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Exoribonuclease R (RNase R) is a 3′ hydrolytic exoribonuclease that can degrade structured RNA. Mutation in RNase R affects virulence of certain human pathogenic bacteria. The aim of this study was to determine whether RNase R is necessary for virulence of the phytopathogen that causes bacterial blight in rice, Xanthomonas oryzae pv. oryzae (Xoo). In silico analysis has indicated that RNase R is highly conserved among various xanthomonads. Amino acid sequence alignment of Xoo RNase R with RNase R from various taxa indicated that Xoo RNase R clustered with RNase R of order Xanthomonadales. To study its role in virulence, we generated a gene disruption mutant of Xoo RNase R. The Xoo rnr– mutant is moderately virulence deficient, and the complementing strain (rnr–/pHM1::rnr) rescued the virulence deficiency of the mutant. We investigated swimming and swarming motilities in both nutrient-deficient minimal media and nutrient-optimal media. We observed that RNase R mutation has adversely affected the swimming and swarming motilities of Xoo in optimal media. However, in nutrient-deficient media only swimming motility was noticeably affected. Growth curves in optimal media at suboptimal temperature (15°C cold stress) indicate that the Xoo rnr– mutant grows more slowly than the Xoo wild type and complementing strain (rnr–/pHM1::rnr). Given these findings, we report for the first time that RNase R function is necessary for complete virulence of Xoo in rice. It is also important for motility of Xoo in media and for growth of Xoo at suboptimal temperature.

Effectors that suppress effector-triggered immunity (ETI) are an essential part of the arms race in the co-evolution of bacterial pathogens and their host plants. Xanthomonas oryzae pv. oryzae uses multiple type III secretion system (T3SS) secreted effectors such as XopU, XopV, XopP, XopG, and AvrBs2 to suppress rice immune responses that are induced by the interaction of two other effectors, XopQ and XopX. Here we show that each of these five suppressors can interact individually with both XopQ and XopX. One of the suppressors, XopG, is a predicted metallopeptidase that appears to have been introduced into X. oryzae pv. oryzae by horizontal gene transfer. XopQ and XopX interact with each other in the nucleus while interaction with XopG sequesters them in the cytoplasm. The XopG E76A and XopG E85A mutants are defective in interaction with XopQ and XopX, and are also defective in suppression of XopQ–XopX-mediated immune responses. Both mutations individually affect the virulence-promoting ability of XopG. These results indicate that XopG is important for X. oryzae pv. oryzae virulence and provide insights into the mechanisms by which this protein suppresses ETI in rice.

Bacterial Blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases in various rice cultivating countries. Xoo secretes a mixture of plant cell wall degrading enzymes (CWDEs) such as cellulases, lipases, xylanases, and proteases to degrade different components of the plant cell wall. LipA; a lipase/esterase, is one such Xoo secreted CWDE and is an important virulence factor of Xoo. Treatment of rice tissue with purified LipA induces immune responses. In this study, a LC-MS based proteomics study was performed to identify the differentially expressed proteins (DEPs) in rice following LipA treatment. A total of 212 proteins were identified in control and 201 proteins in LipA treated samples. There were 151 proteins common between control and treatment. Fold change analysis of these common proteins through SIEVE identified 26 upregulated and 49 downregulated proteins by at least ≥1.5 fold in the LipA treated sample. Pathway analysis indicated that many proteins related to redox regulation, photosynthesis, and translation are differentially expressed after LipA treatment. We also observed that some of the differentially expressed proteins contain translation regulatory elements that may regulate translation after LipA treatment. The comparison of proteomics data with previously performed transcriptome analysis indicated that different sets of genes and pathways are altered in both the analyses.

