Identification And Analysis Of Pathogenicity-related Genes Of Xanthomonas Campestris Pv. Campestris

Xanthomonas campestris pv. campestris(here after Xcc) is the causal agent of the black rot disease of cruciferous plants. The pathogenesis of Xcc involves seven steps including adhering, recognition, penetration, multiplication, colonization, spreading and symptom displaying. It has been known that many pathogenicity genes are involved in the pathogenesis processes of this pathogen. Daniels predicted that the number of pathogenicity genes of Xcc might be 20-100, and more than 250 pathogenicity-related genes were annotated in Xcc8004 genome. Up to date, about 60 pathogenicity-related genes has been identified in Xcc. This information implies that there are many pathogenicity-related genes have not been identified. Generally, three strategies are used for the identification of pathogenicity-related gene: (i) genome-wide mutagenesis followed by virulence tests;(ii) differential expression of genes, useing DNA microarray or GUS reporter to find genes that is only expressed in the host or host-similar conditions;(iii) bioinformatic method, useing the sequence of a targeted gene to blast the NCBI Database to determine the similarity of the target sequence to that of any pathogenicity genes reported in other pathogen. In this study, we used method (i) and (iii) to identify pathogenicity-related genes from Xcc. A genome-wide Tn5gw.sA5 insertional mutant library has been constructed in our lab, and many virulence-reduced mutants have been screened. In this study, we use marker exchange method to reconstruct the mutant of 10 virulence-reduced mutants, and confirmed that only two mutants in which the reduced virulence is actually caused by the Tn5gusA5 insertion. Through further confirmation by non-polar mutant construction and mutant complementation, finally we demonstrated that hgiA and gpdA are pathogenicity-related genes in Xcc. Moreover, we found the homolog of the pathogenicity gene rsmA of Erwinia carotovora csrA is exists in Acc8004 genome;and we further found that the disruption mutant of this gene is unable to cause disease in host plant Chinese radish.hgiA encodes an putative protein belonging to the MarR family transcriptional regulator, which contains a helix-turn-helix motif. MarR was first identified as a regulator of multiple antibiotic resistances in Escherichia coli, and it has been reported that marR family transcriptional regulator played important role in the pathogenesis of several animal or plant pathogens such as Salmonella typhimurium, Yersinia pseudotuberculosis and Erwinia carotovora. In Xcc, the virulence of the non-polar mutant of hgiA on Chinese radish is significant decreased compared to that of wild type strain when inoculation by clipping method with cell concentration of OD₆₀₀ = 0.1. 10 days postinoculation, the lesion length caused by hgiA mutant is 3.25mm, while the lesion length caused by the wild type strain is 12.5mm, and the reduced virulence of the hgiA mutant can be restored by wild type hgiA in trans. This result indicated that hgiA is required for the full virulence of Xcc. We test the virulence of hgiA mutant on different cultivars of cruciferous crops and found that the mutant is unable to cause disease on Chinese cabbageJianChun (Brassica campestris var.JianChun) , Chinese cabbage ZhongBai78 (Brassica campestris var.ZhongBai78) , and Chinese cabbage JingFengNo.l {Brassica campestris var. JingFengNol), but still can cause symptom on radish Huaye (Raphanus sativus L. var. radiculus HuaYe) , Radish BanYeHongPao {Raphanus sativus L. var. radiculus BanYeHongPao) , Radish XianHongXiaoJinZhong(i?ap/ja?w5 sativus L. var. radiculus XianHongXiaoJinZhong), Cabbage WanFeng (Brassica oleracea L. var. WanFeng) and Chinese cauliflower QiuWang70 (Brassica oleracea var. QiuWang70) . Although mutation in hgiA resulted in the reduction in virulence, the growth rate in rich or minimal medium and the production of virulence factors (EPS, extracellular protease, extracellular endoglucannase and extracellular amylase) is not affected. In order to investigate the regulation of hgiA, an hgiA promoter-gw.sv4 transcriptional reporter was constructed and introduced into Xcc strains with different genetic background, and then the GUS activities were determined in different growth conditions. Results show that the expression of hgiA is repressed in the rich medium NYG and induced in minimal medium MMX and in planta, and the expression of hgiA in minimal medium and in planta requires hrpG and hrpX, the two regulator of hrp gene cluster.gpdA encodes a NAD-dependent glycerol-3-phosphate dehydrogenase (GDPH, EC 1.1.1.94), which reversely catalyses dihydroxyacetonephosphate (DHAP) to glycerol 3-Phosphate (G3P) using NADH as co-enzyme. The product of Xcc gpdA shows 40.6% identity and 61.1% similarity to that of E. coli gpsA, which also encodes a NAD-dependent glycerol-3-phosphate dehydrogenase, at amino acid level. The role of gpdA in the pathogenesis of a pathogen has not been reported. In this study, we test the virulence of gpdA mutant on Chinese radish by leaf clipping method with cell concentration of OD6oo = 0.1, 10 days postinoculation, the lesion length caused by gpdA mutant is 1.9 mm, while the lesion length caused by wild type strain is 13.6 mm, and the lesion length caused by complemented strain is 10.9 mm. This result demonstrated that gpdA is required for the full virulence of Xcc. We further tested if mutation in gpdA has any effect on the growth rate in rich and minimal medium and the production of known virulence factors of Xcc, and found that gpdA mutant grows as well as the wild type strain in rich medium but grows very poorly in minimal medium;and the production of virulence factors (EPS, extracellular protease, extracellular endoglucannase and extracellular amylase) is not affected by gpdA mutation. The fact that gpdA mutant is able to grow in the medium with glucose as the sole carbon source indicating that there may exist another gene encoding a NAD-dependent glycerol-3-phosphate dehydrogenase in Jfcc8004 genome.CsrA (carbon storage regulator) , a multifunctional posttranscriptional regulator, was first identified in E.coli as a global regulator that positively regulates glycolysis, and negatively regulates glycogen synthesis and gluconeogenesis. CsrA is widely distributed in different bacterial species and play role in carbon metabolism, biofilm formation, cell motility and quorum sensing. It has been reported that CsrA is involved in the pathogenesis of severalpathogens such as Erwinia carotovora, Helicobacter pylori, Salmonella typhimurium and Yersinia pestis. In Xcc, mutation in csrA gene resulted in several phenotypes changes: (i) lost the virulence on host plant Chinese radish and hypersensitive reaction on non-host plant pepper EC W-1 OR;(ii) reduced the activity of extracellular endoglucannase and extracellular amy lase;(iii) reduction in the production of EPS in solid EPS production medium;(iv) increased the glycogen accumulation inside the cell;(v) lost cell mobility;and (vi) grows in aggregated fashion in LB medium. All these phenotypic changes can be corrected by wild type csrA in trans except the biofilm formation. These results indicated that csrA might act as a global regulator that positively regulates virulence factor production and cell mobility, and negatively regulates biofilm formation and glycogen synthesis in Xcc.