and are likely to share a commonsignal transduction pathway in the defense mechanism ofrice.

3.5 Xoo and JA temporally regulate the expression of

five proteins

Time is a determining element in the expression of R genesduring resistance and susceptible responses. In a resistantresponse, the expression of PR proteins is faster than in asusceptible one. Suharsono et al. [41] observed a delay of 24 hin the expression of PR1 and PBZ1 in rice dwarf1 (d1) sus-ceptible mutant as compared to wild type. The symptoms ofbacterial blight of rice developed earlier in d1 mutant, andthere was a delay of 24 h in the maximum induction of PBZ1after probenazole treatment [42].

To study whether time affects the induction of defenseproteins during the interaction of rice with Xoo, a timecourse experiment was designed. Three-week-old rice seed-lings were inoculated with 108 cfu/mL of Xoo or treated with100 mM JA for 2, 3, and 4 days. Cytosolic and membraneproteins from the leaf blades were fractionated, separated by2-DE, and detected with CBB staining. Transketolase (spot 2)and an undetermined protein (spot 1) were not affected byT7174 inoculation, down-regulated by Xo7435 inoculation

and up-regulated by JA treatment. PR5 (spot 10) and PBZ1(spot 11) increased with time, reaching a maximum 3 daysafter T7174 and Xo7435 inoculation and JA treatment. Addi-tionally, the induction of PR5 and PBZ1 by T7174 was higherthan by Xo7435. OEE2 (spot 18) was not affected by T7174 inall time intervals but decreased 3 days after Xo7435 inocula-tion and 2 days of JA treatment (Fig. 6).

Plant resistance to pathogens depends upon timelyrecognition of the invading pathogen and a rapid activationof a defense response [7]. Resistance is characterized by aslight response in the start that reaches to a maximumthreshold level with time. Birch et al. [43] observed a slightresistance response in the beginning that became visible

Figure 6. Time-dependent protein accumulation in response toXoo inoculation or JA treatment. Three-week-old rice seedlingswere inoculated with T7174 and Xo7435 of Xoo at a concentra-tion of 108 cfu/mL or treated with 100 mM JA. Cytosolic andmembrane proteins were fractionated from leaf blade after 2, 3,and 4 days of Xoo inoculation or JA treatment. Proteins wereseparated by 2-DE and stained with CBB. Upward arrows indicatethe positions of up-regulated proteins and downward arrowsshow the positions of down-regulated proteins, while circlesrepresent the same proteins in control.



3 days after potato plant inoculation with P. infestans. In aresistant response, a relatively small number of epidermalcells died in the beginning when the potato plant was inocu-lated with P. infestans [44]. The accumulation of PBZ1reached the maximum level at 3 days in the wild type riceafter probenazole treatment [42], indicating a time depend-ent increase of PBZ1. From these results, it appears that thelength of time after Xoo inoculation or JA treatment is a keyfactor which affects the differential expression of PR proteinsin association with metabolism and energy-related proteinsin rice. Further, the expression of these proteins in a com-patible interaction differs from the expression in an incom-patible interaction of rice with Xoo or treatment with JA.

3.6 PR5 and PBZ1 are commonly up-regulated by Xoo

and JA

Based on the analysis of Figures 3 and 5, proteins changed byT7174, Xo7435, and JA were identified and categorized. Twoproteins, PR5 (spot 10) and PBZ1 (spot 11) were commonlyinduced by Xoo and JA. Three proteins, transketolase (spot 2),

OEE2 (spot 18), and an undetermined protein (spot 1) wereaffected by JA and Xo7435. Six proteins including SOD(spot 12), RuBisCO LSU (spot 9), GADPH (spot 4), 50 Sribosomal protein (spot 20), an unknown protein (spot 13),and an undetermined protein (spot 8) were commonlyaffected by T7174 and Xo7435 (Fig. 7).

An early event associated with the defense response isthe oxidative burst, leading to generation of reactive oxygenspecies (ROS) [45]. Abbasi and Komatsu [32] determined thatSOD is a protective agent during salt stress of rice when SODwas up-regulated. These results propose that SOD plays amajor role in the host defense by scavenging excess toxicoxides. Under controlled conditions, the level of ROS can bemaintained by the plant, while in disease conditions ROSlevels cannot be controlled, resulting in severe damage to theplant [46]. It is possible that the concentration of ROS isenough to restrict bacteria in the interaction between riceand T7174; however, excessive production of ROS due to thepathological effect of Xo7435 might be the cause of severeplant damage. Previous reports of fragmentation ofRuBisCO LSU may have occurred as a result of stress in the

Figure 7. Commonly expressed proteins by Xoo inoculation and JA treatment. Three-week-old rice seedlings were used and inoculatedwith Xoo at a concentration of 108 cfu/mL or treated with 100 mM JA for 3 days. Ven diagram analysis shows 20 up-regulated and down-regulated proteins by T7174, Xo7435, and JA, as indicated in Figs. 3, 5. Protein spot numbers are indicated in the brackets. Proteinschanged by JA alone were not analyzed as shown by the cross area. Numbers in the parenthesis represent corresponding protein spots.



fungal blast resistant mutant [18]. In this study, the levels of a22 kDa RuBisCO LSU (spot 9) and a 29 kDa RuBisCO LSU(spot 19) increased, indicating possible fragmentation. Basedon these results, it appears that oxidative stress does takeplace during the interaction of rice and Xoo, which may causefragmentation of RuBisCO LSU. However, excessive stressby the compatible strain is beyond the control of defensesystem of the plant. The phenomenon is also evident by anincreased level of RuBisCO LSU fragmentation (Spots 9and 19) and SOD expression (Spot 12) in compatible inter-action than the incompatible one (Figs. 3, 4).

