Identification And Expression Analysis Of Citron C-05 Genes Involved In Defense Responses To Xanthomonas Axonopodis Pv. Citri

Citrus bacterial canker diseases(CBCD) caused by Xanthomonas axonopodis pv. citri (Xac),is a worldwide quarantine disease that has brought great damages to citrus industry but difficult to prevent. Therefore, the flow of citrus materials carrying Xac into non-epizootie area must be strictly prohibited and the detection of Xac must be strengthened. However, it’s just a temporary solution. To fundamentally solve this problem, the most effective and economical approach relies on the selection or breeding of resistant cultivars. After many years screening, an unique resistant citrus genotype, citron C-05 had been identified with hypersensitive reaction (HR) after inoculated by Xac by our group. It is necessary to elucidate the molecular responses of citron C-05 to Xac invasion for identification of resistance associated genes, so then to break through the bottleneck of breeding for disease resistance.On the base of the transcriptome sequencing data of resistant citron C-05 and susceptible cultivar’Bingtang’sweet orange and the result of GO function classification and "pathway" metabolic pathway analysis, this research selected several candidate genes which might be associated with the resistance to citrus canker disease. The protein structure of candidate genes were predicted by bioinformatical approaches. And their expression pattern in two genotypes which inoculated with flg22 or Xac in different concentration were analysed by qPCR. Moreover, the sensitivity of Real-time Quantitative PCR in detecting Xac was also analyzed.The main results were as follows:1. After injection inoculation and spray inoculation with Xac, the expression level of FLS2 increased in citron C-05, while decreased in’Bingtang’sweet orange. The expression of EFR was increased in the two genotypes with the same trend after injection inoculation, but was oppositely changed after spray inoculation.The expression of BAK1 was increased after injection inoculation and decreased after spray inoculation both in the two genotypes with the same trend.2.After injection inoculation with flg22, the expression of FLS2,BAK1,CERK1 and LYP2 were all increased in citron C-05, while decreased in’Bingtang’sweet orange. This uncovered flg22 might have the function of PAMP in mesophyll. And its induction effect to FLS2 and BAK1 was more rapid and efficient than Xac; but to CERK1 and LYP2, it had little impact. After spray inoculation, the 4 genes were all decreased in the two genotypes with the same trend. In terms of this, we considered flg22 could not induce the expression of these candidate genes on the surface of leaves.3.The sensitivity of qPCR was higher than that of conventional PCR by 10-1000 fold and the detection of conventional PCR would be influenced by plant DNA but qPCR won’t. Analyzing the inoculated ’Bingtang’ sweet orange, qPCR could detect Xac at once, while conventional PCR waited until two days later. And with qPCR, we could observe Xac proliferated slowly first and rapidly afterwards. Compared with high concentration, Xac proliferated faster with low inoculum concentration, and compared with citron C05, Xac proliferated faster in ’Bingtang’ sweet orange. After inoculation with different Xac concentration, the expression level of FLS2,EFR and BAK1 were all increased in citron C-05, while decreased in ’Bingtang’ sweet orange.When inoculated with the concentration of 1010 cfu/ml, the expression levels of these candidate genes in citron C-05 were higer than the other two concentration. While if inoculated with the concentration of 108 cfu/ml, the expression levels of these candidate genes in ’Bingtang’ sweet orange were lower than the other two concentration.