Cloning And Expression Arialysis Of Genes Of Citrus Interacting With Canker Pathogen

Citrus canker is a serious quarantine disease. More than100years have passed since it was reported; however, the knowledge of the mechanism about the pathogenesis and resistance is still poor. In this study, several genes related to the interaction between the pathogen and the host were cloned, and their expression was analyzed after inoculation with Xac bacteria. The main results are the following:1. By using monoclonal antibody against to PthA-nls and co-immuno precipitation, a potential protein interacting with PthA, the pathogensis efector, was detected and named CsSRP54. And then, the srp54gene and the imp-a gene, which was reported to interact with PthA, were cloned and sequenced from a susceptible ’Bingtang’ sweet orange and a resistant citron-C05. Both genes’sequences were compared between the two citrus genotypes. Sequence blast results showed that these two different genotypes have98.5%similarity for srp54gene in genomic DNA sequence,99.6%in cDNA, and98.6%in amino acid sequence; for imp-a gene, sweet orange and citron have98.6%similarity in genomic DNA sequence,98.6%in cDNA, and99.44%in amino acid sequence.2. The pathogen stays in the intercellular space of the host for further colonization after the infection. In this first period of infection, the pathogen may cause the PAMP-triggered immunity (PTI). In the transcriptomic analysis, differentially expressed genes were found, among which some were highly expressed after inoculation with Xac in a susceptible genotype while inhibited in a resistant one, or vice versa; or some genes’expression was very high in one genotype but not in another. One of such genes was cerkl, which is reported to inhibit the bacterial propagation in the intercellular space, and introduces PTI. These are basic results for further studies on pathogen-host interaction.3.In order to verify the possible roles of the cloned genes srp54and imp-a and cerkl obtained from previous transcriptomic analysis in the interaction between pathogen and host, dynamic expression analyses of these genes were performed following the inoculation with the pathogen. The results indicated that srp54gene was immediately up-regulated, reaching the maximum on the9th day after inoculation in the susceptible genotype; and imp-a showed the same up-regulated expression, but reduced to a stably lower level soon after; while in the resistant genotype, both genes remained at low level of expression during all the period. It was observed that typical symptoms appeared on inoculated sweet orange leaves on the9th day after inoculation, for the citron C05no typical symptom at all the period, implying that these two genes may related to the pathogenesis. In sweet orange, the expression of cerkl showed down-regulated after inoculation, and then remained at low level during all the rest period; while in citron C05, its expression was up-regulated during the first week after inoculation, and return to lower level then after. It may be suggested that cerkl gene plays a role to inhibit the pathogen propagation and further to stop the canker disease development, while in sweet orange this function does not exist. The results may provide promising basis for studies on the mechanisms of resistance and pathogenesis.