Prediction,Validation And Functional Study Of Candidatus Liberibacter Asiaticus Sec Dependent Secretary Proteins

Citrus huanglongbing（HLB）is one of the destructive diseases threatening citrus industry worldwide.It is spread by citrus psyllids and is associated with three species of phloem-restrictedα-Proteobacteria,Candidatus Liberibacter asiaticus’（CLas）,Candidatus L.africanus and Candidatus L.americanus.The unculturable characteristic of these pathogens,greatly hampered our understanding of the mechanisms for pathogenesis.A complete circular CLas genome has been obtained by metagenomics,the 1.23-Mb genome losts many secretion systems as well as typical free-living or plant colonizing extracellular degradative enzymes,However,CLas contains a complete General Secretory Pathway（Sec Pathway）,which forms a conserved route for the secretion of housekeeping proteins and is essential for bacterial viability.A few of Sec dependent secretary proteins were identified as virulence factors during CLas infection.In this study,we focus on prediction,validation and functional study of the Sec dependent secretary proteins of CLas isolate psy62.（1）Prediction of Sec dependent secretary proteins using CLas psy62 genomeA total of 57 proteins were predicted to be Sec denpendent secretary proteins in CLas psy62,then,further analyses were focused on the transmembrane structure,signal peptides,the way and the sites of signal peptides（SP）cut,and the hypothetical functions of them.To experimentally validate the presence of SP in the predicted Sec dependent secretary proteins,a more efficient PhoA assay system was established.When all the 57 predicted proteins overexpressed via a potato virus X（PVX）-based expression vector in Nicotiana benthamiana respectively,three of them,CLIBASIA＿02470,CLIBASIA＿03875,CLIBASIA＿04405 could suppressed programmed cell death（PCD）triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1.（2）Function study of m03875We show that CLIBASIA＿03875 contains a putative Sec-secretion SP,a 29 amino acid residue located at the N-terminus,with the mature protein（m3875）of 22 amino acids found to localize in multiple subcellular components of leaf epidermal cells of N.benthamiana.When overexpressed via a PVX-based expression vector in N.benthamiana,m3875 suppressed PCD and H₂O₂ accumulation triggered by INF1 and BAX.Overexpression also resulted in a phenotype of dwarf,leaf deformation and mosaic,suggesting that m3875 had roles in the plant immune response,growth and development.Alanine substitution mutagenesis of the charged amino acid（D7,R9,R11 or K22）within m3875 did not recover the phenotypes for PCD and normal growth.Additionally,m3875 may regulate functions or transcriptional levels of N.benthamiana orthologs of CNGCs（Cyclic nucleotide-gated channels）,BI-1（Bax-inhibitor 1）and WRKY33 that are involved in plant defense mechanisms by directly or indirectly interaction.（3）Function study of m04405We experimentally verified the secretory characteristic of a predicted Sec-delivered protein,CLIBASIA＿04405,which is was conserved among the CLas strains and significantly upregulated in citrus relative to psyllids.Transient expression of mature CLIBASIA＿04405 protein（m4405）in N.benthamiana leaves could also suppress PCD and the H₂O₂ accumulation triggered by INF1 and BAX.Alanine-substitution mutagenesis assay showed that different charged amino acids are crucial for suppressing INF1（A24-25、A39-40、A45-47、A52-A54、A64-65、A75-78 and A93-94）and BAX（A45-47 and A64-65）induced PCD.Constitutive expression of m4405 in N.benthamiana producted a phenotype of dwarfing,leaf deformation as well as suppressed Pseudomonas syringae DC3000（hopQ1-1）growth.Furthermore,transcription of photosynthetic pathway related gene,Small heat shock protein（sHSPs）genes and VB6 synthesis related genes were significant changed in two transgenic lines,and m4405 could interacted with N.benthamiana catalase indicating that the basal immune and normal development of these plants may be disturbed by m4405 overexpression.In conclusion,we predicted 57 Sec denpendent secretary proteins in CLas psy62,three of them were identified as PCD repressors.The functions of CLIBASIA＿03875 and CLIBASIA＿04405 were further investigated in our study.The results provide insight as to how CLas attenuates the host immune responses in order to proliferate and cause Huanglongbing disease in citrus plants.