Study On Functions Of The SDE70 And SDE695 Effectors In Interaction Between Candidatus Liberibacter Asiaticus And Citrus

Citrus Huanglongbing（HLB）is currently the most devastating disease in citrus throughout the world,causing great economic losses to the citrus industry.The disease is caused by Candidatus Liberibacter spp,which is a gram-negative bacteria parasitizing exclusively the phloem of plants,and is mainly spread by citrus psyllids and field grafting.Candidatus Liberibacter asiaticus（Las）is the main pathogen of HLB.HLB pathogen still cannot be cultured in vitro so far,which hinders the research progress of the disease,so its pathogenic mechanisms are not well understood.As important virulence factors,effector proteins secreted by bacteria into host cells to help pathogen infection.The current researches considered that Sec-dependent effectors（SDEs）are the main effectors of Las.Here,we reported our progress in exploring the functions and mechanisms of SDE70 and SDE695 in interaction between Las and citrus.SDE70 and SDE695 genes were cloned from Las infected sweet orange.The expression profiles of effectors in different HLB-tolerance citrus,different plant tissues,and different susceptible periods were constructed using quantitative Real-time PCR（q RT-PCR）.Using HLB-susceptible Wanjincheng oranges as explants,transgenic plants overexpressing SDE70 or SDE695were generated by Agrobacterium-mediated transformation to dissect effects of SDE70and SDE695 overexpression on the growth and development,HLB resistance,and gene transcriptions of transgenic plants.The interaction proteins of the effectors in citrus were screened by yeast two-hybrid（Y2H）.The principal research results are as follows:1.Bioinformatics and secretion characteristics analysis of SDE70 and SDE695.Bioinformatics analysis showed that SDE70 and SDE695 had 20 and 29 amino acid residues-long Sec-dependent signal peptides at their N-terminals,respectively.SDE70contained the conserved domains of Lpr I and Yec T,and Lpr I was a lysozyme inhibitor domain;SDE695 did not contained putative conserved domains.Alkaline phosphatase（phoA）assay confirmed that the predicted signal peptides had extracellular secretion function in Escherichia coli,so SDE70 and SDE695 are Sec-dependent secreted proteins.The subcellular location analysis of Nicotiana benthamiana showed that mature proteins of SDE70 and SDE695 were distributed in the nucleus,cytoplasm and cell membrane.2.The expression characteristics of SDE70 and SDE695 in citrusThe expression of SDE70 in symptomatic veins of HLB-susceptible Wanjincheng orange was significantly higher than that of asymptomatic veins,and its expression in symptomatic veins of HLB-tolerant Citrus hystrix（C.hystrix）was significantly lower than that of asymptomatic veins.The expression of SDE70 in roots of Wanjincheng was significantly higher than that in veins,while its expression in roots of C.hystrix was significantly lower than that in veins.The expression of SDE70 in fully mature leaves was significantly higher than that in young leaves in both Wanjincheng and C.hystrix.The expression of SDE695 in symptomatic veins was down-regulated than that in asymptomatic veins in both Wanjincheng and C.hystrix.The expression of SDE695 in roots of Wanjincheng was significantly higher than that in veins,while its expression in roots of C.hystrix was significantly lower than that in veins.The expression of SDE695in old leaves of Wanjincheng was significantly higher than that in young leaves,while its expression in fully mature leaves of C.hystrix was significantly lower than that in young leaves.The data showed that the expression of SDE70 and SDE695 were significantly different in different HLB-resistant varieties,different tissues,different symptom leaves and different maturity leaves.3.Analysis of biological functions of SDE70 and SDE695Plant overexpression vectors containing SDE70 and SDE695 without signal peptide sequence were constructed to generate transgenic Wanjincheng Orange plants overexpressing the target genes.（1）Effects of overexpression of SDE70 and SDE695 on plant growth anddevelopment.Agrobacterium rhizogenes-mediated transformation assays showed that the transformation efficiency of SDE695 was significantly lower than that of control,suggesting that the SDE695 may be involved in the regulation of citrus growth and development.But SDE70 had no effects on efficiency of citrus genetic transformation.One-year greenhouse cultivation and observation did not find any obvious phenotypic difference between transgenic plants and wild type.（2）Effects of overexpression of SDE70 and SDE695 on the resistance of transgenicplants to HLB.The 10-months HLB resistance evaluation showed that mottled yellow symptoms appeared in transgenic plants and wild type control at nine months after Las infection,the symptoms of the transgenic plants were not significantly different from those of the wild type plants.SDE70 overexpression promoted the accumulation of Las in the first month after Las infection,and inhibited the proliferation of Las in the second and fourth months;while the Las contents in SDE695 transgenic plants did not change significantly,compared with wild type control.（3）Effects of SDE70 and SDE695 overexpression on the immune signals intransgenic plants.Compared with the wild-type control,the contents of salicylic acid（SA）and H₂O₂ in SDE70 transgenic lines increased,and the contents of jasmonic acid（JA）decreased;the SA contents in the SDE695 transgenic lines increased,while the JA and methyl salicylate（Me SA）contents decreased.（4）Effects of overexpression of SDE70 and SDE695 on the transcriptome oftransgenic plants.Transcriptome sequencing analysis showed that overexpression SDE70 and SDE695 negatively regulated plant biotic stress and receptor kinase-mediated signal transduction.Function analysis of differential expressed genes showed that the SDE70 and SDE695 mainly down-regulated the expressions of genes involved in pathogen recongnition and host disease resistance.The results suggest that SDE70 and SDE695 may be involved in Las suppression of the citrus immune response.4.Screening and verification of interacting proteins of SDE70 and SDE695 in citrusThe Y2H assay verified that SDE70 interacts with transporter particle complex CsTRAPP,ADP-ribosylation factor CsARF,and ubiquitin-NEDD8-like RUB2 protein CsRub1 which is related to the ubiquitination pathway,suggesting that SDE70 may regulate the transport mediated by the host membrane system and the signal transduction.SDE695 interacts with plastocyanin CsPc and cytochrome b6f protein CsB6f,suggesting that SDE695 may target plant photosynthetic electron transport-related proteins and regulate photosynthesis or some other processes of citrus.