Summary Many bacterial phytopathogens employ effectors secreted through the type‐III secretion system to suppress plant innate immune responses. The Xanthomonas type‐III secreted non‐TAL effector protein Xanthomonas outer protein Q (XopQ) exhibits homology to nucleoside hydrolases. Previous work indicated that mutations which affect the biochemical activity of XopQ fail to affect its ability to suppress rice innate immune responses, suggesting that the effector might be acting through some other pathway or mechanism. In this study, we show that XopQ interacts in yeast and in planta with two rice 14‐3‐3 proteins, Gf14f and Gf14g. A serine to alanine mutation (S65A) of a 14‐3‐3 interaction motif in XopQ abolishes the ability of XopQ to interact with the two 14‐3‐3 proteins and to suppress innate immunity. Surprisingly, the S65A mutant gains the ability to interact with a third 14‐3‐3 protein that is a negative regulator of innate immunity. The XopQS65A mutant is an inducer of rice immune responses and this property is dominant over the wild‐type function of XopQ. Taken together, these results suggest that XopQ targets the rice 14‐3‐3 mediated immune response pathway and that its differential phosphorylation might enable interaction with alternative 14‐3‐3 proteins.

Plant pathogens secrete cell wall-degrading enzymes that degrade various components of the plant cell wall. Plants sense this cell wall damage as a mark of infection and induce immune responses. However, the plant functions that are involved in the elaboration of cell wall damage-induced immune responses remain poorly understood. Transcriptome analysis revealed that a rice (Oryza sativa) receptor-like kinase, WALL-ASSOCIATED KINASE-LIKE21 (OsWAKL21.2), is up-regulated following treatment with either Xanthomonas oryzae pv oryzae (a bacterial pathogen) or lipaseA/esterase (LipA; a cell wall-degrading enzyme of X. oryzae pv oryzae). Overexpression of OsWAKL21.2 in rice induces immune responses similar to those activated by LipA treatment. Down-regulation of OsWAKL21.2 attenuates LipA-mediated immune responses. Heterologous expression of OsWAKL21.2 in Arabidopsis (Arabidopsis thaliana) also activates plant immune responses. OsWAKL21.2 is a dual-activity kinase that has in vitro kinase and guanylate cyclase activities. Interestingly, kinase activity of OsWAKL21.2 is necessary to activate rice immune responses, whereas in Arabidopsis, OsWAKL21.2 guanylate cyclase activity activates these responses. Our study reveals a rice receptor kinase that activates immune responses in two different species via two different mechanisms.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall-degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 A. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N- and C-terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild-type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence-promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.

SummaryXanthomonas oryzaepv.oryzaeuses several type III secretion system (T3SS) effectors, namely XopN, XopQ, XopX, and XopZ, to suppress rice immune responses that are induced following treatment with cell wall degrading enzymes. Here we show that the T3SS secreted effector XopX interacts with two of the eight rice 14-3-3 proteins. Mutants of XopX that are defective in 14-3-3 binding are also defective in suppression of immune responses, suggesting that interaction with 14-3-3 proteins is required for suppression of host innate immunity. However,Agrobacteriummediated delivery of both XopX and XopQ into rice cells results in induction of rice immune responses. These immune responses are not observed when either protein is individually delivered into rice cells. XopQ-XopX induced rice immune responses are not observed in a XopX mutant that is defective in 14-3-3 binding. Yeast two-hybrid and BiFC assays indicate that XopQ and XopX interact with each other. In a screen forXanthomonaseffectors which can suppress XopQ-XopX induced rice immune responses, five effectors were identified, namely XopU, XopV, XopP, XopG and AvrBs2, which were able to do so. These results suggest a complex interplay ofXanthomonasT3SS effectors in suppression of pathogen triggered immunity and effector triggered immunity to promote virulence on rice.Significance statementThis work studies the role of the type III effector XopX in the suppression and induction of rice immune responses, by differential interaction with the 14-3-3 proteins, or with the type III effector XopQ respectively. We have also identified a subset of type III effectors which can suppress this form of immune responses.

