Xoo Virulence Research Articles

The conserved Xanthomonas effector XopM targets allene oxide synthase OsAOS3 and interferes with jasmonate-mediated defense in rice1

Bacterial blight (BB) of rice caused by the phytopathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is a disease of global importance. Xoo utilizes the type III secretion system (T3SS) and its effectors for virulence, and XopM is a conserved T3SS effector in Xanthomonas spp. However, the virulence function of XopM is largely unknown. In this study, we show that XopM contributes to Xoo virulence in rice. We demonstrate that XopM interacts with allene oxide synthase OsAOS3, a key enzyme involved in jasmonic acid (JA) biosynthesis. The expression levels of OsAOS3 and three homologues of OsAOS were elevated after Xoo infection. Knockout mutants of OsAOS3 exhibited decreased JA accumulation and reduced resistance to Xoo and X. oryzae pv. oryzicola. Moreover, JA-related defense genes were downregulated in osaos3 mutants during Xoo infection. Based on our results, we propose a model showing how XopM hijacks OsAOS3 to interfere with JA-mediated defenses, leading to a suppression of rice immunity. Our findings reveal a novel virulence strategy where Xanthomonas pathogens interfere with the JA pathway and modulate the host defense response.

Integrative genomic and transcriptomic analysis of Xanthomonas oryzae pv. oryzae pathotype IV, V, and IX in China reveals rice defense-responsive genes

Bacterial blight of rice is a devastating disease caused by the gram-negative bacteria Xanthomonas oryzae pv. oryzae (Xoo). Chinese Xoo strain pathotypes IV, V, and IX are the major virulent Xoo strain types in South China sequentially from the 1990s to the present. Here, we report the isolation of GD0201 and GD0202, which belong to pathotypes IV and IX, respectively, and the complete genome sequence and transcriptomic analysis of GD0201 (IV), GD1358 (V), and GD0202 (IX). We found that resistance genes xa5, Xa23, and Xa27 confer strong resistance to all three Xoo strains, indicating that they are currently good choices for resistance rice breeding. The genome analysis reveals fewer TAL and non-TAL effector coding genes in GD0202 than in the other two strains, potentially contributing to its strong virulence. Transcriptomic analysis of ZH11 inoculated with the three Xoo strains strongly suggests that three Xoo strains for better infection repress the ethylene response factor (ERF) gene family members. Furthermore, weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) analysis revealed 14 hub genes potentially associated with rice response to the three Xoo strains. The expression of several hub genes was validated to be induced by all three Xoo strains, suggesting its role in bacterial blight disease response to Xoo strains. Genomic analysis of the Xoo strains belonging to pathotypes IV, V, and IX, identification of effectors and genes related to Xoo virulence in rice plants will provide insights into understanding the molecular mechanism underlying rice-Xoo interaction and the gene expression pattern in response to Xoo infection.

Vitamin B6 biosynthesis is essential for the virulence of Xanthomonas oryzae pv. oryzae

AbstractXanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which threatens rice production around the world. Efficient infection of rice leaves by Xoo depends on its ability to obtain nutrients from the plant and to cope with the host defence responses. Vitamin B6 (VB6) is essential to the growth of all organisms, but bacteria and plants use different biosynthesis pathways, meaning VB6 biosynthesis might be attractive as an antibacterial target. In bacteria, deoxyxylose 5‐phosphate (DXP)‐dependent and deoxyxylose 5‐phosphate‐independent biosynthesis pathways can produce VB6. However, the Xoo VB6 biosynthesis pathway has not been experimentally identified, and its role in Xoo virulence remains unclear. Here, we analysed the VB6 biosynthesis pathway in Xoo and its role in host–pathogen interactions. PdxA and PdxJ are key components for DXP‐dependent VB6 biosynthesis; we found that Xoo mutants containing mutations in these two putative genes (pdxA and pdxJ) were fully auxotrophic for VB6, demonstrating that Xoo VB6 de novo biosynthesis is DXP‐dependent. We also found that mutation of these key genes (pdxA and pdxJ) significantly reduced Xoo virulence in rice. These results suggest that VB6 de novo biosynthesis is essential for Xoo virulence.

