Leaf Streak In Rice Research Articles

Pest categorisation of Xanthomonas oryzae pathovars oryzae and oryzicola.

The EFSA Panel on Plant Health performed a pest categorisation for Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc), the causal agents of the bacterial blight and the bacterial leaf streak of rice, respectively. These pathovars are widely distributed in Asia, Africa and Australia. Xoo is also reported in some states of the USA and in some other countries of America. The identity of both pathovars is well established and efficient identification methods are available. The major host is cultivated rice (Oryza sativa), but different Oryza spp. as well as Poaceae weeds are reported as alternative hosts, with some uncertainty concerning the actual host range. Both pathovars are seed associated, despite the fact that seed transmission is still controversial for Xoo. Both pathovars are already regulated in Directives 2000/29/EC, on harmful organisms for plants, and 66/402/EEC, on the marketing of cereal seeds. The main pathway for entry is seed. Should these pathovars enter into EU, they may establish and spread, and they may have an impact on the rice crops, with uncertainties. The knowledge gaps identified are (1) the quantity of EU importation of rice seeds, (2) the risk of introduction through unprocessed rice for consumption, (3) the suitability of the EU growing climate conditions for the bacteria to establish and spread, (4) role of seed transmission (Xoo), (5) the role of weeds in the epidemiology and especially in seed transmission and dispersal, (6) host range of weeds. As none of the pathovars is known to occur in the EU, they do not meet one of the criteria for being considered as Union regulated non‐quarantine pests. Nevertheless, both pathovars meet the criteria assessed by EFSA for consideration as Union quarantine pest.

Insights Into the Roles of Two Genes of the Histidine Biosynthesis Operon in Pathogenicity of Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola is an X. oryzae pathovar that causes bacterial leaf streak in rice. In this study, we performed functional characterization of a nine-gene his operon in X. oryzae pv. oryzicola. Sequence analysis indicates that this operon is highly conserved in Xanthomonas spp. Auxotrophic assays confirmed that the his operon was involved in histidine biosynthesis. We found that two genes within this operon, trpR and hisB, were required for virulence and bacterial growth in planta. Further research revealed that trpR and hisB play different roles in X. oryzae pv. oryzicola. The trpR acts as a transcriptional repressor and could negatively regulate the expression of hisG, -D, -C, -B, -H, -A, and -F. hisB, which encodes a bifunctional enzyme implicated in histidine biosynthesis, was shown to be required for xanthomonadin production in X. oryzae pv. oryzicola. The disruption of hisB reduced the transcriptional expression of five known shikimate pathway-related genes xanB2, aroE, aroA, aroC, and aroK. We found that the his operon in X. oryzae pv. oryzicola is not involved in hypersensitive response in nonhost tobacco plants. Collectively, our results revealed that two genes in histidine biosynthesis operon play an important role in the pathogenicity of X. oryzae pv. oryzicola Rs105.

Overexpression of OsHSP18.0-CI Enhances Resistance to Bacterial Leaf Streak in Rice

BackgroundThe small heat shock proteins represent a large family of proteins that respond to a wide range of abiotic and biotic stresses. OsHsp18.0-CI confers tolerance to salt and cadmium and interacts with viral RNA-dependent RNA polymerase (RdRp). However, the direct function of OsHsp18.0-CI in resistance against biotic stresses remains unclear in rice.ResultsHere, we report that the expression of OsHsp18.0-CI was up-regulated upon inoculation with RS105, a strain of Xanthomonas oryzae pv. oryzicola (Xoc) that causes bacterial leaf streak in rice. In comparison with wild-type, OsHsp18.0-CI overexpression (OE) lines exhibited enhanced resistance to RS105, whereas repression lines exhibited compromised resistance to RS105. In addition, the transcriptional profiles of wild type and OE lines were compared with and without inoculation with RS105. After inoculation with RS105, most of the genes with up-regulated expression were commonly stimulated in the wild type and OE lines, with stronger induction in the OE lines than in wild type.ConclusionOur study reveals that OsHsp18.0-CI positively regulates resistance to Xoc by mediating an enhanced version of the basal defense response in rice.

An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the GA phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid-mediated defense response. Here the functions of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X.oryzae is investigated in over 100 isolates. The Xoc operon leads to production of the bioactive GA4 , an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (>90%), but absent in the other major X.oryzae pathovar. These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice.