Xanthomonas oryzae pv. oryzae (Xoo) is a serious pathogen causing bacterial blight disease in rice. Population genomic studies have revealed that rampant inter-strain rather than inter-lineage differences are contributing to the evolutionary success of this pathogen. Here, we report the complete genome sequence of BXO1, a strain of Xoo belonging to a dominant lineage from India. A complete genome-based investigation revealed the presence of two plasmids, pBXO1-1 (66.7 kb) and pBXO1-2 (25.6 kb). The pBXO1-1 plasmid encodes 71 genes, 38 of which encode hypothetical proteins of unknown function. However, these hypothetical genes possess atypical GC content, pointing towards their acquisition and movement through horizontal gene transfer. Interestingly, pBXO1-2 encodes a type IV secretion system (T4SS), which is known to play an important role in the conjugative transfer of genetic material, and also provides fitness to pathogenic bacteria for their enhanced survival. Neither plasmid has been reported previously in any other complete Xoo genome published to date. Our analysis also revealed that the pBXO1-2 plasmid is present in Xanthomonas albilineans str. GPE PC73, which is known to cause leaf scald, a lethal disease in sugarcane. Our complete genome sequence analysis of BXO1 has provided us with detailed insights into the two novel strain-specific plasmids, in addition to decoding their functional capabilities, which were not assessable when using the draft genome sequence of the strain. Overall, our study has revealed the mobility of a novel T4SS in two pathogenic species of Xanthomonas that infect the vascular tissues of two economically important monocot plants, i.e. rice and sugarcane.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have collected leaf blight-affected samples from wild rice (Oryza nivara) plants growing naturally at 22 locations in five revenue districts (Nalgonda, Ranga Reddy, Medak, Nizamabad, and Adilabad) in the Telangana Region of Andhra Pradesh, India. Pathotype analysis on a set of differential rice cultivars and DNA fingerprinting with two multilocus restriction fragment length polymorphism probes indicated that the X. oryzae pv. oryzae strains from the O. nivara plants belonged to a pathotype and lineage previously widely distributed among cultivated rice in India. This suggests that the lineage may be native to wild rice and may have been transferred subsequently to cultivated rice plants.

Treatment of rice leaves with isolated Xanthomonas oryzae pv. oryzae lipopolysaccharide (LPS) induces the production of callose deposits, reactive oxygen species, and enhanced resistance against subsequent bacterial infection. Expression profiling of X. oryzae pv. oryzae LPS-treated rice (Oryza sativa subsp. indica) leaves showed that genes involved in the biosynthetic pathways for lignins, phenylpropanoids, chorismate, phenylalanine, salicylic acid, and ethylene, as well as a number of pathogenesis-related proteins are up-regulated. Gene ontology categories like cell-wall organization, defense response, stress response, and protein phosphorylation/kinases were found to be upregulated, while genes involved in photosynthesis were down-regulated. Coinfiltration with xanthan gum, the xanthomonas extracellular polysaccharide (EPS), suppressed LPS-induced callose deposition. Gene expression analysis of rice leaves that are treated with an EPS-deficient mutant of X. oryzae pv. oryzae indicated that a number of defense-regulated functions are up-regulated during infection. These transcriptional responses are attenuated in rice leaves treated with an EPS-deficient mutant that is also deficient in the O-antigen component of LPS. Overall, these results suggest that the O-antigen component of X. oryzae pv. oryzae LPS induces rice defense responses during infection and that these are suppressed by bacterial EPS.

Xanthomonas oryzae pv. oryzae secretes a number of plant cell wall–degrading enzymes (CWDEs) whose purified preparations induce defense responses in rice. These defense responses are suppressed by X. oryzae pv. oryzae using type 3 secretion system (T3SS) effectors and a type 3 secretion system mutant (T3SS−) of X. oryzae pv. oryzae is an inducer of rice defense responses. We assessed the role of individual CWDEs in induction of rice defense responses during infection, by mutating them in the genetic background of a T3SS−. We mutated the genes for five different plant CWDEs secreted by X. oryzae pv. oryzae, including two cellulases (clsA and cbsA), one xylanase (xyn), one pectinase (pglA), and an esterase (lipA), singly in a T3SS− background. We have demonstrated that, as compared with a T3SS− of X. oryzae pv. oryzae, a cbsA−T3SS−, a clsA−T3SS−, and a xyn−T3SS− are deficient in induction of rice immune responses such as callose deposits and programmed cell death. In comparison, a lipA− T3SS− and a pglA−T3SS− is as efficient in induction of host defense responses as a T3SS−. Overall, these results indicate that the collective action of X. oryzae pv. oryzae–secreted ClsA, CbsA, and Xyn proteins is required for induction of rice defense responses during infection.