GADPH plays a critical role in the glycogen metabolismpathway and is involved in the synthesis of different meta-bolites and subsequent sources of energy [47]. Microbial-resistant mutants gave differential expression of metabo-lism-related proteins [18]. Ribosomal protein (50 S) wasdown-regulated in the incompatible interaction betweensoybean and P. syringae [36]. In the present study, the down-regulation of GADPH and 50 S ribosomal protein mayreduce metabolic and protein synthesis activities in rice byXoo. It is suggested that the damage to these chloroplast-associated genes might be reversible in a resistance reactionand repaired soon after the mild stress caused by T7174diminishes. Further, this damage might be irreparable due tothe severe stress induced by Xo7435 in the disease condition.

Expression of transketolase was down-regulated whenperiwinkle plants were infected with pathogenic strains ofSpiroplasma citri, while no down-regulation was observed fora nonpathogenic mutant of S. citri [48]. Satoh et al. [49] noteda marked decrease in transketolase when CO2-tolerant algawere treated with chloramphenicol at an elevated CO2 con-centration of 40% that led to decreased photosynthetic activ-ity of the plant. However, the treatment did not affect trans-ketolase when CO2 levels were at 5%. This result suggeststhat the drastic reduction in transketolase by Xo7435 is dueto the severe stress resulting from its pathological effect.OEE2 is required for high levels of oxygen evolution duringphotosynthesis [50]. Photosystem 2, a central feature ofchloroplast is one of the sources of ROS production [36].Based on these results and the present study, severe ROSstress is responsible for the decrease in OEE2 in compatibleinteraction. In contrast, a regulated control of ROS is notlikely to affect OEE2 in an incompatible interaction.

In an HR of potato plant inoculated with P. infestans, PR3and PR5 in association with actin were induced, resulting incytoplasmic aggregation [43]. The isolation of chitinase (PR3)and the observation of an oxidative burst were reported in anincompatible interaction of P. infestans with potato plants[43]. Takahashi et al. [31] noticed a high expression of PBZ1in the highly induced lesion mimic mutants cdr, which isresistant against rice-blast fungus. PR5 and PBZ1 were alsohighly induced in a compatible interaction of rice blast dur-ing the growth of M. grisea within the host [51]. Based onthese results and the present study, it is suggested that theinduction of PR proteins might be an indispensable phe-nomenon of Xoo interaction with rice. The induction of PR5

and PBZ1 by Xoo in association with JA further suggests asignaling role for JA, which may catalyze these proteins inthe defense response of rice against Xoo.

4 Concluding remarks

A proteomic approach was performed to identify proteinsfrom the rice leaf blade that were differentially expressed inresponse to Xoo inoculation. The differential display of theproteome of pathogen-responsive proteins between incom-patible and compatible interactions was studied. Twenty dif-ferentially expressed proteins were analyzed by protein se-quencing and MS. 2-DE analysis clearly demonstrated thatfour defense-related proteins such as PR5, PBZ1, SOD, andperoxiredoxin were induced in leaf blades both by T7174 andXo7435 inoculation, wherein PR5 and PBZ1 were alsoinduced by JA treatment. Further, their expression at differ-ent time intervals after Xoo inoculation or JA treatment wasstudied. The induction of PR5 and PBZ1 was more rapid andhigher in incompatible interactions than the compatible one.PR proteins have been found in many plant species liketobacco, tomato, potato, maize, barley, and rice, in responseto a diverse range of pathogens including viruses, fungi, andbacteria [52]. The developmental stage of the plant at infec-tion is one of the important factors that determine the typesof PR proteins to be induced. In the present study, theinduction of PR5 and PBZ1 in response to Xoo inoculationand JA treatment focused on the seedling stage of rice. Thisis the first report that investigates the temporal and quanti-tative differences in the expression of these defense-orientedproteins and explains a possible role for JA as a signalingmolecule in the bacterial blight of rice. The differentialexpression of PR5 and PBZ1 will be helpful in studying theresistance phenomenon, and may provide rice with a leadingrole in its defense against Xoo.

The authors are thankful to Dr. H. Kaku for providing thebacterial strains and rice cultivar, and extending technical help.Thanks are also due to Mr. T. Nakayama for his valuable sugges-tions in bacterial counting. This work was supported by a grant(Rice Genome Project) from the Ministry of Agriculture, Forestry,and Fisheries (Japan).

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proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

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