Function Of Nicotiana Benthamiana Chloroplast Heat Shock Prtoein 90 In The Process Of Pepper Mild Mottle Virus Infection

Pepper mild mottle virus (PMMoV) is a member of the genus Tobamovirus.PMMoV infected plants exhibit symptoms such as mosaics,which may related to chloroplast damage.Chloroplast-localized Hsp90 acts as a molecular chaperone involved in the import of chloroplast proteins,chloroplast formation and function maintenance.In this study,we analysis the relationship between NbHsp90.5 and PMMoV with Nicotiana benthamiana plants.In this study,we searched the N.benthamiana database with an Arabidopsis chloroplast Hsp90 sequence,and obtained two N.benthamiana Hsp90s with PCR amplification,and named as NbHsp90.5-1,NbHsp90.5-2 respectively.The two NbHsp90.5s showed 86.12% indentity at amino acid level,and less similarity in the approximatedly 60 amino acids segment at the N and C terminal.Evolutionary analysis indicated that NbHsp90.5-1,NbHsp90.5-2 were closely related to Hsp90.5 of Nicotiana tabacum and Nicotiana attenuate.Tobacco rattle virus(TRV)-based virus-induced gene silencing(VIGS) was used to down-regulate the expression of Hsp90.5s in N.benthamiana to explore the function of NbHsp90.5s.Silencing of nbhsp90.5s resulted in chlorosis in the system leaves,and nbhsp90.5-2 silencing plants showed more obvious chlorosis.However,silencing of nbhsp90.5-1/2 had no significant difference in plant height,number of new leaves and leaf size in N.benthamiana.Ultrastructural analysis showed that the degree of leaf chlorosis was positively correlated with the degree of chloroplast structure damage.When these Hsp90.5s-silenced plants were challenged with PMMoV-GFP,this results showed that silencing of nbhsp90.5-2 increased the accumulation of PMMoV-GFP in N.benthamiana,semi-quantitative RT-PCR showed TRV:nbhsp90.5-2 did not cause significant down-regulation of other Hsp90s.Further studies showed that PMMoV-CP was encapsulated around the chloroplast,but no significant colocalization with NbHsp90.5-1/2,and no significant change in the chloroplast localization of NbHsp90.5-1/2.Taken together,the above results indicate that nbhsp90.5-2 silencing lead to significant changes in chloroplast structure,leading to obvious yellowing symptoms,and nbhsp90.5-2 silencing promotes PMMoV-GFP infection.In addition,NbHsp90.5-2 cannot be replaced by NbHsp90.5-1 in chloroplast formation and resistance pathway.