Functional Study Of The ’Candidatus Liberibacter Asiaticus’-encoded Protein Clsp1

Citrus Huanglongbing（HLB）currently is the most destructive disease of citrus worldwide.HLB is mainly transmitted by citrus psyllid,and its causative agent is Gram-negative Candidatus Liberibacter spp.,including Candidatus Liberibacter asiaticus（CLas）,C.Liberibacter americanus（CLam）and C.Liberibacter africanus（CLaf）.Of these three,CLas is the most prevalent and virulent species.However,up to now,the HLB pathogen remains unculturable,thus seriously restricting its pathogen biology research.With the metagenomics method,the scientists completed the genome sequencing of CLas,CLam and CLaf.The CLas genome is about 1.23Mb in size,lacking the typical type III,IV,and VI secretion systems,but has a complete Sec-dependent secretion machinery.CLas encodes～1180 proteins,of which,at least86 ones are Sec-dependent secreted proteins with functions largely unknown.Pathogen secreted proteins,also known as effectors,play important roles during the interaction between pathogens and host plants.Therefore,investigating the biological function of the secreted protein of the HLB pathogen in host cells will help to disclose the molecular pathogenesis of HLB and assist the disease control.Therefore,a secreted protein Clsp1（Gen Bank:ACT57080.1）encoded by the CLas Jiangxi strain（Las-JX）was investigated in this thesis.The resulting data are as follows:（1）Clsp1 is a Sec-dependent secreted proteinBioinformatics analysis revealed that Clsp1 is a Sec-dependent secreted protein.To confirm its secretory characteristics,total DNA was extracted from the periwinkle infected with Las-JX by the CTAB method,and was used as template to amplify the Clsp1 gene.The gene encoding the E.coli BL21 alkaline phosphatase（pho A）was cloned,and p ET-mpho A（pho A lacking its signal peptide）was constructed to detect the Sec-dependent secreted protein.The coding region of the Clsp1 signal peptide was amplified,the p ET-sp1-mpho A was constructed.On pho A assay medium containing BCIP,the E.coli cells containing p ET-sp1-mpho A turned blue,indicating that the sp1::mpho A fusion protein was secreted extracellularly and degraded BCIP in the medium.The data support that Clsp1 is a Sec-dependent secreted protein.（2）mClsp1 suppresses programmed cell death in plantaThe pGR-mClsp1 was constructed and transformed into Agrobacterium tumefaciens GV3101.The Nicotiana benthamiana plants were then infiltrated with the Agrobacterium containing pGR-mClsp1,alcohol decolorization and DAB staining showed that mClsp1 suppressed programmed cell death（PCD）caused by INF1 and Bax and significantly decreased H₂O₂accumulation during cell death.Bioinformatics analysis indicated that the central region of mClsp1 contains a DUF1311 domain and the C-terminus has a zinc finger.To clarify if the two putative domains function in the PCD suppression,the DUF1311 mutant mClsp1-4c and the zinc finger mutant mClsp1-2c were constructed respectively.In addition,the mClsp1homologous genes AAM43049.1 and AQS60992.1 was cloned from Xanthomonas campestris and A.rhizogenes,respectively.These genes were ligated with pGR107,respectively,and transformed into GV3101.The mClsp1-4c,mClsp1-2c,AAM43049.1 and AQS60992.1,when infiltrated in N.benthamiana plants,all inhibit INF1 and Bax-induced PCD,suggesting that both domains of zinc finger and DUF1311 were not involved in PCD suppression.To study the effect of mClsp1 on the pathogenicity of potato virus X（PVX）.Agrobacterium cells containing pGR107,pGR-mClsp1,pGR-mClsp1-2c,pGR-mClsp1-4c,pGR-m AQS60992.1 and pGR-m AAM43049.1 were infiltrated into3-week N.benthamiana plants.At 10 dpi,pGR-mClsp1,pGR-mClsp1-2c and pGR-mClsp1-4c all caused scattered necrosis in the upper leaves,but pGR-m AQS60992.1 and pGR-m AAM43049.1 did not induce any different symptoms compared with pGR107.Therefore,overexpression of mClsp1 may affect the pathogenicity of PVX,but the domains of zinc finger and DUF1311 were not involved in this function.Northern-blot analysis indicated that mClsp1 did not promote the PVX replication,although it inhibited the host PCD.（3）Alteration of mClsp1 sublocalization does not disturb its role in the PCD suppressionTo analyze the sublocalization of mClsp1,pCAM-mClsp1-GFPwas constructed.The mClsp1::GFP was transiently expressed in of epidermal cells of N.benthamiana via Agro-infiltration,and documented with laser confocal microscopy.The results showed that mClsp1 was in multiple localizations including nucleus,cell membrane and cytoplasm.To determine if the nuclear sublocalization of mClsp1 associates with its role of PCD suppression,the nuclear localization signal（NLS）of SV40 and the nuclear export signal（NES）of thermostable inhibitor（PKI）of c APK was fused with the N-terminus of mClsp1,respectively,resulting in pCAM-NLS-mClsp1-GFP,pCAM-NES-mClsp1-GFP.As expected,NLS-mClsp1 was mainly located in the nucleus,but NES-mClsp1 was almost out of the nucleus.Subsequently,the pGR-NLS-mClsp1 and pGR-NES-mClsp1 were constructed,and transformed into GV3010.Agro-infiltration of N.benthamiana showed that both fusion proteins suppressed INF1-and Bax-induced PCD,suggesting changing subcellular localization of mClsp1did not affect its function in PCD.（4）Interaction between mClsp1 and CsNOT2The pGADT7-mClsp1 was constructed,and used to screen a small yeast-two hybrid library based on Arabidopsis thaliana,and At NOT2a was identified to interact with mClsp1.The homologous gene of At NOT2a in sweet orange was cloned,and named as CsNOT2.The subsequent assay with yeast two-hybrid demonstrated the interaction between mClsp1 and CsNOT2.With luciferase bimolecular fluorescence complementation system,this interaction was further confirmed.