Xanthomonas oryzae pv. oryzae ( Xoo) is a serious pathogen of rice causing bacterial leaf blight disease. Resistant varieties and breeding programs are being hampered by the emergence of highly virulent strains. Herein we report population based whole genome sequencing and analysis of 100 Xoo strains from India. Phylogenomic analysis revealed the clustering of Xoo strains from India along with other Asian strains, distinct from African and US Xo strains. The Indian Xoo population consists of a major clonal lineage and four minor but highly diverse lineages. Interestingly, the variant alleles, gene clusters and highly pathogenic strains are primarily restricted to minor lineages L-II to L-V and in particularly to lineage L-III. We could also find the association of an expanded CRISPR cassette and a highly variant LPS gene cluster with the dominant lineage. Molecular dating revealed that the major lineage, L-I is youngest and of recent origin compared to remaining minor lineages that seems to have originated much earlier in the past. Further, we were also able to identify core effector genes that may be helpful in efforts towards building durable resistance against this pathogen.

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, produces siderophores only under iron-limiting conditions. We screened 15 400 mTn5-induced mutants of X. oryzae pv. oryzae and isolated 27 mutants that produced siderophores even under iron-replete conditions. We found that the mTn5 insertions in 25 of these mutants were in or close to six genes. Mutants with insertions in five of these genes [colS, XOO1806 (a conserved hypothetical protein), acnB, prpR and prpB] exhibited a deficiency for growth on iron-limiting medium and a decrease in virulence. Insertions in a sixth gene, XOO0007 (a conserved hypothetical protein), were found to affect the ability to grow on iron-limiting medium, but did not affect the virulence. Targeted gene disruptants for colR (encoding the predicted cognate regulatory protein for ColS) also exhibited a deficiency for growth on iron-limiting medium and a decrease in virulence. colR and colS mutants were defective in the elicitation of hypersensitive response symptoms on the nonhost tomato. In addition, colR and colS mutants induced a rice basal defence response, suggesting that they are compromised in the suppression of host innate immunity. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that a functional ColRS system is required for the optimal expression of several genes encoding components of the type 3 secretion system (T3SS) of X. oryzae pv. oryzae. Our results demonstrate the role of several novel genes, including colR/colS, in the promotion of growth on iron-limiting medium and the virulence of X. oryzae pv. oryzae.

Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 Å from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall–degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. We have used enhanced green fluorescent protein-tagged X. oryzae pv. oryzae cells in conjunction with confocal microscopy to monitor the role of several adhesin-like functions in bacterial adhesion to leaf surface and early stages of leaf entry. Mutations in genes encoding either the Xanthomonas adhesin-like protein A (XadA) or its paralog, Xanthomonas adhesin-like protein B (XadB), as well as the X. oryzae pv. oryzae homolog of Yersinia autotransporter-like protein H (YapH), exhibit deficiencies in leaf attachment or entry. A mutation in the X. oryzae pv. oryzae pilQ gene, which is predicted to encode the type IV pilus secretin, appears to have no effect on leaf attachment or entry. The xadA– mutant is deficient in the ability to cause disease following surface inoculation while the XadB, YapH, and PilQ functions are less important than XadA for this process. The xadA– and xadB– mutants have no effect on virulence following wound inoculation whereas the yapH– and pilQ– mutants are always virulence deficient following wound inoculation. Overall, these results indicate that multiple adhesin-like functions are involved in promoting virulence of X. oryzae pv. oryzae, with preferential involvement of individual functions at different stages of the disease process.

Samba Mahsuri (BPT5204) is a medium slender grain indica rice variety that is very popular with farmers and consumers across India because of its high yield and excellent cooking quality. However, the variety is susceptible to several diseases and pests, including bacterial blight (BB). We have used PCR based molecular markers in a backcross-breeding program to introgress three major BB resistance genes (Xa21, xa13 and xa5) into Samba Mahsuri from a donor line (SS1113) in which all the three genes are present in a homozygous condition. At each backcross generation, markers closely linked to the three genes were used to select plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were used to select plants that have maximum contribution from the recurrent parent genome (background selection). A selected BC4F1 plant was selfed to generate homozygous BC4F2 plants with different combinations of BB resistance genes. The three-gene pyramid and two-gene pyramid lines exhibited high levels of resistance against the BB pathogen. Under conditions of BB infection, the three-gene pyramid lines exhibited a significant yield advantage over Samba Mahsuri. Most importantly, these lines retain the excellent grain and cooking qualities of Samba Mahsuri without compromising the yield as determined in multi-location trials. This work demonstrates the successful application of marker-assisted selection for targeted introgression of multiple resistance genes into a premium quality rice variety.