The MinCDE Cell Division System Participates in the Regulation of Type III Secretion System (T3SS) Genes, Bacterial Virulence, and Motility in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) in rice, which is one of the most severe bacterial diseases in rice in some Asian countries. The type III secretion system (T3SS) of Xoo encoded by the hypersensitive response and pathogenicity (hrp) genes is essential for its pathogenicity in host rice. Here, we identified the Min system (MinC, MinD, and MinE), a negative regulatory system for bacterial cell division encoded by minC, minD, and minE genes, which is involved in negative regulation of hrp genes (hrpB1 and hrpF) in Xoo. We found that the deletion of minC, minD, and minCDE resulted in enhanced hrpB1 and hrpF expression, which is dependent on two key hrp regulators HrpG and HrpX. The minC, minD, and minCDE mutants exhibited elongated cell lengths, and the classic Min system-defective cell morphology including minicells and short filamentations. Mutation of minC in Xoo resulted in significantly impaired virulence in host rice, swimming motility, and enhanced biofilm formation. Our transcriptome profiling also indicated some virulence genes were differentially expressed in the minC mutants. To our knowledge, this is the first report about the Min system participating in the regulation of T3SS expression. It sheds light on the understanding of Xoo virulence mechanisms.

XopZ and ORP1C cooperate to regulate the virulence of Xanthomonas oryzae pv. oryzae on Nipponbare

ABSTRACT Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) has always been considered to be one of the most severe worldwide diseases in rice. Xoo strains usually use the highly conserved type III secretion system (T3SS) to deliver virulence effectors into rice cells and further suppress the host’s immunity. Previous studies reported that different Xanthomonas outer protein (Xop) effectors include XopZ from one strain appear to share functional redundancies on suppressing rice PAMP-triggered immunity (PTI). But only xopZ, except other xop genes, could significantly impaire Xoo virulence when individually deleting in PXO99 strains. Thus, the XopZ effector should not only suppress rice PTI pathway, but also has other unknown indispensable pathological functions in PXO99–rice interactions. Here, we also found that ∆xopZ mutant strains displayed lower virulence on Nipponbare leaves compared with PXO99 strains. We identified an oxysterol-binding related protein, ORP1C, as a XopZ-interacting protein in rice. Further studies found that rice ORP1C preliminarily played a positive role in regulating the resistance to PXO99 strains, and XopZ–ORP1C interactions cooperated to regulate the compatible interactions of PXO99-Nipponbare rice. The reactive oxygen species (ROS) burst and PTI marker gene expression data indicated that ORP1C were not directly relevant to the PTI pathway in rice. The deeper mechanisms underlying XopZ–ORP1C interaction and how XopZ and ORP1C cooperate for regulating the PXO99–rice interactions require further exploration.

Interplay between the cyclic di-GMP network and the cell-cell signalling components coordinates virulence-associated functions in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes a serious disease of rice known as bacterial leaf blight. Several virulence-associated functions have been characterized in Xoo. However, the role of important second messenger c-di-GMP signalling in the regulation of virulence-associated functions still remains elusive in this phytopathogen. In this study we have performed an investigation of 13 c-di-GMP modulating deletion mutants to understand their contribution in Xoo virulence and lifestyle transition. We show that four Xoo proteins, Xoo2331, Xoo2563, Xoo2860 and Xoo2616, are involved in fine-tuning the in vivo c-di-GMP abundance and also play a role in the regulation of virulence-associated functions. We have further established the importance of the GGDEF domain of Xoo2563, a previously characterized c-di-GMP phosphodiesterase, in the virulence-associated functions of Xoo. Interestingly the strain harbouring the GGDEF domain deletion (ΔXoo2563GGDEF ) exhibited EPS deficiency and hypersensitivity to streptonigrin, indicative of altered iron metabolism. This is in contrast to the phenotype exhibited by an EAL overexpression strain wherein, the ΔXoo2563GGDEF exhibited other phenotypes, similar to the strain overexpressing the EAL domain. Taken together, our results indicate a complex interplay of c-di-GMP signalling with the cell-cell signalling to coordinate virulence-associated function in Xoo.