A resistance locus in the American heirloom rice variety Carolina Gold Select is triggered by TAL effectors with diverse predicted targets and is effective against African strains of Xanthomonas oryzae pv. oryzicola.

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X.oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X.oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes.

The polygalacturonase-inhibiting protein 4 (OsPGIP4), a potential component of the qBlsr5a locus, confers resistance to bacterial leaf streak in rice.

OsPGIP4 overexpression enhances resistance to bacterial leaf streak in rice. Polygalacturonase-inhibiting proteins are thought to play important roles in the innate immunity of rice against fungi. Here, we show that the chromosomal location of OsPGIP4 coincides with the major bacterial leaf streak resistance quantitative trait locus qBlsr5a on the short arm of chromosome 5. OsPGIP4 expression was up-regulated upon inoculation with the pathogen Xanthomonas oryzae pv. oryzicola strain RS105. OsPGIP4 overexpression enhanced the resistance of the susceptible rice variety Zhonghua 11 to RS105. In contrast, repressing OsPGIP4 expression resulted in an increase in disease lesions caused by RS105 in Zhonghua 11 and in Acc8558, a qBlsr5a resistance donor. More interestingly, upon inoculation, the activated expression of pathogenesis-related genes was attenuated for those genes involved in the salicylic acid pathway, while the activated expression of jasmonic acid pathway markers was increased in the overexpression lines. Our results not only provide the first report that rice PGIP could enhance resistant against a bacterial pathogen but also indicate that OsPGIP4 is a potential component of the qBlsr5a locus for bacterial leaf streak in rice.

Confirmation of Bacterial Leaf Streak of Rice Caused by Xanthomonas oryzae pv. oryzicola in Vietnam

Confirmation of Bacterial Leaf Streak of Rice Caused by <i>Xanthomonas oryzae</i> pv. <i>oryzicola</i> in Vietnam

TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

New multilocus variable-number tandem-repeat analysis tool for surveillance and local epidemiology of bacterial leaf blight and bacterial leaf streak of rice caused by Xanthomonas oryzae.

Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales.

Correction: Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene

Correction: Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene

Identification of 17 HrpX-Regulated Proteins Including Two Novel Type III Effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola

The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.

Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene

Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

Suppression of expression of the putative receptor-like kinase gene NRRB enhances resistance to bacterial leaf streak in rice

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important disease of rice, which is responsible for the economic losses worldwide. Functional investigation of differentially expressed protein genes (DEPGs) from rice (Oryza sativa L.) upon Xoc infection provides insight into the molecular mechanism of rice-Xoc interactions. Here, we show that one of DEPGs designated NRRB plays a role in rice-Xoc interactions. NRRB, a receptor-like cytoplasmic kinase gene was preferentially expressed in leaf blades and leaf sheaths where the pathogen colonized. Its transcription was depressed by two defense-signal compounds salicylic acid and 1-aminocyclopropane-1-carboxylic-acid, but was activated by wounding and abscisic acid. Additionally, a plenty of cis-elements associated with stress responses were discovered in the promoter region of NRRB. These data suggest that NRRB is involved in stress responses. More importantly, the NRRB-suppressing rice plants exhibited enhanced resistance against BLS, with the markedly shorter average lesion length than that of the wild type. Furthermore, transcription of some salicylic acid synthesis-related and pathogenesis-related genes including PAD4, PR1a and WRKY13 in transgenic plants was activated, implying that enhanced resistance to BLS might be mediated by the activation of the SA signaling pathway. In conclusion, NRRB gene is involved in various stress responses and regulating resistance to BLS, therefore it might be one of useful genes for rice improvement in future.

Requirement of the Lipopolysaccharide O-Chain Biosynthesis Gene wxocB for Type III Secretion and Virulence of Xanthomonas oryzae pv. Oryzicola

Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak of rice. A mutant disrupted in wxocB, predicted to encode an enzyme for lipopolysaccharide (LPS) synthesis, was previously shown to suffer reduced virulence. Here, we confirm a role for wxocB in virulence and demonstrate its requirement for LPS O-chain assembly. Structure analysis indicated that wild-type LPS contains a polyrhamnose O chain with irregular, variant residues and a core oligosaccharide identical to that of other Xanthomonas spp. and that the wxocB mutant lacks the O chain. The mutant also showed moderate impairment in exopolysaccharide (EPS) production, but comparison with an EPS-deficient mutant demonstrated that this impairment could not account entirely for the reduced virulence. The wxocB mutant was not detectably different from the wild type in its induction of pathogenesis-related rice genes, type II secretion competence, flagellar motility, or resistance to two phytoalexins or resveratrol, and it was more, not less, resistant to oxidative stress and a third phytoalexin, indicating that none of these properties is involved. The mutant was more sensitive to SDS and to novobiocin, so increased sensitivity to some host-derived antimicrobials cannot be ruled out. However, the mutant showed a marked decrease in type III secretion into plant cells. This was not associated with any change in expression of genes for type III secretion or the ability to attach to plant cells in suspension. Thus, virulence of the wxocB mutant is likely reduced due primarily to a direct, possibly structural, effect of the loss of the O chain on type III delivery of effector proteins.