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S¯) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S¯ T3S¯ double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA¯ mutant or a ClsA¯ LipA¯ double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. We demonstrated earlier that the type II secretion system (T2S) is important for virulence of X. oryzae pv. oryzae and that several proteins, including a xylanase, are secreted through this system. In this study, the xynB gene encoding for the secreted xylanase was cloned as a 6.9-kb Eco RI fragment (pRR7) that also included a paralog called xynA. As in X. oryzae pv. oryzae, xynA and xynB are adjacent to each other in X. axonopodis pv. citri, whereas only the xynA homolog is present in X. campestris pv. campestris. Mutations in xynB but not xynA affect secreted xylanase activity. Western blot analysis using anti-XynB antibodies on exudates from infected rice leaves indicated that this xylanase is expressed during in planta growth. Another T2S-secreted protein was identified to be a lipase/esterase (LipA) based on the sequence tags obtained by tandem mass spectrometry analysis and biochemical assays. Mutations in either xynB or lipA partially affected virulence. However, a lipA-xynB double mutant was significantly reduced for virulence, and the pRR7 clone containing an intact xynB gene could complement the virulence-deficient phenotype of the lipA-xynB mutant. Our results suggest that there is functional redundancy among the T2S secreted proteins of X. oryzae pv. oryzae in promoting virulence on rice.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. A mutation was isolated in the ferric uptake regulator (fur) gene of X. oryzae pv. oryzae and it was shown to result in the production of siderophores in a constitutive manner. The fur mutant is hypersensitive to the metallo-antibiotic streptonigrin, a phenotype that is indicative of intracellular free-iron overload, and also exhibits a slow growth phenotype on rich medium. The fur mutant is virulence deficient, hypersensitive to hydrogen peroxide, and exhibits reduced catalase activity. Exogenous supplementation with ascorbic acid (an antioxidant) rescues the growth deficiency of the fur mutant in rice leaves. The virulence deficiency of the X. oryzae pv. oryzae fur mutant is proposed to be due, at least in part, to an impaired ability to cope with the oxidative stress conditions that are encountered during infection.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a novel virulence deficient mutant (BXO1691) of X. oryzae pv. oryzae that has a Tn5 insertion in an open reading frame (phyA; putative phytase A) encoding a 373-amino acid (aa) protein containing a 28-aa predicted signal peptide. Extracellular protein profiles revealed that a 38-kDa band is absent in phyA mutants as compared with phyA+ strains. A BLAST search with phyA and its deduced polypeptide sequence indicated significant similarity with conserved hypothetical proteins in Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and limited homology to secreted phytases of Bacillus species. Homology modeling with a Bacillus phytase as the template suggests that the PhyA protein has a similar six-bladed β-propeller architecture and exhibits conservation of certain critical active site residues. Phytases are enzymes that are involved in degradation of phytic acid (inositol hexaphosphate), a stored form of phosphate in plants. The phyA mutants exhibit a growth deficiency in media containing phytic acid as a sole phosphate source. Exogenous phosphate supplementation promotes migration of phyA X. oryzae pv. oryzae mutants in rice leaves. These results suggest that the virulence deficiency of phyA mutants is, at least in part, due to inability to use host phytic acid as a source of phosphate. phyA-like genes have not been previously reported to be involved in the virulence of any plant pathogenic bacterium.

Summary Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1265 amino acids that exhibits significant similarity to non-fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti-XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a β-helix conformation-like pertactin, a non-fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non-fimbrial adhesin-like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ−/pHM1::xopQD116A and xopQ−/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ−/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ−/pHM1::xopQD116A and xopQ−/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl β-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.

Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas . We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig − Vir − Aro − ). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli . The nucleotide sequence of the 2.9-kb region containing aroE revealed that a putative DNA helicase gene is located adjacent to aroE . Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production in X. oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. Spontaneous mutants which are deficient for virulence and extracellular polysaccharide (Eps) production accumulate in large numbers in stationary-phase cultures of this bacterium, a phenomenon which we have called stationary-phase variation. A clone (pSD1) carrying the Eps biosynthetic gene ( gum ) cluster of X. oryzae pv. oryzae restored Eps production and virulence to several spv (for stationary-phase variation) mutants. Data from localized recombination analysis, Southern hybridization, PCR amplification, and sequence analysis showed that the mutations are due to insertion of either one of two novel endogenous insertion sequence (IS) elements, namely, IS Xo1 and IS Xo2 , into gumM , the last gene of the gum gene cluster. The results of Southern analysis indicate the presence of multiple copies of both IS elements in the genome of X. oryzae pv. oryzae. These results demonstrate the role of IS elements in stationary-phase variation in X. oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae causes a serious disease of rice called bacterial leaf blight. It produces copious amounts of extracellular polysaccharide (EPS). An EPS- and virulence-deficient mutant of X. oryzae pv. oryzae was isolated by Tn5 mutagenesis. The mutant allele in this strain was cloned by transposon tagging in the Escherichia coli vector pBluescript and the DNA sequences flanking the transposon insertion site were determined. Computer-based similarity searches in the DNA database using the BLAST algorithm showed these sequences to be 78% identical at the nucleotide level to a gene, gumG, in the gum cluster, which is required for EPS biosynthesis in Xanthomonas campestris pv. campestris. A 36-kb X. oryzae pv. oryzae genomic clone containing the putative EPS biosynthetic gene cluster of X. oryzae pv. oryzae restored both EPS production and virulence proficiency to the gumGXo::Tn5 mutant. The results suggest that EPS is an important virulence factor of X. oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant-microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 A and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 A, beta = 90.8 degrees . Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 A, beta = 92.6 degrees and diffract to 1.86 A. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Spontaneous loss of virulence in X. oryzae pv. oryzae is associated with mutants that produce reduced levels of extracellular polysaccharide. Evidence is presented that these mutants accumulate in long-term stationary phase cultures but are not obtained from exponentially growing cultures. A number of independent spv (stationary-phase variation) mutants have been isolated and characterized. These mutants differ from each other with respect to the stability of the mutant phenotype, extent of loss of virulence, ability to outcompete wild type cells in stationary-phase cocultures, and hypersensitivity to hydrogen peroxide.Key words: stationary-phase variation, spontaneous loss of virulence, Xanthomonas oryzae.

Innate immune responses are induced in plants and animals through perception of Damage Associated Molecular Patterns. These immune responses are suppressed by pathogens during infection. A number of studies have focussed on identifying functions of plant pathogenic bacteria that are involved in suppression of Pathogen Associated Molecular Pattern induced immune responses. In comparison, there is very little information on functions used by plant pathogens to suppress Damage Associated Molecular Pattern induced immune responses. Xanthomonasoryzae pv. oryzae, a gram negative bacterial pathogen of rice, secretes hydrolytic enzymes such as LipA (Lipase/Esterase) that damage rice cell walls and induce innate immune responses. Here, we show that Agrobacterium mediated transient transfer of the gene for XopN, a X. oryzae pv. oryzae type 3 secretion (T3S) system effector, results in suppression of rice innate immune responses induced by LipA. A xopN (-) mutant of X. oryzae pv. oryzae retains the ability to suppress these innate immune responses indicating the presence of other functionally redundant proteins. In transient transfer assays, we have assessed the ability of 15 other X. oryzae pv. oryzae T3S secreted effectors to suppress rice innate immune responses. Amongst these proteins, XopQ, XopX and XopZ are suppressors of LipA induced innate immune responses. A mutation in any one of the xopN, xopQ, xopX or xopZ genes causes partial virulence deficiency while a xopN (-) xopX (-) double mutant exhibits a greater virulence deficiency. A xopN (-) xopQ (-) xopX (-) xopZ (-) quadruple mutant of X. oryzae pv. oryzae induces callose deposition, an innate immune response, similar to a X. oryzae pv. oryzae T3S(-) mutant in rice leaves. Overall, these results indicate that multiple T3S secreted proteins of X. oryzae pv. oryzae can suppress cell wall damage induced rice innate immune responses.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Upon clip inoculation of rice leaves, Xoo causes typical V-shaped lesions whose leading edge moves through the mid-veinal region. We have isolated a virulence deficient mutant of Xoo, referred to as BXO808 that causes limited lesions which primarily extend through the side-veinal regions of rice leaves. Functional complementation studies identified a clone, pSR19, from a cosmid genomic library that restored wild-type virulence and lesion phenotype to BXO808. Transposon mutagenesis of the pSR19 clone, marker exchange experiments, and targeted mutagenesis, revealed that the BXO808 phenotype is due to mutation in the gltB/D genes of Xoo, which encode glutamate synthase subunits α and β, respectively. The gltB/D mutants that were generated in this study also exhibited virulence deficiency, an altered lesion phenotype and growth deficiency on minimal medium with low levels of ammonium as a sole nitrogen source. This is the first report that mutations in the gltB/D genes of Xoo cause virulence deficiency.