Proteomic and Transcriptomic Analyses Provide Novel Insights into the Crucial Roles of Host-Induced Carbohydrate Metabolism Enzymes in Xanthomonas oryzae pv. oryzae Virulence and Rice-Xoo Interaction

BackgroundXanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a devastating rice disease. The Xoo-rice interaction, wherein wide ranging host- and pathogen-derived proteins and genes wage molecular arms race, is a research hotspot. Hence, the identification of novel rice-induced Xoo virulence factors and characterization of their roles affecting rice global gene expression profiles will provide an integrated and better understanding of Xoo-rice interactions from the molecular perspective.ResultsUsing comparative proteomics and an in vitro interaction system, we revealed that 5 protein spots from Xoo exhibited significantly different expression patterns (|fold change| > 1.5) at 3, 6, 12 h after susceptible rice leaf extract (RLX) treatment. MALDI-TOF MS analysis and pathogenicity tests showed that 4 host-induced proteins, including phosphohexose mutase, inositol monophosphatase, arginase and septum site-determining protein, affected Xoo virulence. Among them, mutants of two host-induced carbohydrate metabolism enzyme-encoding genes, ΔxanA and Δimp, elicited enhanced defense responses and nearly abolished Xoo virulence in rice. To decipher rice differentially expressed genes (DEGs) associated with xanA and imp, transcriptomic responses of ΔxanA-treated and Δimp-treated susceptible rice were compared to those in rice treated with PXO99A at 1 and 3 dpi. A total of 1521 and 227 DEGs were identified for PXO99A vs Δimp at 1 and 3 dpi, while for PXO99A vs ΔxanA, there were 131 and 106 DEGs, respectively. GO, KEGG and MapMan analyses revealed that the DEGs for PXO99A vs Δimp were mainly involved in photosynthesis, signal transduction, transcription, oxidation-reduction, hydrogen peroxide catabolism, ion transport, phenylpropanoid biosynthesis and metabolism of carbohydrates, lipids, amino acids, secondary metabolites, hormones, and nucleotides, while the DEGs from PXO99A vs ΔxanA were predominantly associated with photosynthesis, signal transduction, oxidation-reduction, phenylpropanoid biosynthesis, cytochrome P450 and metabolism of carbohydrates, lipids, amino acids, secondary metabolites and hormones. Although most pathways were associated with both the Δimp and ΔxanA treatments, the underlying genes were not the same.ConclusionOur study identified two novel host-induced virulence factors XanA and Imp in Xoo, and revealed their roles in global gene expression in susceptible rice. These results provide valuable insights into the molecular mechanisms of pathogen infection strategies and plant immunity.

Reciprocal adaptation of rice and Xanthomonas oryzae pv. oryzae: cross-species 2D GWAS reveals the underlying genetics.

A 1D/2D genome-wide association study strategy was adopted to investigate the genetic systems underlying the reciprocal adaptation of rice (Oryza sativa) and its bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo) using the whole-genome sequencing and large-scale phenotyping data of 701 rice accessions and 23 diverse Xoo strains. Forty-seven Xoo virulence-related genes and 318 rice quantitative resistance genes (QR-genes) mainly located in 41 genomic regions, and genome-wide interactions between the detected virulence-related genes and QR genes were identified, including well-known resistance genes/virulence genes plus many previously uncharacterized ones. The relationship between rice and Xoo was characterized by strong differentiation among Xoo races corresponding to the subspecific differentiation of rice, by strong shifts toward increased resistance/virulence of rice/Xoo populations and by rich genetic diversity at the detected rice QR-genes and Xoo virulence genes, and by genome-wide interactions between many rice QR-genes and Xoo virulence genes in a multiple-to-multiple manner, presumably resulting either from direct protein–protein interactions or from genetic epistasis. The observed complex genetic interaction system between rice and Xoo likely exists in other crop–pathogen systems that would maintain high levels of diversity at their QR-loci/virulence-loci, resulting in dynamic coevolutionary consequences during their reciprocal adaptation.