A Highly-Conserved Single-Stranded DNA-Binding Protein in Xanthomonas Functions as a Harpin-Like Protein to Trigger Plant Immunity

Harpins are produced by Gram-negative phytopathogenic bacteria and typically elicit hypersensitive response (HR) in non-host plants. The characterization of harpins in Xanthomonas species is largely unexplored. Here we demonstrate that Xanthomonas produce a highly conserved single-stranded DNA-binding protein (SSBX) that elicits HR in tobacco as by harpin Hpa1. SSBX, like Hpa1, is an acidic, glycine-rich, heat-stable protein that lacks cysteine residues. SSBX-triggered HR in tobacco, as by Hpa1, is characterized by the oxidative burst, the expression of HR markers (HIN1, HSR203J), pathogenesis-related genes, and callose deposition. Both SSBX- and Hpa1-induced HRs can be inhibited by general metabolism inhibitors actinomycin D, cycloheximide, and lanthanum chloride. Furthermore, those HRs activate the expression of BAK1 and BIK1 genes that are essential for induction of mitogen-activated protein kinase (MAPK) and salicylic acid pathways. Once applied to plants, SSBX induces resistance to the fungal pathogen Alternaria alternata and enhances plant growth. When ssbX was deleted in X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak in rice, the resulting ssbXoc mutant was reduced in virulence and bacterial growth in planta, but retained its ability to trigger HR in tobacco. Interestingly, ssbXoc contains an imperfect PIP-box (plant-inducible promoter) and the expression of ssbXoc is regulated by HrpX, which belongs to the AraC family of transcriptional activators. Immunoblotting evidence showed that SSBx secretion requires a functional type-III secretion system as Hpa1 does. This is the first report demonstrating that Xanthomonas produce a highly-conserved SSBX that functions as a harpin-like protein for plant immunity.

AsnB, regulated by diffusible signal factor and global regulator Clp, is involved in aspartate metabolism, resistance to oxidative stress and virulence in Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak in rice, which is a destructive disease worldwide. Xoc virulence factors are regulated by diffusible signal factor (DSF) and the global regulator Clp. In this study, we have demonstrated that asnB (XOC_3054), encoding an asparagine synthetase, is a novel virulence-related gene regulated by both DSF and Clp in Xoc. A sequence analysis revealed that AsnB is highly conserved in Xanthomonas. An asnB mutation in Xoc dramatically impaired pathogen virulence and growth rate in host rice, but did not affect the ability to trigger the hypersensitive response in nonhost (plant) tobacco. Compared with the wild-type strain, the asnB deletion mutant was unable to grow in basic MMX (-) medium (a minimal medium without ammonium sulphate as the nitrogen source) with or without 10 tested nitrogen sources, except asparagine. The disruption of asnB impaired pathogen resistance to oxidative stress and reduced the transcriptional expression of oxyR, katA and katG, which encode three important proteins responsible for hydrogen peroxide (H(2)O(2)) sensing and detoxification in Xanthomonas in the presence of H(2)O(2), and nine important known Xoc virulence-related genes in plant cell-mimicking medium. Furthermore, the asnB mutation did not affect extracellular protease activity, extracellular polysaccharide production, motility or chemotaxis. Taken together, our results demonstrate the role of asnB in Xanthomonas for the first time.

