Preliminary Study On The Pathogenic Mechanism Of The Secreted Protein Clsp33 Of ’Candidatus Liberibacter Asiaticus’

Citrus Huanglongbing（HLB）is an important quarantine disease caused by the gram-negative bacterium Candidatus Liberibacter spp..The pathogens are divided into three subspecies:“Candidatus Liberibacter asiaticus”（CLas）,“Ca.L.africanus”（CLaf）and“Ca.L.americanus”（CLam）.Among them,CLas and CLam are spread by Asian citrus psyllid（Diaphorina citri）,while CLaf is spread by African citrus psyllid（Trioza erytreae）.Due to the lack of resistant/tolerant commercial citrus varieties and effective cure approaches to the pathogen,the integrated strategies,including controlling the spread of psyllid at wide-area level,removal of the infected trees,and using disease-free seedlings of HLB,are recommended to control HLB.HLB pathogens can not be cultured in vitro so far,so it is difficult to carry out traditional molecular and genetic manipulations,thus greatly restricting the understanding of the virulence mechanism of HLB pathogens.With the development of sequencing technologies and bioinformatics,the whole genome sequence of the first CLas strain Psy62 was released in2009,which laid the foundation for subsequent research.Effector proteins are a class of important proteins encoded by pathogens and can be classified into apoplastic effectors and intracellular effectors according to their localization in host cells.In some cases,it is also called virulence factor,which is an important weapon for pathogens to infect the host.Bioinformatics analysis showed that the genome of CLas had a complete Sec secretion pathway.Of the 166 putative effector proteins,86 proteins were confirmed to contain signal peptides by experiments,44 of were differentially expressed in plants and psyllids,indicating that these proteins maybe participate in the response of the pathogen-host interactions.So far,there have been only few reports on the secreted proteins of the Sec-dependent pathway,more secretion protein functions are deserved to be dissected for laying theoretical basis for the prevention and control of the disease.In this study,the candidate secreted protein Clsp33 was selected for further research.Based on the bioinformatics analysis and the validation of extracellular secretion system,Clsp33 was identified as a secretory protein;the relative expression of Clsp33 in citrus and psyllid was analyzed by RT-qPCR;the subcellular localization and biological function of Clsp33 were analyzed by constructing plant expression vectors;its interaction protein was verified by yeast two-hybrid technique and bimolecular fluorescence complementation（BiFC）.The main findings obtained are as follows:1.Blast analysis using the nucleic acid sequence and amino acid sequence of Clsp33 from the genome of Candidatus Liberibacter asiaticus str.psy62 showed that Clsp33 was highly conserved.The transmembrane domain and signal peptide prediction of Clsp33 using TMHMM Server v.2.0 and signal 4.1 Server online site showed that the protein did not contain a transmembrane domain but contained a typical signal peptide with a length of 20 aa.2.The extracellular secretion verification system was successfully constructed using the alkaline phosphatase（phoA）secretion characteristics.Clsp33 signal peptide was fused to phoA with the deletion of signal peptide and successfully expressed.After transformation into E.coli BL21,the blue colonies showed in LB selection culture,similar to the colonies expressing phoA intact protein.Clsp33 signal peptide successfully promoted the secretion of phoA,indicating that Clsp33 has secretory ability.3.The relative expression of Clap33 in CLas-infected sweet orange and psyllid by RT-qPCR showed that the expression level of this gene in sweet orange was about 6 times that in psyllid,suggesting the candidate protein may be a potential effector in plant hosts.4.The subcellular localization of Clsp33 in N.benthamiana epidermal cells showed that Clsp33 was widely distributed in the nucleus and cytoplasm at 25℃,but the nuclear accumulation of Clsp33 was greatly restricted after rising temperature to 32℃.The mutations of five tyrosines K₅₂,K₆₃,K₆₄,K₇₁ and K₇₂ in the Clsp33 nuclear localization signal sequence had no significant effect on subcellular localization,whereas the introduction of exogenous NLS or NES had a significant effect on the subcellular localization of Clsp33.At the same time,the biological function analysis of Clsp33 was carried out by PVX vector indicated that Clsp33was pathogenic to N.benthamiana,which was presumed to be a potential virulence factor.Simultaneous mutation of five tyrosines K₅₂,K₆₃,K₆₄,K₇₁ and K₇₂ in the Clsp33 nuclear localization signal sequence affected its pathogenicity to N.benthamiana,but the single mutation or the two mutations had no significant effect on pathogenicity.The introduction of exogenous NLS or NES had a significant effect on the pathogenicity of Clsp33,speculating that the pathogenicity of Clsp33 was related to its amount entering the nucleus.5.A preliminary screening of Arabidopsis thaliana plasmid library by yeast two-hybrid technique revealed that Clsp33 has strong interaction with PSR1-Interacting Protein 1（PINP1）.The weak interaction between Clsp33 and the homologous protein CsPINP1 in sweet orange was confirmed by X-β-gel detection,ONPG method and BiFC.It was speculated that CsPINP1may be a potential virulence target of Clsp33.