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Xanthomonas oryzae pv. oryzae ( Xoo) is a Gram-negative bacterium that causes bacterial leaf blight in rice. In this study, we identified a putative TrpR-like protein, PXO_TrpR (PXO_00831), in Xoo. This protein contains a tryptophan (Trp) repressor domain and is highly conserved in Xanthomonas. Auxotrophic assays and RT-qPCR confirmed that PXO_TrpR acts as a Trp repressor, negatively regulating the expression of Trp biosynthesis genes. Pathogenicity tests showed that PXO_trpR knockout in Xoo significantly reduced lesion development and disease symptoms in the leaves of susceptible rice. RNA-seq analysis and phenotypic tests revealed that the PXO_trpR mutant exhibited impaired cell motility and was more sensitive to H2O2 oxidative stress than the wild-type strain. Furthermore, we found that the sigma 70 factor RpoD controlled the transcription of PXO_trpR by directly binding to its promoter region. This study demonstrates the biological function and transcriptional mechanism of PXO_TrpR as a Trp repressor in Xoo and evaluates its novel pathogenic roles by regulating flagellar motility and the oxidative stress response.

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the top ten bacterial plant diseases worldwide. Serotonin N-acetyltransferase (SNAT) is one of the key rate-limiting enzymes in melatonin (MT) biosynthesis. However, its function in pathogenic bacteria remains unclear. In this study, a Xoo SNAT protein (xoSNAT3) that showed 27.39% homology with sheep SNAT was identified from a collection of 24 members of GCN5-related N-acetyltransferase (GNAT) superfamily in Xoo. This xoSNAT3 could be induced by MT. In tobacco-based transient expression system, xoSNAT3 was found localized on mitochondria. In vitro studies indicated that xoSNAT3 showed the optima enzymatic activity at 50 °C. The recombinant enzyme showed Km and Vmax values of 709.98 μM and 2.21 nmol/min/mg protein, respectively. Mutant △xoSNAT3 showed greater impaired MT biosynthesis than the wild-type strain. Additionally, △xoSNAT3 showed 14.06% less virulence and 26.07% less biofilm formation. Collectively, our results indicated that xoSNAT3 services as a SNAT involved in MT biosynthesis and pathogenicity in Xoo.

Protein glycosylation is an essential process that plays an important role in proteome stability, protein structure, and protein function modulation in eukaryotes. However, in bacteria, especially plant pathogenic bacteria, similar studies are lacking. Here, we investigated the relationship between protein glycosylation and pathogenicity by using Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight in rice, as a well-defined example. In our previous work, we identified a virulence-related hypothetical protein, PXO_03177, but how this protein regulates X. oryzae pv. oryzae virulence has remained unclear. BLAST analysis showed that most homologous proteins of PXO_03177 are glycoside hydrolase family 99-like domain-containing proteins. In the current study, we found that the outer membrane integrity of ΔPXO_03177 appeared to be disrupted. Extracting the outer membrane proteins (OMPs) and performing comparative proteomics and sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel staining analyses revealed that PXO_03177 deletion altered the protein levels of 13 OMPs. Western blot analyses showed that the protein level and glycosylation modification of PXO_02523, a related OmpA family-like protein, was changed in the ΔPXO_03177 mutant background strain. Additionally, the interaction between PXO_03177 and PXO_02523 was confirmed by coimmunoprecipitation. Both PXO_03177 and PXO_02523 play important roles in regulating pathogen virulence and viability in stressful environments. This work provides the first evidence that protein glycosylation is necessary for the virulence of plant pathogenic bacteria.

Bacteria can withstand various types of environmental osmostress. A sudden rise in osmostress affects bacterial cell growth that is countered by activating special genes. The change of osmostress is generally a slow process under the natural environment. However, the collective response of bacteria to low osmostress remains unknown. This study revealed that the deletion of

N6-methylated adenine (m

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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.

Lysobacter enzymogenes OH11 is currently considered to be a novel biocontrol agent for various plant fungi diseases. At present, only heat-stable antifungal factor (HSAF) has been isolated and identified in culture, although other active compounds also showed antifungal activity. In the present study, a novel active compound, alteramide B (ATB), which exhibits broad-spectrum antagonistic activity against phytopathogenic fungi and oomycetes, was isolated. The genes responsible for ATB biosynthesis were also determined. In addition, a strain producing ATB with minimal HSAF production was successfully generated by redirecting metabolic flux, namely L. enzymogenes OH57. Furthermore, ATB production increased to 893.32 ± 15.57 mg/L through medium optimization and precursor supply strategy, which was 24.36-fold higher than that of 10% tryptic soy broth (36.67 ± 1.63 mg/L). Taken together, this study indicates ATB has great development value as a biopesticide because of its bioactivity and high production.