Epv, Encoding a Hypothetical Protein, Is Regulated by DSF-Mediating Quorum Sensing as Well as Global Regulator Clp and Is Required for Optimal Virulence in Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak in rice, a destructive disease worldwide. In this study, six putative hypothetical secreted proteins, which were absent in X. oryzae pv. oryzae, were detected from X. oryzae pv. oryzicola strain BLS256. Disruption-based mutagenesis study revealed that one of them, Xoc_15235, named as extracellular polysaccharide and virulence-related gene (epv), was required for the optimal virulence in host rice but not for the induction of a hypersensitive reaction in nonhost tobacco. Sequence analysis revealed that epv was highly conserved in Xanthomonas spp. (except X. oryzae pv. oryzae). In-frame deletion of epv in X. oryzae pv. oryzicola dramatically impaired pathogen virulence and extracellular polysaccharide (EPS) production, one of the important known virulence-associated functions in Xanthomonas spp. Quantitative real-time reverse-transcription polymerase chain reaction showed that expression of both gumB (a gene encoding exopolysaccharide xanthan biosynthesis export protein) and a known virulence-related gene, pgk (encoding phosphoglycerate kinase), were obviously reduced in the epv-deletion mutant compared with the wild-type strain Rs105. In addition, we observed that epv was positively regulated by both diffusible signal factor and global regulator Clp in X. oryzae pv. oryzicola. Taken together, the novel roles and genetics of epv of X. oryzae pv. oryzicola in the EPS production and virulence were investigated for the first time.

Identification and Characterization of a Novel Gene, hshB, in Xanthomonas oryzae pv. oryzicola Co-Regulated by Quorum Sensing and clp

Virulence factors of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in rice, are regulated by a diffusible signal factor (DSF)-dependent quorum-sensing (QS) system. In this study, a novel pathogenicity-related gene, Xoryp_010100018570 (named hshB), of X. oryzae pv. oryzicola was characterized. hshB encodes a hydrolase with a putative signal peptide, which is a homolog of imidazolonepropionase. Bioinformatic analysis showed that hshB is relatively conserved in the genus Xanthomonas but the homologous gene of hshB was not found in X. oryzae pv. oryzae. Reverse-transcription polymerase chain reaction (PCR) analysis showed that hshB and its upstream gene, Xoryp_010100018565 (named hshA), are co-transcribed in X. oryzae pv. oryzicola. Subsequent experimental results indicated that mutation of hshB remarkably impaired the virulence, extracellular protease activity, extracellular polysaccharide production, growth in minimal medium, and resistance to oxidative stress and bismerthiazol of X. oryzae pv. oryzicola. Mutation of clp, encoding a global regulator, resulted in similar phenotypes. Real-time PCR assays showed that hshB transcription is positively regulated by clp and DSF, and induced by poor nutrition. Our study not only found a novel gene hshB regulated by DSF-dependent QS system and clp but also showed that hshB was required for virulence of X. oryzae pv. oryzicola.

Identification of HrpX regulon genes in Xanthomonas oryzae pv. oryzicola using a GFP visualization technique

Xanthomonas oryzae pv. oryzicola is the causal agent of bacterial leaf streak in rice and injects repertoires of T3S effectors (T3SEs), which are normally regulated by a global regulator HrpX, into plant cells to suppress plant innate immunity for disease development. To establish a visualization technique to identify HrpX regulon genes in this pathogen, we chose six known or unknown T3SE genes of X. oryzae pv. oryzicola (strain RS105) as the targets in this report. The promoters of these candidates, whether or not containing a PIP-box, were fused with the gfp (green fluorescent protein) reporter gene, and the gfp reporters were introduced into the wild-type RS105 and the hrpX mutant RΔhrpX. GFP expression in the wild-type strain could be observed under fluorescence microscopy, but fluorescence was not observed in the hrpX mutant. Consequently, transcription and secretion detection demonstrated that these HrpX regulon members were repressed when hrpX was mutated and were not secreted into the hrp-inducing medium XOM3 because of a mutation in hrcV (T3SS deficient). These data suggest that the gfp reporter system is a feasible visualization tool for the identification of HrpX regulon genes and discovery of new T3SEs in the X. oryzae pv. oryzicola-rice pathosystem.

A Non-Marker Mutagenesis Strategy to Generate Poly- hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola (Xoc), the critical pathogen causing bacterial leaf streak in rice, possesses a hrp cluster that is responsible for triggering hypersensitive response (HR) in non-host tobacco and pathogenicity in host rice, and is considered to be one of the model pathogens in the rice model plant. Here, we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1. It was used to generate single or poly-gene mutants of hpa1, hpa2, hrcV, hrpE, hpaB, and hrpF gene for functional analysis. In total, five single, four double, and two triple hrp gene mutants were constructed. The double and triple hrp gene deletion mutants triggered novel phenotypes in planta. Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.