Functional Analysis Of Effector Proteins AGH17470 And AGH17488 In The Prophage Region Of "Candidatus Liberibacter Asiaticus"

Citrus Huanglongbing（HLB）is a devastating disease in citrus production,and it has been prevalent in more than 50 countries or regions,including China,the United States and Brazil.So far,HLB is present in 10 provinces in mainland China.HLB can cause phloem necrosis of citrus,blockage of sieve tubes or root rot,and small and sour fruits such as green fruit or red nose fruit,which seriously affects the yield and quality of citrus,and threatens the safety of Chinese citrus industry.The causal agents of HLB,which are believed to be Gram negative non culturable bacteria,belong to genus“Candidatus Liberibacter”.So far,three species of the bacterium have been identified:“Ca.L.asiaticus”（CLas）,“Ca.L.americanus”（CLam）,and“Ca.L.africanus”（CLaf）.The pathogen of HLB in China is CLas.Due to the inability to culture CLas in vitro,the pathogenicity of CLas remains largely unknown.The genome of CLas is about 1.23 Mb in size and consists of conserved genomic regions and prophage regions with large variation.Currently,three representative prophage types,SC1,SC2 and P-JXGC-3,have been identified from released CLas genomes.The highly reduced genome still confers the ability to secrete numerous effector proteins into the host through the type I secretion system（T1SS）and the Sec-dependent secretion system.In recent years,the prediction,screening and functional study of secretary proteins have become the hotspots of HLB research.In this study,a genome-wide identification of secretory proteins from representative CLas prophage types was conducted and two secretory proteins were chose for further study.1.Screening of secreted proteins in the prophage region of CLasThe Signal P,Phobius,TMHMM 2.0 and Secretome P 2.0 were used to predict classical and non-canonical secreted proteins encoded by prophage P-gxpsy-1,P-gxpsy-2 and P-JXGC-3.Five secreted proteins which can cause/inhibit necrosis were identified by the transient expression of Nicotiana benthamiana mediated by potato virus X（PVX）.Among them,the transient expression of AGH17470 protein can induce necrosis of N.benthamiana leaves,and AGH17460,AGH17488,AGH17492and ARB06709 proteins can inhibit the hypersensitive response（HR）triggered by INF1 and Bax.RT-qPCR analysis indicated that the gene expression level of three secretary proteins,AGH17470,AGH17488 and AGH17460 were significantly higher in CLas-infected citrus than those in psyllids.The in silico prediction combined with the alkaline phosphatase（PhoA）validation supported the secretion ability of these proteins.2.Identification and functional analysis of non-classical secretary protein AGH17470Through the PhoA assay,it was confirmed that the first 50 and last 50 amino acid sequences of AGH17470 protein jointly determined its secretion.Transient expression of AGH17470 by PVX can trigger HR,ion leakage and accumulation of reactive oxygen species（ROS）in N.benthamiana leaves.The truncation analysis revealed that the integrity of the AGH17470 protein is critical for triggering necrosis in N.benthamiana leaves.To determine its interacting target,yeast two-hybrid library screening,co-immunoprecipitation combined with mass spectrometry（IP-MS）analysis suggested multiple interacting potential proteins in citrus or N.benthamiana.However,no interacting proteins were identified via yeast two-hybrid（Y2H）.RT-qPCR showed that Nb HIN1,PR family genes（Nb PR1,Nb PR2,Nb PR3,Nb PR4 and Nb PR5）and SA pathway signal transduction genes Nb NPR1,Nb TGA and Nb EDS1 were significantly up-regulated after transient expression of AGH17470.AGH17470 could induce the up-regulated expression of Cs NPR1,Cs TGA,Cs PR1,Cs PR2,Cs PR3 and Cs PR5 in citrus transgenic shoots of AGH17470.High performance liquid chromatography analysis showed that the content of free salicylic acid（SA）was significantly accumulated in the shoots of the AGH17470-transgenic citrus and the leaves of N.benthamiana and citrus transiently expressing AGH17470.Symptoms observation and bacterial counts showed that transient expression of AGH17470 improved the resistance of citrus to Xanthomonas citri subsp.citri（Xcc）.3.Functional analysis of AGH17488-APX6 interactionTransient expression of AGH17488 in N.benthamiana suppressed programmed cell death（PCD）,ROS accumulation and ion leakage induced by the Phytophthora infestans elicitin INF1,pro-apoptotic mouse protein Bax and AGH17470.PhoA assay revealed that AGH17488 is an extracellular secreted protein,and subcellular localization showed that AGH17488 was localized to the cell membrane and nucleus of N.benthamiana leaf cells.Y2H,BiFC and Co-IP confirmed that AGH17488 interacts with citrus L-ascorbate peroxidase 6（CsAPX6）and N.benthamiana L-ascorbate peroxidase 6（NbAPX6）in vitro and in vivo.Subcellular localization showed that CsAPX6 was localized in the chloroplast,and NbAPX6 was localized in the chloroplast and membrane.AGH17488 co-localized with NbAPX6/CsAPX6 in the cell membrane,which was consistent with the result that the interaction between AGH17488 and APX6 only occurred in the cell membrane by BiFC.When AGH17488was transiently expressed,the expression level of NbAPX6 and CsAPX6 did not change significantly.Prokaryotic expression of AGH17488,CsAPX6 and NbAPX6 for APX enzyme activity assay showed that CsAPX6 and NbAPX6 have ascorbate peroxidase activity.APX activity and ROS detection showed that the AGH17488 can promote APX6 activity and reduce the accumulation of hydrogen peroxide（H₂O₂）,superoxide anion（O₂^-）and lipid oxidation end product malondialdehyde（MDA）after transient expression of AGH17488 and CsAPX6 in citrus leaves.Transient expression of NbAPX6 also inhibited AGH17470,INF1 and Bax-induced plant cell necrosis.Transient expression of AGH17488,NbAPX6 or CsAPX6 in N.benthamiana and citrus promote the proliferation of Pseudomonas syringae pv.tomato DC3000（Pst DC3000）and Xcc,and enhance the susceptibility of the host.The proliferation of Pst DC3000was significantly reduced in the NbAPX6-silenced plants,indicating that NbAPX6 is involved in biotic stress response.In conclusion,the function analysis of the secretary proteins AGH17470 and AGH17488 of CLas prophage region demonstrated that overexpression of AGH17470in N.benthamiana and citrus plants upregulated the transcription of PR family and salicylic acid（SA）-signaling pathway genes,promoted SA accumulation and thus enhance citrus resistance to Xcc.AGH17488 interacts with APX6 protein and reduces ROS accumulation by promoting APX activity which further to enhance the susceptibility to the host.This study reveals a novel mechanism by which AGH17488promotes bacterial proliferation by interfering with ROS accumulation.