Protein glycosylation is an essential process that plays an important role in proteome stability, protein structure, and protein function modulation in eukaryotes. However, in bacteria, especially plant pathogenic bacteria, similar studies are lacking. Here, we investigated the relationship between protein glycosylation and pathogenicity by using

Background Anthracnose caused by Colletotrichum fructicola is one of the most important diseases in pear fruit, resulting in huge economic losses. Public awareness of protecting the environment and food safety, together with pathogen resistance to many key fungicides have led to an urgent need to develop alternative strategies for controlling fruit diseases. Here, the antifungal activity of a natural product, dihydromaltophilin [heat-stable antifungal factor (HSAF)], against C. fructicola in vitro and in vivo was investigated to determine its efficacy for anthracnose management. Results HSAF exhibited pronounced antifungal activity against in vitro mycelial growth of C. fructicola, with a half-inhibition concentration of 0.43 mg L-1 . Hyphae treated with HSAF showed defects such as hyperbranching, swelling and depolarized growth. Conidia germination in the pathogen was inhibited by HSAF in a dose-dependent manner. In the presence of 4 mg L-1 HSAF, conidia germination was significantly delayed, and germ tube growth was inhibited. HSAF at 8 mg L-1 completely blocked conidia germination in C. fructicola. In addition, HSAF disrupted coordination of cytokinesis with growth and nuclear division, induced reactive oxygen species production in conidia, and damaged the integrity of the conidia cell wall. Moreover, an in vivo test confirmed that 50 mg L-1 HSAF significantly reduced the development of anthracnose decay in pear fruit caused by C. fructicola. Conclusion HSAF was highly effective in reducing pear anthracnose caused by C. fructicola and has great potential to become a new type of fruit preservative.

Lysobacter species are emerging as novel sources of antibiotics, but the regulation of their physiological metabolism is still poorly understood. In this work, we extracted AHL (acyl-homoserine lactone) autoinducers, identified the structures of AHLs and described the AHL quorum-sensing system in Lysobacter brunescens OH23. AHLs were isolated from the supernatant of L. brunescens OH23, and ESI-MS/MS (electrospray ionization mass spectrometry) analysis revealed biosynthesis of three different AHL chemical structures by L. brunescens OH23: N-(3-oxohexanoyl)- homoserine lactone (HSL), 3-OH-C10-HSL and C8-HSL. The growth rate of AHL quorum-sensing knockout mutants was dramatically increased compared to that of wildtype. Sucrose consumptions were also twice as high in AHL quorum-sensing knockout mutants than that in wildtype in early-log phase. Additionally, expression of key genes related to sucrose metabolism α-glucosidase was enhanced in AHL quorum-sensing knockout mutants, which indicated that AHL quorum sensing negatively regulates sucrose uptake and metabolism which further affects the growth rate of L. brunescens. Furthermore, autolysis was strongly induced in AHL quorum-sensing knockout mutants compared to wildtype, suggesting that AHL quorum sensing plays a negative regulatory role in cell autolysis. Moreover, compared to wildtype, XSAC (Xanthomonas-specific antibiotic compound) production was significantly increased in AHL knockout mutants in the early-log and late-log phases, and surface motility capabilities are also enhanced also in AHL knockout mutants; the normalized data of XSAC production and surface motility and expressions of key genes related to these two phenotypes reveal that growth rare and autolysis strongly affects XSAC biosynthesis and surface motility rather than AHL quorum-sensing system. Our results show that the AHL quorum-sensing system negatively regulates cell growth and autolysis, and further maintain nutrition homeostasis and population stability in L. brunescens.

Lactococcus lactis subsp. lactis is a pathogenic bacterium causing postharvest decay of the cultivated mushroom Pleurotus eryngii, whose pathogenic mechanism is little known. Sequencing of its complete genome is a prerequisite for revealing the molecular mechanism of infection. In this research, the complete genome of SLPE1-3 was obtained using the Single Molecular Real Time (SMRT) sequencing strategy. The genome was analyzed both structurally and functionally. The complete genome of SLPE1-3 consists of a single, circular chromosome (2,522,493 bp; 34.91% GC content) without any plasmid. The results showed the feasibility and superiority of SMRT in bacterial complete-genome research. The genome of SLPE1-3 has the specific features of L. lactis subsp. lactis not just in the phylogenesis and genome structure, but also in functional classification. Compared with L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris MG1363 and L. lactis subsp. lactis KF147, 23 peculiar genes were identified in SLPE1-3 which were involved in lipid metabolism, cell wall biogenesis and some functional enzymes. In addition, 37 potential genes relating to antifungal function were filtered for further mechanism research.