A number of rice resistance genes, called Xa genes, have been identified that confer resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. An understanding of pathotype diversity within the target pathogen population is required for identifying the Xa genes that are to be deployed for development of resistant rice cultivars. Among 1024 isolates of Xoo collected from 20 different states of India, 11 major pathotypes were distinguished based on their reaction towards ten Xa genes (Xa1, Xa3, Xa4, xa5, Xa7, xa8, Xa10, Xa11, xa13, Xa21). Isolates belonging to pathotype III showing incompatible interaction towards xa8, xa13 and Xa21 and compatible interaction towards the rest of Xa genes formed the most frequent (41%) and widely distributed pathotype. The vast majority of the assayed Xoo isolates were incompatible with one or more Xa genes. Exceptionally, the isolates of pathotype XI were virulent on all Xa genes, but have restricted distribution. Considering the individual R-genes, Xa21 appeared as the most broadly effective, conferring resistance against 88 % of the isolates, followed in decreasing order by xa13 (84 %), xa8 (64 %), xa5 (30 %), Xa7 (17 %) and Xa4 (14 %). Fifty isolates representing all the eleven pathotypes were analyzed by southern hybridization to determine their genetic relatedness using the IS1112 repeat element of Xoo. Isolates belonging to pathotype XI were the most divergent. The results suggest that one RFLP haplotype that is widely distributed all over India and is represented in strains from five different pathotypes might be an ancestral haplotype. A rice line with xa5, xa13 and Xa21 resistance genes is resistant to all strains, including those belonging to pathotype XI. This three gene combination appears to be the most suitable Xa gene combination to be deployed in Indian rice cultivars.

The bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. The secreted exoglucanase CbsA is an important virulence factor of this pathogen. It belongs to the glycosyl hydrolase 6 family of proteins based on the carbohydrate-active enzyme (CAZY) classification. In this study, CbsA has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.86 Å and belonged to space group P2(1)2(1)2(1). It contained one monomer per asymmetric unit, with a solvent content of 45.8%.

Xanthomonas oryzae pv. oryzae causes bacterial blight, a serious disease of rice. Our analysis revealed that the X. oryzae pv. oryzae genome encodes genes responsible for iron uptake through FeoB (homolog of the major bacterial ferrous iron transporter) and a siderophore. A mutation in the X. oryzae pv. oryzae feoB gene causes severe virulence deficiency, growth deficiency in iron-limiting medium, and constitutive production of a siderophore. We identified an iron regulated xss gene cluster, in which xssABCDE ( X anthomonas s iderophore s ynthesis) and xsuA ( X anthomonas s iderophore u tilization) genes encode proteins involved in biosynthesis and utilization of X. oryzae pv. oryzae siderophore. Mutations in the xssA , xssB , and xssE genes cause siderophore deficiency and growth restriction under iron-limiting conditions but are virulence proficient. An xsuA mutant displayed impairment in utilization of native siderophore, suggesting that XsuA acts as a specific receptor for a ferric-siderophore complex. Histochemical and fluorimetric assays with gusA fusions indicate that, during in planta growth, the feoB gene is expressed and that the xss operon is not expressed. This study represents the first report describing a role for feoB in virulence of any plant-pathogenic bacterium and the first functional characterization of a siderophore-biosynthetic gene cluster in any xanthomonad.