Genome-scale metabolic reconstruction and in silico analysis of therice leaf blight pathogen, Xanthomonas oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a vascular pathogen that causes leaf blight in rice, leading to severe yield losses. Since the usage of chemical control methods has not been very promising for the future disease management, it is of high importance to systematically gain new insights about Xoo virulence and pathogenesis, and devise effective strategies to combat the rice disease. To do this, we reconstructed a genome‐scale metabolic model of Xoo (iXOO673) and validated the model predictions using culture experiments. Comparison of the metabolic architecture of Xoo and other plant pathogens indicated that the Entner–Doudoroff pathway is a more common feature in these bacteria than previously thought, while suggesting some of the unique virulence mechanisms related to Xoo metabolism. Subsequent constraint‐based flux analysis allowed us to show that Xoo modulates fluxes through gluconeogenesis, glycogen biosynthesis, and degradation pathways, thereby exacerbating the leaf blight in rice exposed to nitrogenous fertilizers, which is remarkably consistent with published experimental literature. Moreover, model‐based interrogation of transcriptomic data revealed the metabolic components under the diffusible signal factor regulon that are crucial for virulence and survival in Xoo. Finally, we identified promising antibacterial targets for the control of leaf blight in rice by using gene essentiality analysis.

Biosynthesis of Amino Acids in Xanthomonas oryzae pv. oryzae Is Essential to Its Pathogenicity

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which causes a large reduction in rice production. The successful interaction of pathogens and plants requires a particular nutrient environment that allows pathogen growth and the initiation of both pathogen and host responses. Amino acid synthesis is essential for bacterial growth when bacteria encounter amino acid-deficient environments, but the effects of amino acid synthesis on Xoo pathogenicity are unclear. Here, we systemically deleted the essential genes (leuB, leuC, leuD, ilvC, thrC, hisD, trpC, argH, metB, and aspC) involved in the synthesis of different amino acids and analyzed the effects of these mutations on Xoo virulence. Our results showed that leucine, isoleucine, valine, histidine, threonine, arginine, tryptophan, and cysteine syntheses are essential to Xoo infection. We further studied the role of leucine in the interaction between pathogens and hosts and found that leucine could stimulate some virulence-related responses and regulate Xoo pathogenicity. Our findings highlight that amino acids not only act as nutrients for bacterial growth but also play essential roles in the Xoo and rice interaction.

Identification of c-di-GMP Signaling Components in Xanthomonas oryzae and Their Orthologs in Xanthomonads Involved in Regulation of Bacterial Virulence Expression.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. Xoo produces a range of virulence-related factors to facilitate its pathogenesis in rice, however, the regulatory mechanisms of Xoo virulence expression have been not fully elucidated. Recent studies have revealed that virulence factor production is regulated via cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway that is well-conserved in Xoo and other Xanthomonas species. A set of GGDEF, EAL, HD-GYP, and PilZ domain proteins with diverse signal sensory domains for c-di-GMP synthesis, hydrolysis, and binding is encoded in the Xoo genome. Bioinformatic, genetic, and biochemical analysis has identified an array of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), as well as degenerate GGDEF/EAL, PilZ domain proteins along with a transcription regulator. These signaling components have been characterized to regulate various bacterial cellular processes, such as virulence, exopolysaccharide (EPS) production, biofilm formation, motility, and adaptation at the transcriptional, post-translational, and protein-protein interaction levels. This review summarized the recent progress in understanding the importance and complexity of c-di-GMP signaling in regulating bacterial virulence expression, highlighting the identified key signal elements and orthologs found in Xanthomonads, discussing the diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs, and effectors, and further exploration of the new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in regulation of bacterial pathogenesis.

