Identification And Functional Analysis Of Pathogenicity-Related Genes Of Xanthomonas Oryzae Pv. Oryzicola

Xanthomonas oryzae pv. oryzicola （Xoc） is one of two pathovars in Xanthomonas oryzae, causing serious bacterial leaf streak （BLS） in rice which becomes the fourth destructive rice diseases recently in China. The pathogen enters through leaf stomata or wounds and colonizes the parenchyma apoplast, causing interveinal lesions that appear as water soaked initially and then develop into translucent, yellow-to-white streaks. As the susceptible hybrid rice grows in large scale and particularly there is no simply inherited gene for resistance to the disease, rice production encounters a serious threat of security. In recent years, a number of pathogenicity-related genes, such as hrp, avr, rpf and other candidate virulence genes, have been discovered in X. oryzae pv. oryzicola, suggesting that genome-widely identification of virulence genes is necessary for fully understanding the bacterial pathogenicity and for developing biotechnology to control the disease.Currently, it is accepted that construction of a Tn5-tagged mutant library is an effective approach to mine pathogenicity-related genes at functional genomic level in various plant pathogens. Therefore, to genome-widely mine pathogenesis-related genes of X. oryzae pv. oryzicola, a Tn5transposon-mediated mutation library was generated using the wild-type RS105as a recipient in this study. Twenty-five thousand transformants were produced by inserting the Tn5transposon, appropriately corresponding to5×ORF coverage of the genome. Southern Blot analysis suggested that Tn5-mediated mutant library is stable and random. Each transformant of the library is inoculated into susceptible host rice （cv. Shanyou63） to screen candidate virulence genes. Totally, in comparison to the wild-type, there were1,753mutants with virulence reduced or lost after the first screening. In order to accurately confirm the phenotypes achieved, we re-inoculated those mutants into rice seedlings. After the second screening,114mutants were obtained with virulence reduced or lost compared to the wild-type. Of those, there were14mutants completely losing pathogenicity in rice. Finishing the virulence assay in rice, we then injected114mutants into tobacco （cv. Xanthi） for HR induction assay. There were23 mutants with the ability of HR induction reduced or lost, compared to the wild-type RS105. Of these, there were15mutants completely losing the ability to trigger HR in tobacco. The mutants identified above may provide a basis for analysis of pathogenesis-related genes in X. oryzae pv. oryzicola.In order to rapidly and accurately identify Tn5insertion positions of corresponding genes in each mutants of this library, thermal asymmetric interlaced PCR （TAIL-PCR） was adopted to isolate Tn5-tagged genes of114mutants. The results suggest that random insertions of Tn5targets set at a large number of genes associated with the pathogenicity, including hrp, avr, rpf, wxoc, metabolism-related genes, genes encoding conserved hypothetical proteins, and other known or unknown virulence factors. Novel pathogenicity-related genes were revealed that they play important roles in pathogenesis of X. oryzae pv. oryzicola in rice at molecular level.Identification of key virulence genes/factors is prerequisite for understanding pathogenesis of X. oryzae pv. oryzicola in rice. Therefor,27Tn5insertional mutants that either completely lost pathogenicity or reduced virulence in rice were thorough analysed. Specifically,14mutants completely lost pathogenicity in rice and the ability to trigger HR in tobacco （classified as Pth-/HR-） and13reduced the full virulence in rice but kept HR induction in tobacco （Vir-/HR+）. Sequencing analysis of the Tn5-tagged genes indicated that these14Pth-/HR-mutants include hrcC （2mutants）, hrcT, hrcV （4mutants）, hpaP, hrcQ （2mutants）, hrpF, hrpG and hrpX （2mutants） genes. The13Vir-/HR+mutants include tal-C10c-like （encodes a TAL effector）, rpfC （a regulator of pathogenicity factors）, oxyP （an oxidative stress transcriptional regulator）, dsbC （a disulfide isomerase）, opgH （a glucans biosynthesis glucosyltransferase H）, rfbA （a glucose-1-phosphate thymidylyltranssferase）, amtR （an aminotransferase）, purF （an amidophosphoribosyl-transferase）, thrC （a threonine synthase）, trpA （a tryptophan synthase alpha subunit）, as well as3hypothetical protein coding genes Xoryp₀2235, Xoryp₀0885and Xoryp₂2910. Together, these27Tn5insertions are located in21different open reading frames （ORFs）. Reduced virulence by the thirteen mutants above was complemented to the wild-type level in trans by the present of the corresponding wild-type genes above. These results suggested that amtR, purF, thrC, trpA, Xoryp₀2235, Xoryp₀0885and Xoryp₂2910are novel virulence genes involved in X. oryzae pv. oryzicola pathogenesis in rice. Real-time PCR demonstrated that the seven novel virulence genes were significantly （P=0.01, t test） induced in planta, although their precise functions during X. oryzae pv. oryzicola pathogenesis await further investigation.One important aspect of interactions between plant pathogens and their hosts is the ability of the pathogen to obtain nutrients within the plant tissue. The ability to acquire carbohydrate is essential for a pathogen to propagate and establish an infection relationship in host plant successfully. Although metabolic pathways are generally not considered to be virulence factors, elucidation of the mechanism to acquire and metabolize carbohydrates is critically important for fully understanding the pathogenicity of X. oryzae pv. oryzicola.Fructose-bisphophate aldolase （FbaB）, is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of X. oryzae pv. oryzicola, led