Background Xanthomonas oryzae pv. oryzae causes bacterial blight of rice (Oryza sativa L.), a major disease that constrains production of this staple crop in many parts of the world. We report here on the complete genome sequence of strain PXO99A and its comparison to two previously sequenced strains, KACC10331 and MAFF311018, which are highly similar to one another. Results The PXO99A genome is a single circular chromosome of 5,240,075 bp, considerably longer than the genomes of the other strains (4,941,439 bp and 4,940,217 bp, respectively), and it contains 5083 protein-coding genes, including 87 not found in KACC10331 or MAFF311018. PXO99A contains a greater number of virulence-associated transcription activator-like effector genes and has at least ten major chromosomal rearrangements relative to KACC10331 and MAFF311018. PXO99A contains numerous copies of diverse insertion sequence elements, members of which are associated with 7 out of 10 of the major rearrangements. A rapidly-evolving CRISPR (clustered regularly interspersed short palindromic repeats) region contains evidence of dozens of phage infections unique to the PXO99A lineage. PXO99A also contains a unique, near-perfect tandem repeat of 212 kilobases close to the replication terminus. Conclusion Our results provide striking evidence of genome plasticity and rapid evolution within Xanthomonas oryzae pv. oryzae. The comparisons point to sources of genomic variation and candidates for strain-specific adaptations of this pathogen that help to explain the extraordinary diversity of Xanthomonas oryzae pv. oryzae genotypes and races that have been isolated from around the world.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a Tn5-induced virulence-deficient mutant (BXO1704) of X. oryzae pv. oryzae. The BXO1704 mutant exhibited growth deficiency in minimal medium but was proficient in inducing a hypersensitive response in a non-host tomato plant. Sequence analysis of the chromosomal DNA flanking the Tn5 insertion indicated that the Tn5 insertion is in the purH gene, which is highly homologous to purH genes of other closely related plant pathogenic bacteria Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris. Purine supplementation reversed the growth deficiency of BXO1704 in minimal medium. These results suggest that the virulence deficiency of BXO1704 may be due to the inability to use sufficient purine in the host.Key words: auxotroph, plant pathogen, resistance.

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas . We have characterized a genetic locus ( pig ) from Xanthomonas oryzae pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X. oryzae pv. oryzae. We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus. We estimate that more than 100 copies of this element might be present in the genome of X. oryzae pv. oryzae and other xanthomonads.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.

Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 51.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.

Xanthomonas oryzae pathovar oryzae causes a serious disease of rice in India and is endemic in all of the major rice-growing areas of the country. Sixty-seven X. oryzae pv. oryzae strains, collected mostly in 1994 and 1995, from 18 locations in India were analyzed by DNA fingerprinting methods using two separate repeat element probes from the X. oryzae pv. oryzae genome. These results show that strains belonging to a single pathogen lineage can be isolated from 16 of the 18 locations sampled; many of these locations are separated from each other by hundreds of kilometers and represent ecologically diverse rice-growing areas. Pathotyping analysis indicated that the strains in this lineage belong to pathotype 1b of X. oryzae pv. oryzae.

In animal pathogenic bacteria, horizontal gene transfer events (HGT) have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide (LPS). As a result, different strains of the same pathogen can have substantially different lps biosynthetic gene clusters. Since LPS is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. Although LPS has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium. We report here the complete sequence of a 12.2 kb virulence locus of Xanthomonas oryzae pv. oryzae (Xoo) encoding six genes whose products are homologous to functions involved in LPS biosynthesis and transport. All six open reading frames (ORFs) have atypical G+C content and altered codon usage, which are the hallmarks of genomic islands that are acquired by horizontal gene transfer. The lps locus is flanked by highly conserved genes, metB and etfA, respectively encoding cystathionine gamma lyase and electron transport flavoprotein. Interestingly, two different sets of lps genes are present at this locus in the plant pathogens, Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas axonopodis pv. citri (Xac). The genomic island is present in a number of Xoo strains from India and other Asian countries but is not present in two strains, one from India (BXO8) and another from Nepal (Nepal624) as well as the closely related rice pathogen, Xanthomonas oryzae pv. oryzicola (Xoor). TAIL-PCR analysis indicates that sequences related to Xac are present at the lps locus in both BXO8 and Nepal624. The Xoor strain has a hybrid lps gene cluster, with sequences at the metB and etfA ends, being most closely related to sequences from Xac and the tomato pathogen, Pseudomonas syringae pv. tomato respectively. This is the first report of hypervariation at an lps locus between different strains of a plant pathogenic bacterium. Our results indicate that multiple HGT events have occurred at this locus in the xanthomonad group of plant pathogens.