Genome-wide Screening to Identify Responsive Regulators Involved in the Virulence of Xanthomonas oryzae pv. oryzae.

Two-component systems (TCSs) are critical to the pathogenesis of Xanthomonas oryzae pv. oryzae (Xoo). We mutated 55 of 62 genes annotated as responsive regulators (RRs) of TCSs in the genome of Xoo strain PXO99A and identified 9 genes involved in Xoo virulence. Four (rpfG, hrpG, stoS, and detR) of the 9 genes were previously reported as key regulators of Xoo virulence and the other 5 have not been characterized. Lesion lengths on rice leaves inoculated with the mutants were shorter than those of the wild type and were significantly restored with gene complementation. The population density of the 5 mutants in planta was smaller than that of PXO99A at 14 days after inoculation, but the growth curves of the mutants in rich medium were similar to those of the wild type. These newly reported RR genes will facilitate studies on the function of TCSs and of the integrated regulation of TCSs for Xoo pathogenesis.

Contribution of the cold shock protein CspA to virulence in Xanthomonas oryzae pv. oryzae.

Summary Xanthomonas oryzae pv. oryzae (Xoo) causes a damaging bacterial leaf blight disease in rice. Cold shock proteins (Csps) are highly conserved nucleic acid‐binding proteins present in various bacterial genera, but relatively little is known about their functions in Xanthomonas. Herein, we identified four Csps (CspA–CspD) in the Xoo PXO99A strain. Deletion of cspA decreased cold adaptation and a few known pathogenic factors, including bacterial pathogenicity, biofilm formation and polysaccharide production. Furthermore, we performed transcriptomic and chromosome immunoprecipitation (ChIP) experiments to identify direct targets of CspA and to determine its DNA‐binding sequence. Integrative data analysis revealed that CspA directly regulates two genes, PXO_RS11830 and PXO_RS01060, by binding to a conserved CCAAT sequence in the promoter region. We generated single‐deletion mutants of each gene and the results indicate that both are responsible for Xanthomonas pathogenicity. In addition, quantitative real‐time polymerase chain reaction and western blotting showed that CspA suppressed the expression of its direct targets. In summary, our study clarifies the characteristics of Csps in Xanthomonas and greatly advances our understanding of the mechanisms underlying the contribution of CspA to bacterial virulence.

DNA Sequencing Analysis of African Xanthomonas oryzae pv. oryzae Virulence Gene (AXaVrg) DNA Marker

Abstract Global rice production is constrained by bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo). BLB disease incidence in West Africa was between 70–85% and yield loss in farmers’ fields was in the range of 50–90% from 2005 to 2010. In the present study, African Xoo virulence gene OPP-172000 DNA marker was identified and purified using randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) products from 50 Xoo isolates. Genomic DNA of 50 Xoo isolates were analyzed using OPP-17 primer in RAPD-PCR during which African Xoo virulence gene OPP-172000 DNA marker was identified, purified, cloned, and sequenced. Cloning and DNA sequencing of African Xoo virulence gene OPP-172000 DNA generated a 1953 bp nucleotide sequence consequently tagged as AXaVrg-1953. BLAST homologous analysis of the AXaVrg-1953 sequence provides comprehensive identification of the type II secretion genes and secreted proteins, type III secretion genes and secreted proteins in African Xoo virulence gene. Phylogenetic unweighted pairgroup method arithmetic (UPGMA) analysis revealed the African AXaVrg-1953 sequence was distinct from the other Xoo virulence gene sequences from China, Japan, Korea, Germany, and the United States. This information is potentially useful for effective management of BLB disease in West Africa.

