Interaction Study Between WYMV CP And Host FtsH2

WYMV(Wheat yellow mosaic virus)is a member of the genus of Bymorirus and family of Potyviridae.There are few studies about the formation mechanism of the symptoms of WYMV.In our previous work.the cDNA library of wheat was screened via yeast two-hybrid system,with the WYMV coat protein(CP)as bait protein,and the fragment of the wheat FtsH2 was identified as interacting with CP.CP,the only structural protein of WYMV,is involved in the process of viral packaging and movement and the formation of symptoms.FtsH2 protein,a member of the AAA protease families,is involved in plant chloroplast photodamage repair and thylakoid development.In order to study the interaction between TaFtsH2 and WYMV CP and explore its physiological functions of this interaction in the process of viral infection and symptom formation,the following works were taken:1.Bioinformatical analysis of TaFts H2The CDS of wheat TaFtsH2 was cloned and the similarity and structural domains of TaFtsH2 were analysed with bioinformatical softwares.The results showed that TaFtsH2 possessed a N-temminal transmembrane domain,a intermediate AAA domain and a C-terminal Peptidase_M41 domain.TaFtsH2 shared the highest sequence similarity of 100%with Aegilops chinensis,followed by 95.25%with Brachypodium serrata:the amino acid sequence similarities with corn,rice and sorghum are also greater than 90%,which were 91%,92.02%,91%,respectively.2.Interaction between WYMV CP and TaFtsH2The recombinant plasmids pGADT7-TaFtsH2 and pGBKT7-CPWYMV were co-transfected into yeast,and the results showed that WYMV CP interacted with TaFtsH2 protein in yeast.The agrobacterium containing FtsH2-YFPn and CP-YFPc recombinant plasmids were co-infiltrated into Nicotiana benthamiana,the green fluorescence observed under laser confocal microscopy indicated that WYMV CP interacted with TaFtsH2 protein in vivo.3.Co-localization analysis of WYMV CP and TaFtsH2The CDS of WYMV CP and TaFtsH2 were cloned into fluorescent expression vector.At 48 h post infiltration,CP-GFP was mainly found in the cytoplasm,while the TaFtsH2-GFP was mainly found in the cytoplasm and nucleus,and a small amount was located in the chloroplast.When CP-GFP and TaFtsH2-RFP proteins were co-expressed,no significant change were observed in the case of their subcellular localizations,and they were mainly co-located in the cytoplasm.4.Effect of WYMV infection on TaFtsH2 expressionThe qPCR showed that the mRNA of TaFtsH2 increased firstly and then decreased along with the disease severity of leaves:the mRNA of TaFtsH2 in the leaves of level 1 and level 2 of disease severity increases 1.72 times and 2.81 times compared with the mRNA of TaFtsH2 in healthy leaves,while the mRNA of TaFtsH2 in level 3 and level 4 leaf falled back to the level of healthy leaves.5.Effect of NbFtsH2 on virus infectionThe CDS of NbFtsH2 was cloned,and the similarity of amino acid sequence between NbFtsH2 and TaFtsH2 reached to 82.88%.After knocking down NbFtsH2 by VIGS,the leaf showed the phenomenon of chlorosis.The expression of TuMV was significantly decreased when NbFtsH2was knocked down,suggesting that the expression of FtsH2 gene is related to symptoms,and the silencing of NbFtsH2 gene is harmful for TuMV infection.