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Background Xanthomonas 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. Results Using 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. Conclusion Our 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.

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.

Xanthomonas oryzae pv. oryzicola causes leaf streak disease of rice. The gene hshB is a newly identified virulence-associated gene that is co-regulated by diffusible signal factor signaling and global regulator Clp in X. oryzae pv. oryzicola. Our previous study showed that mutation of hshB remarkably impaired the virulence, extracellular protease activity, extracellular polysaccharide production and resistance to oxidative stress of X. oryzae pv. oryzicola. In this study, the regulatory role of hshB in X. oryzae pv .oryzicola was expanded. Results showed that hshB was also required for cell swimming motility. Transcriptome analysis showed that 305 genes were significantly differentially expressed after deletion of hshB in X. oryzae pv. oryzicola. Further analysis of transcriptome data indicated that the differentially expressed genes focused on two aspects: namely, cell motility and cell signal transduction. This finding strongly identified the closely related function of hshB to cell motility and signal transduction. In addition, the mutation of hshB of X. oryzae pv. oryzicola enhanced biofilm formation. Collectively, the study showed novel functions of gene hshB in cell motility and biofilm formation by transcriptome analysis, thus expanding our understanding of the roles of gene hshB in the pathogenic X. oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzae (Xoo) causes rice bacterial blight (BB), one of the most widespread and destructive diseases in rice-growing regions worldwide. Melatonin enhances pathogen resistance by inducing plant innate immunity, but the direct effect of melatonin on plant pathogenic bacteria is poorly understood. In this study, we investigated the direct effects of melatonin on Xoo. Exogenous melatonin at 200 μg/mL significantly inhibited the proliferation of Xoo and reduced the mRNA expression of five genes involved in cell division. This concentration of melatonin also inhibited the motility and biofilm formation of Xoo. Notably, melatonin was observed to alter the length of Xoo cells. To provide deeper insights into the mechanisms underlying this antibacterial activity, we examined global gene expression changes in Xoo strain PXO99 in response to the application of 200 μg/mL melatonin using RNA sequencing (RNA-Seq). A wide range of differentially expressed genes (DEGs) related to catalytic activity and metal-binding activity were downregulated in Xoo cells in response to the melatonin treatment. In addition, DEGs responsible for carbohydrate and amino acid metabolism were also downregulated. These results suggest that the inhibitory mechanism of melatonin on Xoo proliferation may involve the regulation of cell division in combination with a reduction in the concentration or activity of enzymes involved in metabolism.

Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae are two pathovars of X. oryzae that cause leaf streak and blight in rice, respectively. These two bacterial pathogens cause different disease symptoms by utilizing different infection sites on rice. Compared with X. oryzae pv. oryzae, the molecular virulence mechanism of X. oryzae pv. oryzicola remains largely unknown. Previously, we identified a unique diffusible signal factor (DSF)-controlled virulence-related gene (hshB) in X. oryzae pv. oryzicola Rs105 located in the nodB-rghB locus, which is absent in X. oryzae pv. oryzae PXO99A. In the present study, we identified two additional genes within this locus (hshA and hshC) that were unique to X. oryzae pv. oryzicola Rs105 compared with X. oryzae pv. oryzae PXO99A, and we found that the transcription of these genes was regulated by DSF signaling in X. oryzae pv. oryzicola. The mutation of these genes impaired the virulence of the wild-type Rs105 when using a low inoculation density of X. oryzae pv. oryzicola. In contrast to hshB, the mutation of these genes did not have any visible effect on characterized virulence-related functions, including in vitro growth, extracellular polysaccharide production, extracellular protease activity, and antioxidative ability. However, we found that mutation of hshA or hshC significantly reduced the in planta growth ability and epiphytic survival level of X. oryzae pv. oryzicola cells, which was the probable mechanisms of involvement of these two genes in virulence. Collectively, our studies of X. oryzae pv. oryzicola have identified two novel DSF-controlled virulence-associated genes (hshA and hshC), which will add to our understanding of the regulatory mechanisms of conserved DSF virulence signaling in Xanthomonas species.

Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular proteins controlled by DSF signal remain largely unclear. In the present study, 33 DSF-regulated extracellular proteins, whose functions include small-protein mediating QS, oxidative adaptation, macromolecule metabolism, cell structure, biosynthesis of small molecules, intermediary metabolism, cellular process, protein catabolism, and hypothetical function, were identified by proteomics in Xoc. Of these, 15 protein encoding genes were in-frame deleted, and 4 of them, including three genes encoding type II secretion system (T2SS)-dependent proteins and one gene encoding an Ax21 (activator of XA21-mediated immunity)-like protein (a novel small-protein type QS signal) were determined to be required for full virulence in Xoc. The contributions of these four genes to important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, and antioxidative ability, are presented. To our knowledge, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xanthomonas.

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.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, a serious bacterial disease of rice in Asia and parts of Africa. The virulence mechanisms of Xoc are not entirely clear and control measures for BLS are poorly developed. The solo LuxR proteins are widespread and shown to be involved in virulence in some plant associated bacteria (PAB). Here, we have cloned and characterized a PAB LuxR solo from Xoc, named as XocR. Mutation of xocR almost completely impaired the virulence ability of Xoc on host rice, but did not alter the ability to trigger HR (hypersensitive response, a programmed cell death) on non-host (plant) tobacco, suggesting the diversity of function of xocR in host and non-host plants. We also provide evidence to show that xocR is involved in the regulation of growth-independent cell motility in response to a yet-to-be-identified rice signal, as mutation of xocR impaired cell swimming motility of wild-type Rs105 in the presence but not absence of rice macerate. We further found that xocR regulated the transcription of two characterized virulence-associated genes (recN and trpE) in the presence of rice macerate. The promoter regions of recN and trpE possessed a potential binding motif (an imperfect pip box-like element) of XocR, raising the possibility that XocR might directly bind the promoter regions of these two genes to regulate their transcriptional activity. Our studies add a new member of PAB LuxR solos and also provide new insights into the role of PAB LuxR solo in the virulence of Xanthomonas species.

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.

Xanthomonas oryzae pv. oryzicola (Xoc), which caused bacterial leaf streak in rice, is a bacterial pathogen limited to the apoplast of the mesophyll tissue. The rpfF that encodes diffusible signal factor (DSF) synthase, played a key role in the virulence of many plant pathogenic bacteria. In this study, the rpf gene cluster was cloned, and the rpfF was deleted in Xoc. It was observed that the rpfF mutant lost the ability to produce DSF molecular, and exhibited a significant reduction of virulence in rice compared to the wild-type strain. Furthermore, the mutation of rpfF impaired EPS production, and led to Xoc cell aggregation. To analyze the differences of proteome expression between Xoc wild type and rpfF mutant, a comparative proteome analysis was performed by two-dimensional gel electrophoresis (2-DE). The results clearly revealed that 48 protein spots were differentially expressed above the threshold ratio of 1.5. Among them, 18 proteins were identified by MS, which were involved in nitrogen transfer, protein folding, elimination of superoxide radicals and flagellar formation. Our results indicated that DSF might play an important role in virulence and growth of Xoc by mediating expression of proteins.