Label-free quantitative secretome analysis of Xanthomonas oryzae pv. oryzae highlights the involvement of a novel cysteine protease in its pathogenicity

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases resulting in a huge loss of the total rice productivity. The initial interaction between rice and Xoo takes place in the host apoplast and is mediated primarily by secretion of various proteins from both partners. Yet, such secretory proteins remain to be largely identified and characterized. This study employed a label-free quantitative proteomics approach and identified 404 and 323 Xoo-secreted proteins from in vitro suspension-cultured cells and in planta systems, respectively. Gene Ontology analysis showed their involvement primarily in catalytic, transporter, and ATPase activities. Of a particular interest was a Xoo cysteine protease (XoCP), which showed dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence. Besides, a parallel analysis of in planta rice-secreted proteins resulted in identification of 186 secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. Identified secretory proteins were exploited to shed light on their possible role in the rice-Xoo interaction, and that further deepen our understanding of such interaction. Biological significanceXanthomonas oryzae pv. oryzae (Xoo), causative agent of bacterial blight disease, results in a huge loss of the total rice productivity. Using a label-free quantitative proteomics approach, we identified 727 Xoo- and 186 rice-secreted proteins. Functional annotation showed Xoo secreted proteins were mainly associated with the catalytic, transporter, and ATPase activities while the rice secreted proteins were mainly associated with the catalytic, antioxidant, and electron carrier activities. A novel Xoo cysteine protease (XoCP) was identified, showing dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence.

A thiadiazole reduces the virulence of Xanthomonas oryzae pv. oryzae by inhibiting the histidine utilization pathway and quorum sensing.

Thiazole, isothiazole, thiadiazole and their derivatives are widely thought to induce host defences against plant pathogens. In this article, we report that bismerthiazol, a thiadiazole molecule, reduces disease by inhibiting the histidine utilization (Hut) pathway and quorum sensing (QS). Bismerthiazol provides excellent control of bacterial rice leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), but does not greatly inhibit Xoo growth in vitro. According to RNA-sequencing analysis, the transcription of the Hut pathway genes of Xoo ZJ173 was inhibited after 4.5 and 9.0 h of bismerthiazol treatment. Functional studies of hutG and hutU indicated that the Hut pathway had little effect on the growth and bismerthiazol sensitivity of Xoo in vitro, but significantly reduced the aggregation of Xoo cells. Deletion mutants of hutG or hutU were more motile, produced less biofilm and were less virulent than the wild-type, indicating that the Hut pathway is involved in QS and contributes to virulence. The overexpression of the hutG-U operons in ZJ173 reduced Xoo control by bismerthiazol. Bismerthiazol did not inhibit the transcription of Hut pathway genes, QS or virulence of the bismerthiazol-resistant strain 2-1-1. The results indicate that bismerthiazol reduces Xoo virulence by inhibiting the Hut pathway and QS.

DgcA, a diguanylate cyclase from Xanthomonas oryzae pv. oryzae regulates bacterial pathogenicity on rice

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ΔdgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases.

Secretome analysis of rice suspension-cultured cells infected by Xanthomonas oryzae pv.oryza (Xoo).

