| Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most bacterial disease of citrus and vastly threatens to the citrus industry worldwide through weaken the plant growing and influence the quality of fruit. Xcc secrets many effectors through the type III secretion system (T3SS) and interacts with citrus host. TALe (transcription activator-like effector) exists in all Xcc strains as the main effector. This research analyzed the genetic diversity of tale genes from Chinese citrus canker isolates using Southern blot with conservative Bam HI fragment as the probe, and investigated the distribution of tale genes and the dominant genotypes in Xcc. In the same time, each tale gene were mutated by the technology of no marker gene knock-out in the strain Xcc049, and then were inoculated onto the susceptible citrus varieties Sweet orange (Citrus Sinensis) along with Xcc049(wild-type) and every relative complement strain, respectively. After24hrs, we analyzed the expression difference of genes in citrus induced by different tale genes through deep sequencing of the tested citrus mRNA.We collected105Xcc strains from nine citrus growing provinces of China on six citrus strains were classified into14different genotypes (designated A to N) based on the number and size of pthA homologues (a member of tale gene family, the most important virulent factor of Xcc). Genotypes G and B represented62%and19%of the isolate collection, respectively. Genotypes J and L, lacked pthA or a pthA-hybridizing fragment were less virulent on grapefruit (C. paradisi) and sweet orange (C. sinensis) compared to strains containing pthA or a pthA homologue. The virulence of genotypes J and L was increased when the wild-type pthA was introduced. Genotype I, which was isolated from sweet orange in Jiangxi province, caused typical canker symptoms and may contain a novel pthA-like. gene. To our knowledge, this is the first description of genetic diversity in Chinese Xcc strains based on tale gene analysis, which will faciliate our understanding the pathological mechnism of Xcc.Wild-type strain (Xcc049A), tale gene knock-out strains (Xcc049B,Xcc049C, Xcc049D and Xcc049E), and pthA complementary stain (Xcc049E+pthA) were inoculated into sweet citrus, respectively. Then all the inoculated samples were collected24hrs post inoculation, and total RNA was purified from each sample and subjected to deep sequencing. The results showed that the strain Xcc049E+pthA induced the most differentially expressed genes among all the tested strains. Compared with Xcc049E, a total of112genes, most of which were relevant to cell division, DNA replication and programmed cell death, differed in expression level, including101up-regulated and11down-regulated genes. It was found that the up-regulated expression of11genes could be induced by either tal-C or pthA, which indirectly indicated the similar gene function of tal-C and pthA. The strains Xcc049A, Xcc049B and Xcc049C induced the close symptoms phenotype, however, tal-A and tal-B induced less differentially expressed genes, which indicated that tal-A and tal-B did not contribute greatly to the virulence of Xcc and were suggested to be redundant members of tale gene family in Xcc. As the aa12and13of the repeated units encoded by tale gene could bind to the gene promoters in planta, and initiate resistant/susceptible reactions. So excavation of the PthA-targeted sequences in citrus will be very important to citrus canker-resistant breeding. |
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