Study On Pathogenic Mechanism Of The Effector CaLasSDE115 Of Candidatus Liberibacter Asiaticus In Citrus

Citrus Huanglongbing(HLB),which is caused by ’Candidatus Liberibacter asiaticus’(Ca Las),is the most serious bacterial disease in the world citrus industry.Ca Las causes systemic diseases through colonizing the phloem of plants,but also spread by citrus psyllids(Diaphorina citri)among plants in field.Ca Las cannot be artificially cultured so far,and no dramtic progress has been made in understanding its pathogenic molecular mechanism.Ca Las has a complete SEC-dependent secretory system,which can secrete SEC-dependent effectors(SDEs)into host cells to participate in pathogen infection.It has been suggested that the CLIBASIA＿05115(CaLasSDE115)effector protein encoded by Ca Las may be a pathogenic factor,but its roles in Ca Las infections is still unclear.In this study,we characterized the CaLasSDE115 effector from bioinformatics,gene expression,and subcellular localization in prokaryotic and eukaryotic cells.Further,functions of CaLasSDE115 in Ca Las infections were determined through overexpressing CaLasSDE115 in Wanjincheng oranges(Citrus sinensis Osbeck)which is a HLB-susceptible cultivars.Finally,we dissected the molecular mechanisms of CaLasSDE115 in Ca Las infections using RNA-seq.The study lays the foundations for further exploring Huanglongbing’s pathgoneisis and unearthing resistant genes to generate HLB-resistant varieties in citrus in future.The main findings are as follows:1.Bioinformatics and subcellular localization analysis of CaLasSDE115Bioinformatics analyses showed that CaLasSDE115 was 100% conserved in all reported Ca Las strains but had sequence differences compared with orthologs from other ‘Candidatus liberibacter’.Prediction of protein structures suggested that the crystal structure of CaLasSDE115 was very close to that of the invasion-related protein B(Ial B),a virulence factor from Bartonella henselae.Alkaline phosphatase(Pho A)assay in E.coli confirmed that CaLasSDE115 had 32 amino acid residues-long Sec-dependent signal peptides at its N-terminal,which indicated that CaLasSDE115 can be secreted extracellularly to function.Tobacco subcellular localization showed that the mature protein of CaLasSDE115 was mainly distributed in the cytoplasm and nucleus of plants.2.Expression characteristics of CaLasSDE115Expression levels of CaLasSDE115 in Ca Las-infected Asian citrus psyllid(ACPs)were much higher(～45-fold)than those in Ca Las-infected Wanjincheng oranges,with the expression in symptomatic leaves being significantly higher than that in asymptomatic ones.Our data implied that CaLasSDE115 had a role in Ca Las colonization of citrus psyllid and symptomic development of citrus plants.3.Effects of CaLasSDE115 on HLB-resistance of citrus(1)Generation of transgenic Wanjincheng oranges overexpressing m SDE115To study the function of CaLasSDE115 involved in regulation of Ca Las infection in citrus,a plant expression vector overexpressing m SDE115 without signal peptide sequence was constructed.Using the epicotyls from HLB-susceptible Wanjingcheng oranges as explants,this gene was introduced into citrus genome through Agrobacterium-mediated transformation.transgenic plants were identified using GUS histochemical staining and PCR and q RT-PCR.Here,four m SDE115-overexpressing transgenic plants were obtained by q RT-PCR analysis.The phenotypes of transgenic plants were not significantly different from that of wild-type plants in greenhouse.(2)Effects of m SDE115 overexpression on the resistance of transgenic plants to HLBThe resistance of transgenic plants to Ca Las was evaluated using a grafting inoculation in greenhouse.After six-month of infection,chlorosis or mottled yellow symptoms were first detected in the new leaves of transgenic plants while no visible symptoms appeared in the WT plants.Ca Las content determination showed that overexpression of m SDE115 favored Ca Las proliferation during the early stage(two months)of infection.Microscopic observation showed that the phloem of transgenic plants was obviously thickened,and the deposition of callose were aggravated,compared to WT control after Ca Las infection.The results showed that CaLasSDE115 contributed to the early proliferation of Ca Las and the development of symptoms in citrus.4.Molecular mechanisms of CaLasSDE115 in regulation of HLB resistance in citrus(1)RNA-seq analysis of transgenic plants overexpressing m SDE115To understand molecular mechanisms of CaLasSDE115 in regulation of HLB resistance in citrus,a representative transgenic plant was subjected to RNA-seq analysis.1,011 differentially expressed genes(DEGs)were obtained in this test.Among them,329 and 682 DEGs were up-and down-regulated by m SDE115 overexpression.Map Man analysis showed that 280 of the 386 DEGs related to biotic stress were down-regulated,indicating that overexpression of m SDE115 significantly inhibited the transcriptional activity of citrus biotic stress.Further,KEGG enrichment analysis showed that eight up-regulated DEGs were significantly enriched in ABC transporters pathway while 27 and 23 down-regulated DEGs were significantly enriched in Phenylpropanoid biosynthesis and MAPK signaling pathways,respectively.Based on these data,we speculated that CaLasSDE115 negatively regulates citrus defense responses through interfering Phenylpropanoid biosynthesis,MAPK signaling and ABC transporters pathways.(2)Effects of overexpressing m SDE115 on SAR response in transgenic plants.Salicylic acid(SA),jasmonic acid(JA),and methyl salicylate(Me SA)are signal molecules of systemic-acquired resistance in plants.Compared with the WT control,SA and JA levels in all the transgenic plants were downregulated by m SDE115 overexpression while Me SA was upregulated.The expression levels of SAR genes Cs PR1,Cs PR2,Cs PR5,WRKY45,and WRKY70 were significantly suppressed in transgenic plants.Cs PR1,Cs PR2 and Cs PR5 are the SAR marker genes,suggesting that CaLasSDE115 is involved in Ca Las-induced supresssion of citrus SAR response.