BackgroundRice bacterial blight (BB) caused by Xanthomonas oryzae pv.oryzae (Xoo) is one of the most devastating bacterial diseases in rice-growing regions worldwide. The rice-Xoo interaction is a classical model for studying the interaction between plants and pathogens. Secreted proteins play important roles in plant-bacterial interactions, but are poorly studied in the rice-Xoo system. Rice cv. Nipponbare is highly susceptible to Xoo. Here, we used two-dimensional difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF/TOF mass spectrometry (MS), to investigate secreted proteins in Nipponbare embryo cell suspension culture infected by Xoo.ResultsA total of 32 protein spots changed significantly (p < 0.05) by more than 1.5 fold in gel intensity after Xoo inoculation, and were identified by MS. They represent protein products of 11 unique genes, seven from rice and four from Xoo. Of the rice proteins, six up-regulated proteins are involved in cell wall modification, the TCA cycle, glycolysis and redox, while a down-regulated protein, CHIT16, is involved in plant defense. Quantitative Real-Time PCR showed that transcript levels were not correlated with secreted protein levels. Of the Xoo proteins, three of them were possibly located in the extracellular space as shown by transient expression assays in rice protoplasts. Two of the Xoo proteins were previously reported to be likely involved in pathogenicity, and the third gene, Xoo3654, is likely a negative regulator of Xoo virulence as its overexpression reduced Xoo pathogenicity in our study.ConclusionAmong the secreted proteins that responded to Xoo inoculation, we identified rice proteins involved in cell defense and Xoo proteins involved in pathogenicity. Our study also showed that Xoo3654 (X2) protein is likely a novel negative regulator of Xoo virulence. These results not only help us better understand the interaction between susceptible rice and Xoo, but also serve as a reference for studying the interaction between other plants and their pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0091-z) contains supplementary material, which is available to authorized users.

Role of DetR in defence is critical for virulence of Xanthomonas oryzae pv. oryzae

Like other bacteria, Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight disease in rice, possesses intracellular signalling systems, known as two-component regulatory systems (TCSs), which regulate pathogenesis and biological processes. Completion of the genome sequences of three Xoo strains has facilitated the functional study of genes, including those of TCSs, but the biological functions of most Xoo TCSs have not yet been uncovered. To identify TCSs involved in Xoo pathogenesis, we generated knockout strains lacking response regulators (RRs, a cytoplasmic signalling component of the TCS) and examined the virulence of the RR knockout strains. This study presents a knockout strain (detR(-) ) lacking the PXO_04659 gene which shows dramatically reduced virulence relative to the wild-type. Our studies to elucidate detR function in Xoo pathogenesis revealed a reduction in extracellular polysaccharide (EPS), intolerance to reactive oxygen species (ROS) and deregulation of iron homeostasis in the detR(-) strain. Moreover, gene expression of regulatory factors, including other RRs and transcription factors (TFs), was altered in the absence of DetR protein, as determined by reverse transcription-polymerase chain reaction (RT-PCR) and/or real-time quantitative RT-PCR analyses. All evidence leads to the conclusion that DetR is essential for Xoo virulence through the regulation of the Xoo defence system including EPS synthesis, ROS detoxification and iron homeostasis, solely or cooperatively with other regulatory factors.

The thiG Gene Is Required for Full Virulence of Xanthomonas oryzae pv. oryzae by Preventing Cell Aggregation.

Bacterial blight of rice is an important serious bacterial diseases of rice in many rice-growing regions, caused by Xanthomonas oryzae pv. oryzae (Xoo). The thiG gene from Xoo strain ZJ173, which is involved with thiazole moiety production in the thiamine biosynthesis pathway, is highly conserved among the members of Xanthomonas. The thiG deletion mutant displayed impaired virulence and growth in thiamine-free medium but maintained its normal growth rate in the rice tissues, indicating that the thiG gene is involved in Xoo virulence. Compared to the wild type strain, the formation of cell-cell aggregates was affected in thiG deletion mutants. Although biofilm formation was promoted, motility and migration in rice leaves were repressed in the thiG mutants, and therefore limited the expansion of pathogen infection in rice. Quorum sensing and extracellular substance are two key factors that contribute to the formation of cell-cell aggregates. Our study found that in the thiG mutant the expression of two genes, rpfC and rpfG, which form a two-component regulatory signal system involved in the regulation of biofilm formation by a second messenger cyclic di-GMP is down-regulated. In addition, our study showed that xanthan production was not affected but the expression of some genes associated with xanthan biosynthesis, like gumD, gumE, gumH and gumM, were up-regulated in thiG mutants. Taken together, these findings are the first to demonstrate the role of the thiazole biosynthsis gene, thiG, in virulence and the formation of aggregates in Xanthomonas oryzae pv. oryzae.