Identification Of Novel Effectors Depended On Type Ⅲ Secretion System In Xanthomonas Campestris PV. Campestris

The Xanthomonas genus comprises large amount of various species and pathovars that could infect 392 kinds of plants including gramineous, leguminous and cruciferous, causing great economic losses world wide each year. Xanthomonas campestris pv. campestris (Xcc) is the causalagent of cruciferious black rot and one of the model bacteria for studying the plant-microbial interaction. It is demonstrated that the type III secretion system (T3SS) is the key apparatus for conquering the hostcell to cause disease in most gram negative plant and animal pathogens and once the T3SS was disrupted, the pathogen will lose its ability to cause disease. The so-called effectors including avirulence proteins, virulence factors and Hrp outer proteins (Hops) were translocated directly into the plant cells via the T3SS pathway. The whole genome sequencing made it possible to identify the effectors genome wide, for example, screening the candidate effector genes based on the promoter activity, the promoter related sequence motif (PIP-box,hrp_â…¡box) and proteins having domains with characteristics of eukaryotic proteins, such as, leucine-rich repeats (LRRs). In this work, all the annotated eight avr genes and two genes encoding Leucine-rich proteins of Xcc 8004 were selected as candidate effector genes based on sequence analysis and the expressional file analysis of hrpG and hrpX using DNA microarray. These candidate genes were characterized by mutagenesis, expression analysis and translocation test by using the methods of virulence test, gus reporter, reverse transcriptional PCR (RT-PCR), Western blotting and hypersensitive reaction (HR) assay. The main results are as follow.1. The sequence analysis of the promoter region (25-500 bp upstream of start codon) of whole Xcc 8004 genome showed that 66 genes have PIP-box (TTCGCN15, N16TTCGC) and 123 genes have hrp_â…¡ box (TTCG...n15, n16...TTCG). Among the candidate genes, avrBs2 and XC4273 have perfect PIP-box and avrXccB, avrXccC, avrXccE1 have hrp_â…¡ box. The alignment (BLASTp) results indicated that avrBs2 is high conserved in several sequenced Xanthomonas strains (X. campestris pv. vesicatoria, X. axonopodis pv. citri, X. oryzae pv. oryzae), whereas the other avr genes are not. XC1553 and XC4273 share low similarity with HpaF and PopC from X. axonopodis pv. citri and Ralstonia solanacearum respectively.2. The Tn5gusA5 insertion mutants of candidate genes were confirmed by PCR and their single crossover integration mutants were constructed. The virulence test on host plants Chinese radish and cabbage indicated that avrBs2 was a pathogenicity related gene. The mutant of XC4273 reduced virulence significantly and could be restored completely in trans.3. avrXccC is a host special gene and it confers the resistance to host plant Mustard Guangtou. After avrXccC was mutated, the bacteria became virulent on Mustard Guangtou. Expression of avrXccC in plasmid could make the mutant lose its virulence.4. XC1553 mutant gave virulence symptom when inoculated on resistant ecotype Arabidopsis thaliana Col-0. This avirulence-like phenotype could be complemented by XC1553 gene in trans. We suggest that XC1553 is an avirulence gene.5. The avr-hrpG double mutant of avr genes and the gus reporter of XC1553 and XC4273 were constructed. The results indicated that the expression of avrBs1.1,avrBs1,avrXccB,avrXccC,avrXccE1,XC1553 and XC4273 were regulated by hrpG. Except avrBs1 and XC4273, the hrpG-regulated genes were also regulated by hrpX.6. Xcc 8004^* confering the constitutive expression of hrp genes was attempted to detect the secretion of candidate effectors through Western blotting.7. It was confirmed that AvrBs1 was responsible for Xcc to elicit hypersensitive reaction on nonhost plant pepper ECW-10R. XC0241, a homologous gene of XopN from Xanthomonas campestris pv. vesicatoria (Xcv), and XC1553 were demonstrated as T3SS effectors respectively by using AvrBs1 function region as a reporter. Using the function region of known effetor AvrBs3 from Xcv as a reporter, AvrXccC was also demonstrated as a T3SS effector.In summary, the virulence, expressional regulation and secretion/ transiocation of the genes annotated as Avr and LRR proteins in Xcc 8004 were studied. Two genes involved in host specificity were identified, and the encoding products of three genes were demonstrated as the T3SS dependent effectors.