the pathogen not only to be unable to use pyruvate and malate for growth and delay its growth when fructose was used as the sole carbon source, but also to reduce extracellular polysaccharide （EPS） production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box （plant-inducible promoter）（TTCGT-N9-TTCGT）. The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, real-time PCR suggested that the expression of fbaB in X. oryzae pv. oryzicola RS105strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in R△fbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.In addition, two genes encoding glucose6-phosphate dehydrogenase, which catalyzes glucose6-phosphate into gluconate6-phosphate and plays an important role in Entner-Doudoroff pathway （ED pathway） and pentose phosphate pathway, were identified from the mutant library of X. oryzae pv. oryzicola. In order to clarify the functions of these two genes, we took zwf （Xoryp₁2765） as our target since it is reported that it plays a dominant role in the pathways above in Xanthomonas species. The deletion mutation in zwf of X. oryzae pv. oryzicola could significantly reduce the ability of pathogen to use glucose, fructose, sucrose, mannose and galactose for growth other than pyruvate and malate. These suggest that the zwf mutation may hinder the pentose phosphate pathway and ED pathway instead of glycolysis. Simultaneously, the expression of zwf was strongly induced by glucose, sucrose, fructose, mannose and galactose at least3times higher than that by the medium without sugar. Interestingly, The mutagenesis in this unique zwf gene of X. oryzae pv. oryzicola also led the alteration in the expression of key genes in DSF signaling pathway, such as rpfF, rpfG and clp, suggesting that zwf may be involved in regulation of virulence factors in the downstream of DSF signaling. In addition, the deletion mutation in zwf resulted in impairment of bacterial virulence in planta, and reduction of motility and extracellular polysaccharide （EPS）, but enhanced the activity of extracellular protease. Bacterial virulence and motility and EPS production in RAzwf mutant were completely restored to the wild-type level by the presence of zwf in trans. All these results indicate that zwf is required for the full virulence of X. oryzae pv. oryzicola in rice.Glucose6-phosphate isomerase, reversibly converting glucose6-phosphate to fructose6-phosphate, plays an important role in carbohydrate metabolism. There is only one Xoryp₁0540（pgi） gene, encoding6-phosphate glucose isomerase, in the genome of X. oryzae pv. oryzicola. The mutagenesis in pgi led the pathogen unable to use fructose, sucrose, mannose and pyruvate for growth effectively, but did not affect its growth when glucose or galactose as the sole carbon source. It is worth mentioning that the mutation in pgi also resulted in alteration of key genes in DSF signaling pathway, such as rpfF, rpfG and clp. Furthermore, detection of DSF signal showed the mutant significantly reduced DSF production compared to the wild-type RS105. These results indicated that, the mutation in pgi may have impacts on the downstream of DSF signal regulatory. Similarly, the deletion in pgi not only impaired bacterial virulence in planta, but also reduced bacterial motility and EPS production. Bacterial virulence and motility and EPS production in RApgi mutant were completely restored to the wild-type level by the presence of pgi in trans. All these results indicate that pgi is essential for EPS production and full virulence of X. oryzae pv. oryzicola.Xanthomonas oryzae pv. oryzae （Xoo） is another pathovar of Xanthomonas oryzae that also possesses a type-Ⅲ secretion system （T3SS） to deliver repertoires of T3SS effectors into plant cell to cause serious bacterial blight （BB） in rice. The pathogen invades rice leaves through hydathode openings on leaf tip and leaf margin, and then colonizes the vascular tissues by propagating in the xylem. Based on microarray data in our laboratory, an energy metabolism related-gene, PXO₀3531（ketoglutarate transport protein, kgtP）, in X. oryzae pv. oryzae was found that it was remarkably downregulated in hrpX or hrpG mutant. Bioinformatic analysis revealed that there is an imperfect PIP-box （TTCGA-N21-TTCGC） in the promoter region of kgtP and the first50amino acids of N-terminal in KgtP possesses a typical T3SS signal. All the above indicated that kgtP may be a HrpX regulon and a T3SS effector in X. oryzae pv. oryzae. RT-PCR assay demonstrated that the kgtP expression was positively regulated by the HrpX and HrpG cassette. Southern Blot assay indicated that the KgtP was secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99A strain. Subcellular localization showed that KgtP could be located into plant cell membrane, suggesting that it binds to plant cell membrane to transport a-ketoglutarate from plant cells into bacterial cells. The deletion mutation in kgtP reduced not only bacterial virulence and growth in planta, but also the growth in a minimal medium without a-ketoglutaric acid or sodium succinate. The reduced virulence and the impairment of a-ketoglutaric acid utilization in the kgtP mutant were restored to the wild-type levels by the presence of kgtP in trans. The kgtP expression was strongly induced when the pathogen either grew in the minimal medium supplemented with a-ketoglutaric acid or interacted with rice cells, suggesting the expression of kgtp is induced. Importantly, the expression of OsIDH, which is responsible for the synthesis of a-ketoglutaric acid in rice, was enhanced when kgtP presents in the pathogen. Taken together, we hypothesize that KgtP transports a-ketoglutaric acid to X. oryzae pv. oryzae from outside substrates for saprophytic growth and from rice cells for parasitism.