Study On Biological Functions Of Protein Interactions Between Citrus Tristeza Virus And Its Host And Identification Of Host Genes Targeted By The Virus Derived SiRNA

Citrus tristeza virus(CTV)naturally infects citrus and citrus relatives in the Rutaceae family and is the causal agent of citrus tristeza disease,which is considered as the most devastating viral disease in citrus industry.Nowadays,only a few studies on the interaction between CTV and its host have been reported.In a study previously performed in our laboratory,two proteins cold-regulated 15 k Da protein(COR15)and FK506 banding protein 13-2(FKBP13-2)potentially interacting with CTV proteins p20 and p23 were identified from a library of Citrus sinensis by using a yeast two-hybrid screening.In this study,the interactions between p20 and COR15,and between p23 and FKBP13-2 and the biological function of these interactions were characterized,meanwhile,the genes of citrus targeted by CTV-derived small interfering RNAs(vsi RNAs)were identified.The main results are listed as follows:(1)Yeast split-ubiquitin(membrane-based MYTH assay)and bimolecular-fluorescence complementation(BIFC)assays confirmed the strong interaction between p20 and Ca COR15 of Citrus sinensis and Na COR15 of Nicaniana benthamiana.The effects of the interaction on the suppression activity of CTV RNA silencing suppressor P20 was evaluated on N.benthamiana 16 c leaves.Results showed that the expression of Ca COR15 protein enhanced the gfp gene silencing,and reduced the RNA silencing suppression efficiency of p20.Deep sequencing for small RNAs in infiltrated patches of N.benthamiana line 16 c leaves showed that expression of Ct COR15 protein increased the amount of gfp-derived s RNAs,and in contrast,the expression of p20 protein reduced the number of gfp-derived s RNAs and lowered nucleotide ‘U’ frequencies at the 5’ terminal of gfp-derived s RNAs by 21%.The results suggested that Ct COR15 and p20 might affect the function of SGS3 involved in the RNA silencing amplification process.Further study showed that p20/Ct COR15 interaction complex was indeed co-localized with SGS3 of N.benthamiana,most of the interaction complexes were positioned near nuclear.Bi FC assays further confirmed that p20 and Ct COR15 could respectively interact with SGS3 by forming small florescence bodies.VIGS-based silencing of the endogenous Nb COR15 in N.benthamiana led to 1.8-8 fold increased the accumulation of CTV.Western blot analysis showed that the expression of p20 caused the expression level of SGS3 protein,indicating that p20 might induced the degradation of SGS3.Further analysis found that the interaction complexes of p20 and SGS3 colocalized with an autophagosome marker ATG8 f in N.benthamiana leaf cells.Electronic microscopy observation for the ultra-thin section of the infiltrated N.benthamiana leaves showed that p20 expression induced the production of autophagosomes.These results suggest that silencing suppressor p20 might mediate degradation of Nb SGS3 by regulating autophagy process in N.benthamiana cells.(2)The interactions between p23 and Ct FKBP13-2 of Citrus sinensis and between p23 and Nb FKBP13-2 of Nicaniana benthamiana were confirmed by using membrane-based MYTH and Bi FC assays.In Bi FC assays,extensive YFP fluorescence signals of p23 /Ca FKBP13-2 and p23 /Nb FKBP13-2 interactions appeared at the plasmodesma(PD)of agroinfiltrated cells at 48 hours post infiltration(hpi).The subcellular localization of p23 and Nb FKBP17-2 in the epidermal cells of N.benthamiana leaves was determined,the fluorescence signal of p23 accumulated preferentially as parallel spots at the plasmodesmata,and FKBP13-2 fluorescence signal located in chloroplast.Co-expressing the two proteins p23 and FKBP13-2 showed that the viral p23 can translocate FKBP13-2 from chloroplasts to the PD of epidermal cells of N.benthamiana leaves.Further Bi FC assays showed that FKBP13-2 also could interact with the coat protein(CP)of CTV,and the complex FKBP13-2/CP could rapidly move in the cytoplasm.Moreover,p23 could guide the FKBP13-2/CP complexes to move along cell membrane.The knocked-down expression level of Nb FKBP13-2 m RNA by virus-induced gene silencing resulted in a decrease of CTV titer in N.benthamiana plants inoculated with a CTV infectious clone.The presented results indicated that FKBP13-2 involved in the movement of CTV CP and the virus replication.(3)Small RNA,degradome and transcriptome sequencing were performed for CTV-S45 infected Citrus sinensis leaf samples.Results showed that CTV infection changed the composition of s RNA in Citrus sinensis,in CTV-infected samples that 21-and 22-nt s RNAs were relatively abundant,while in mock-inoculated samples the 24-nt s RNAs were abundant.Totally,594 citrus genes potentially targeted by 662,051CTV-S45 derived vsi RNA were retrieved by s RNA analysis.These targeted genes are mainly involved in protein processing in endoplasmic reticulum,endocytosis,and phenylpropanoid biosynthesis.Transcriptome sequencing showed that the expression of phenylalanine lyase(PAL),involved in lignin synthesis pathway,was down-regulated 249-fold after CTV-S45 infection.The combination analysis of degradome data and vsi RNAs showed that vsi RNA18868 of CTV-S45 might target to citrus gene Cs8g05320,which encodes a UDP glucosyltransferase involved in the disease resistance of plants.RNA ligase-mediated rapid amplification of c DNA ends(RLM-RACE)and a luciferase reporter assays further confirmed the vsi RNA18868 cleaved the m RNA of Cs8g05320 at positions 1132-1133 and 1133-1134.In addition,transcriptome results showed the expression of Cs8g05320 gene was down-regulated 6.77-fold by CTV infection.It is speculated that CTV could down-regulate the expression of Cs8g05320 through its vsi RNA18868,leading to a reduction of disease resistance of citrus.