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.

Additional file 1: Table S1. Primers used for RT-PCR to detect the mRNA expressions of cell division related genes.

Bacillus amyloliquefaciens BGP20 is a promising antagonist in controlling post-harvest bacterial soft rot of vegetables caused by Erwinia carotovora subsp. carotovora (Ecc). The objective of this study was to screen a kind of natural and safe additive which could enhance the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper. The results of this study indicated that the additive natamycin had stronger inhibition against the pathogen Ecc compared with bamboo vinegar and chitosan in the 2× Yeast extract and Tryptone (2YT) medium. However, natamycin had a slight negative effect on the growth of BGP20 in the 2YT medium. In preventative treatments, natamycin significantly improved the bio-efficacy of BGP20, and enhanced its competitive position against Ecc in the wounds of green pepper. Compared with the treatment with BGP20 alone, the viable count of BGP20 after 72 h of incubation increased by 115.8% in the wounds of green pepper treated with BGP20 and 0.1% natamycin...

Background Heat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has reported on the fermentation process involved, representing huge obstacles for large-scale industrial production. Results Medium capacity, culture temperature, and fermentation time were identified as the most significant factors affecting the production of HSAF and employed for further optimization through statistical methods. Based on the analysis of kinetic parameters at different temperatures, a novel two-stage temperature control strategy was developed to improve HSAF production, in which the temperature was increased to 32 °C during the first 12 h and then switched to 26 °C until the end of fermentation. Using this strategy, the maximum HSAF production reached 440.26 ± 16.14 mg L− 1, increased by 9.93% than that of the best results from single-temperature fermentation. Moreover, the fermentation time was shortened from 58 h to 54 h, resulting in the enhancement of HSAF productivity (17.95%) and yield (9.93%). Conclusions This study provides a simple and efficient method for producing HSAF that could be feasibly applied to the industrial-scale production of HSAF. Electronic supplementary material The online version of this article (10.1186/s12896-018-0478-2) contains supplementary material, which is available to authorized users.

Alternaria alternata (Fries) Keissler is a lethal pear pathogen that causes leaf black spot disease of pear in Southern China. Heat-stable activity factor (HSAF) is a polycyclic tetramate macrolactam (PTM) produced by Lysobacter enzymogenes and many other microbes with a broad-spectrum antifungal activity against many filamentous fungi. In this study, we evaluated the antifungal effect of HSAF against A. alternata and proposed its antifungal mechanism in A. alternata. We report that HSAF inhibited the mycelial growth of A. alternata in a dose-dependent manner. Transcriptomics analysis revealed that HSAF treatment resulted in an expression alteration of a wide range of genes, with 3729 genes being up-regulated, and 3640 genes being down-regulated. Furthermore, we observed that HSAF treatment disrupted multiple signaling networks and essential cellular metabolisms in A. alternata, including the AMPK signaling pathway, sphingolipid metabolism and signaling pathway, carbon metabolism and the TCA (tricarboxylic acid) cycle, cell cycle, nitrogen metabolism, cell wall synthesis and a key hub protein phosphatase 2A (PP2A). These observations suggest that HSAF breaches metabolism networks and ultimately induces increased thickness of the cell wall and apoptosis in A. alternata. The improved understanding of the antifungal mechanism of HSAF against filamentous fungi will aid in the future identification of the direct interaction target of HSAF and development of HSAF as a novel bio-fungicide.

Valsa pyri, an ascomycete pathogen that is a member of the Valsaceae family (Sordariomycetes, Diaporthales), which causes pear or apple canker and leads to tree death and massive yield losses. Here, we selected two V. pyri isolates (Vp14 and Vp297) that exhibited different invasion abilities for transcriptomics analyses. Compared toVp297, Vp14 had stronger virulence and spread faster on host-like nutrients. Four samples, including mycelium or infectious mycelium, of the two isolates were sequenced. Clean reads were mapped to the V. pyri genome, and 12490 transcripts and 178 new genes were identified. There were dramatically fewer differentially expressed genes (DEGs) in Vp14 than in Vp297. According to GO and COG annotations, there were both more up- and down-regulated genes in Vp297 than in Vp14 except for genes involved in amino acid transport and metabolism, carbohydrate transport and metabolism, peroxidases and so on. Specific up-regulated DEGs, including genes encoding cell wall degrading enzymes and genes involved in nitrogen metabolism and peroxidases which play crucial roles in virulence and infectious growth, were especially enriched inVp14. These results indicate that the Vp14 isolate may infect its host and take up nutrition more efficiently, reflecting a stronger ability for invasion or infectious growth. Our analysesindicate that a successful V. pyri infection involves multiple instances of transcriptome remodeling to regulate gene functions. Comparative transcriptomics between isolates of V. pyri may aid in our understanding of the virulence mechanism of this pathogen.

