| Citrus Huanglongbing (HLB) is one of the most serious and devastating citrusdiseases in the world. It is caused by any of three uncultured species of α-Proteobacteria,Candidatus Liberibacter asiaticus (CLas), Ca. L. americanus (CLam), and Ca. L. africanus(CLaf), and CLas is the only pathogen responsible for HLB in China. In recent years,detection, identification and genetic variability of HLB pathogen were extensively studied.However, research involving in occurrence mechanism of HLB was seldom carried out. Inthis paper, transcriptomes of different citrus cultivars upon infection of CLas werecomparatively analyzed, and the resistance gene analogs (RGAs) from citrus were cloned.The main objective of this paper is to understand the molecular mechanism of HLBresistance, and to lay a foundation for exploiting the HLB-resistant genes in citrus. Mainresearch contents and their results are as follows:1. Screening of HLB source plants. CLas, Phytoplasma, and Citrus tristeza virus(CTV) were detected from ten leaf samples of qicheng, which came from ten suspectedHLB citrus plants respectively in Dayu county, Jiangxi province. Results showed that twoplants (plant No.4and plant No.5) were infected with CLas, not with Phytoplasma andCTV. These two plants were considered as the ideal HLB source plants, and we selectedplant No.4for using in the subsequent experiments.2. Indoor resistance identification of different citrus cultivars against HLB. Budsfrom above No.4HLB source plant were grafted on plants of different citrus cultivars.HLB Infection status among them were evaluated in regular time intervals (monthly) byPCR method. It was found that CLas was present in Ponkan and guanximiyou after threeand four months of grafting respectively, whereas no CLas has detected in Citrus unshiu.Meanwhile, evident, mild, and no HLB symptoms were observed on Ponkan, guanximiyouand Citrus unshiu. respectively. The results showed that to HLB, Ponkan was highlysusceptible, guanximiyou came second, and Citrus unshiu was highly resistant.3. Analysis of gene differential expression. Using microarray technology,gene differential expression of leaves from Ponkan, guanximiyou, and their healthycontrols were analyzed.1106differentially expressed genes were found in Ponkan, ofwhich564genes were up-regulated, and542genes were down-regulated, whereas910differentially expressed genes were found in guanximiyou, of which491genes wereup-regulated, and419genes were down-regulated. Gene pp2, related to phloem blocking,was up-regulated in both Ponkan and guanximiyou. Genes involving in synthesis ofcysteine proteinase were all up-regulated in guanximiyou, but were up or down regulatedin Ponkan. However, genes in a number of metabolism pathways including photosynthesis,sugar metabolism, chlorophyll, pathogenesis related protein and GDSL-lipase weredifferentially expressed at different levels in the two cultivars. These data were helpful to elucidate the HLB resistance differences between Ponkan and guanximiyou.4. Cloning of resistance gene analogs. Resistance gene analogs (RGAs) from threecitrus cultivars were cloned using degenerate primers located in conserved regions ofresistance gens, and the PCR products were directly sequenced and the sequence wereanalyzed.26nucleotide sequences of RGAs were obtained, of which23sequences couldbe translated into continuous amino acid sequences. They all had several conserved regionscontained in NBS-LRR catalogue of resistance genes. Sequence W-10and W-5weregrouped together with CTV4gene (resistant to Citrus tristeza virus, CTV) in citrus andI2C gene in tomato respectively. It is speculated that these two RGAs were highly relatedto resistance for CTV and fungus of Fusarium. |
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