Identification And Function Analysis Of Citrus Genes Involved In Defense Responses To Xanthomonas Citri Subsp. Citri

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc), is a quarantine disease that has brought great damages to citrus industry worldwide. So far, no effective method can be used to remove or completely control this disease. Consequently, the most effective and economical approach for controlling canker disease relies on the selection or breeding of resistant cultivars. After many years screening, an unique resistant citrus genotype, citron C-05 has been identified with hypersensitive reaction (HR) after inoculated by Xcc. It is necessary to elucidate the molecular responses of citron C-05 to Xcc invasion for identification of resistance associated genes, so then to break through the bottleneck of breeding for disease resistance.In order to verify the relationship between the expression levels of the genes with the pathogen infection, three defense related genes may be involved in the PAMP-triggered immunity (PTI) and two genes may be involved in the effector-triggered immunity (ETI) were confirmed by quantitative PCR of the same two genotypes after inoculation. Furthermore, the candidate resistance related genes which differential expressed between citron C-05 and susceptible cultivar’Bingtang’sweet orange were chosen for further functional analysis. The candidate genes and its promoters were amplified and sequenced, and the transcripts of those genes were verified by using high resolution melting analysis. The protein structure and promoter of candidate genes were predicted by bioinformatical approaches and then functional verification were done by transient expression analysis systems. Moreover, the protein-protein interactions were analyzed by using bimolecular fluorescence complementation. The main results were as follows:1. The expression levels of LYP’2 and CERK1 increased after inoculation in citron C-05, while decreased in ’Bingtang’ sweet orange; the expression of LYP1 and imp-a were both increased in the two genotypes with the same trend; the expression of SRP54 has no regular pattern. Six representative defense marker genes, namely, β-glu, PAL-1, PAL-2, MLO-1, MLO-2 and WRKY13 were all increased in citron C-05, while had no difference in ’Bingtang’sweet orange.2. The cDNA and gDNA sequence comparison between the corresponding genes revealed that CsLYP2 and CmLYP2 genes both contained four introns splitting their open reading frame into five exons. The gDNA full length of CsLYP2 and CmLYP2 is 3324 bp and 3318 bp, respectively; the cDNA full length of both genotype are 1077 bp and encodes 358 amino acids. CsCERK1 and CmCERK1 have no intron and the cDNA full length are 2010 bp and encodes 669 amino acids. The homology of gDNA, cDNA, amino acid sequence were 98.11%,99.07% and 97.77% respectively between CsLYP2 and CmLYP2,98.46%,98.46% and 96.41% respectively between CsCERK1 and CmCERKl. The theoretical Mw and pi of the CsLYP2/CmLYP2 and CsCERK1/CmCERK1 protein is 38.99 kDa,7.82/38.98 kDa,8.07 and 74.20 kDa,6.73/74.22 kDa,7.19 respectively. The CERK1 protein was indicated as a signal peptide, LysM motif, transmembrane region, tyrosine kinase domain-containing (STYKc domain) protein. LYP2 contained a signal peptide and two LysM motifs, without transmembrane region. There were minor differences in secondary structural between CsCERK1 and CmCERKl protein, but the tertiary structure of CERKls have no difference, the same as LYP2s.3. The ratio of the two LYP2 transcripts was 3:2 in’Bingtang’sweet orange after inoculation, while only one type of transcript in citron C-05; the ratio of the two CERK1 transcripts was 3:2 and 3:1 in ’Bingtang’ sweet orange and in citron C-05 after inoculation, respectively, but there was no necessary relation between the induced transcript and expression levels of LYP2 and CERK1 gene in the two genotypes.4. The LYP2 promoters from sweet orange (pCsLYP2) and citron C-05 (pCmLYP2) were 1607 bp and 1956 bp in length, respectively. Cis-acting regulatory element essential for salicylic acid induction (TCA element), cis-acting element involved in MeJA induction (CGTCA-motif), cis-acting element involved in defence and stress responsiveness (W-box) etc were all in common in pCsLYP2 and pCmLYP2. Three cis-acting elements or DNA motif were present in pCsLYP2, but not in pCmLYP2, as follows:cis-acting regulatory element essential for anaerobic induction, ARE motif, cis-acting element involved in defence and stress responsiveness, TC-rich repeats and Enhancer-like element involved in anoxic specific inducibility, GC-motif. Regulation element of genes involved in defence mechanism, TCCACCT-motif was just present in pCmLYP2, but not in pCsLYP2.5. The pCsLYP2 and pCmLYP2 could both drive the downstream reporter gene expression with function, and the activity of pCmLYP2 was enhanced when host leaf was infected by Xcc, but pCsLYP2 was not enhanced by Xcc infection. It seems that the promoter of LYP2 in citron C-05 might play some role in the resistance to canker disease, it is necessary to further investigate the function of the promoter elements and boxes.6. CsLYP2 and CmLYP2 could form homo-dimers, but could not interact with CsCERK1 and CmCERKl. CsCERK1 and CmCERKl could not form homo-dimers. It is necessary to confirm that CERK1 could dimerized when induced by peptidoglycan or chitin.7. The CERK1 and LYP2 proteins could inhibit the growth of Xcc in the ’Bingtang’ sweet orange leaves but CsLYP2-1. Moreover, this confirmed that the differential expression of LYP2 might be due to its structure difference in promoter sequence.