Table S1. Experimental design and responses of PBD. Table S2. Experimental design and responses of BBD. Table S3. Regression analysis of BBD. Figure S1. Response surface curves showing the interface of the medium capacity, culture temperature, and fermentation time. (DOCX 667 kb)

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.

Lactic acid bacteria (LAB) are a group of important beneficial microorganisms for human, but their growth is restricted to the habitats with rich nutrients. In order to develop a simple, low-cost and efficient medium based on the mushroom Pleurotus eryngii, this study evaluated the effects of different treatment methods for the mushroom, concentration of the mushroom, buffers, tween 80, MgSO4·7H2O, MnSO4·4H2O, CuSO4·5H2O, riboflavin and ascorbic acid on the growth of Lactococcus lactis subsp. lactis SLPE1-3. An optimized medium was developed, which was composed of the mushroom at 200 g/L, the buffer sodium acetate at 5 g/L, and riboflavin at 0.5 mg/L. The mushroom was ground, boiled and filtered for the filtrate in advance. In this optimized medium which was named as PSR medium, the population density of SLPE1-3 sharply reached 2.13 × 10(9) CFU/mL within 18 h of incubation, and still maintained 1.17 × 10(8) CFU/mL at 120 h. In addition, this study found that 6 kinds of LAB could grow almost well, and maintained high survival in PSR medium compared to M17 or MRS medium, including Lactococcus lactis subsp. lactis, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, Lactobacillus paracasei, Pediococcus pentosaceus and Lactobacillus rhamnosus. These results showed that PSR medium was a simple, low cost and eurytopic medium for the cultivation of LAB, and could replace MRS or M17 medium in the food industry, biomedicine and laboratory.

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.

To elucidate the biological functions of a two-component system RpfCxoc/RpfGxoc in Xanthomonas oryzae pv. oryzicola (Xoc).Based on the genome template from Xoc wild-type strain Rs105, the rpfCxoc and rpfGxoc genes were amplified by polymerase chain reaction (PCR). The in-frame deletion mutations of rpfCxoc, rpfGxoc and rpfGCxoc (rpfCxoc and rpfGxoc double genes) were performed by the suicide vector pK18mobsacB, and determined diffusible signal factor (DSF) biosynthesis, pathogenicity in host rice, biofilm, extracellular polysaccharide (EPS) production and cell morphology.rpfCxoc and rpfGxoc were cloned from the genomic DNA of Rs105. PCR analysis demonstrated that the rpfCxoc, rpfGxoc and rpfGCxoc genes were in-frame deleted successfully. Compared to the wild-type strain Rs105, DSF were overproduced in deltarpfCxoc and deltarpfGCxoc, but DSF production was remarkably decreased in deltarpfGxoc. The DSF production of these mutants was restored by introducing the complemented cosmid pUFR-rpfCxoc, pUFR-rpfGxoc and pUFR-rpfGCxoc, respectively. Subsequent experimental results indicated that mutation of rpfCxoc, rpfGxoc and rpfGCxoc resulted in pathogenicity loss of Xoc in host rice, and decreased biosynthesis level of EPS at 34.1%-48.5% compared to that of Rs105. In L medium (Tryptoen, 10 g/L; yeast extract, 5 g/L; sodium chloride, 5 g/L; D-glucose, 1 g/L; pH7.0) , Rs105 was growing at planktonic pattern, but the mutation of rpfCxoc and rpfGxoc led to Xoc cell aggregation at the wall of the flaks at the air-liquid interfaces, and deltarpfGxoc generated reticulation biofilm at the bottom of the flaks. But deltarpfGCxoc only generated reticulation biofilm at the bottom of the flaks.The two-component system RpfCxoc/RpfGxoc modulated DSF biosynthesis, EPS production and biofilm dispersal of Xoc, which was required for the pathogenicity of Xoc in host rice.

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.