Screening Of Host Factors Interacting With P10 Protein Of Southern Rice Black Stripe Dwarf Virus And Its Effect On Chloroplast Structure And Function

Southern rice black-streaked dwarf virus(SRBSDV)is a doublestranded RNA virus transmitted by the insect vector Sogatella furcifera(WBPH),which causes rice black-streaked dwarf disease in rice.SRBSDV-infected plants are dwarfed and severely affects crop yield.It is unclear that the roles of the coat protein P10 viral pathogenisis.Therefore,this study aims to screen the host factors interacting with P10 through the yeast two-hybrid system,and study their roles in the SRBSDV infection.In this study,we constructed the bait plasmid p BT3-SUC-P10,which was used a bait to screening the yeast DUAL membrane system rice c DNA library to find the host factors interacting with P10.Three chloroplastrelated genes were obtained and further confirmed their interaction by one-by-one yeast two hybrids methods.Using Co-IP technology,it was verified that Os PS1-L,Os PT3,Os LPA1 can interact with P10 protein in N.benthamiana leaves.It was observed that P10 protein and Os PS1-L are localized on the chloroplast and cytoplasm in N.benthamiana leaves,and are co-located with Os PT3 and Os LPA1 on the cytoplasm,endoplasmic reticulum and nuclear membrane.The expression of these proteinsand the degradation and functional structural proteins of chlorophyll synthesis were analyzed in transgenic rice OE-P10.The results indicated that expression of P10 proteins in rice has significantly affected the expression of chloroplast-related genes.Detection of chlorophyll fluorescence parameters of OE-P10 rice leaves showed that the maximum fluorescence yield(Fm)and the maximum PSII light energy conversion efficiency(Fv/Fm)showed a downward trend,indicating that the original light energy conversion efficiency of the leaves in OE-P10 was markly lower and the electron transfer capacity of the photosynthetic mechanism was obviously reduced,compared with those in wildtype plant ZH11.Meanwhile,the efficiency of light energy conversion in PS II reaction centers is affected in P10-expressed plants.Examination of the photochemical pathways of leaves Y(II),Y(NPQ)and Y(NO)foundthat compared with control rice,OE-P10 rice leaves Y(II)decreased significantly,while Y(NPQ)and Y(NO)increased,indicating that OEP10 releases light energy through its own regulation mechanism and heat dissipation,reducing photosynthetic efficiency.The chloroplast ultrastructure of OE-P10 leaves of transgenic rice was observed.Compared with that of wild-type rice ZH11,the thylakoid structure of OE-P10 was unclear.The thylakoids are not arranged neatly as compared with controls and the number of amyloids is increased.Therefore,it is speculated that the expression of P10 proteins in plants affects the host's chloroplast structure and function,which is not conducive to plant photosynthesis.The expression of chloroplast-related genes in SRBSDV-infected rice was also detected by RT-q PCR.The results indicated that SRBSDV infection significantly affected the expression of chloroplast-related genes in rice.Analysis of chlorophyll fluorescence parameters of rice leaves showed that the maximum fluorescence yield(Fm)and the maximum PSII light energy conversion efficiency(Fv/Fm)in the leaves of SRBSDV infected plants showed a downward trend compared with that in wildtype plants,indicating that the conversion efficiency has been reduced.The conversion of photochemical pathways into energy Y(II)was significantly reduced,and PS II regulated energy dissipation Y(NPQ)and non-regulated energy dissipation Y(NO)were significantly increased.These results indicated that photoinhibition occurs in susceptible rice and its absorption light energy is released through its own regulation mechanism and heat dissipation,and photosynthetic efficiency decreases.Transmission electron microscopy observations showed that after SRBSDV infection,the most obvious changes in chloroplasts in mesophyll cells were destruction of chloroplasts,degradation of the outer membrane,incomplete structure,and thylakoid sheets dispersed in the cells.In this study we screened host factors that interacted with the SRBSDV coat protein P10,and further verified their interactions by Y2 H and Co-IP.The chloroplast structure and function of the transgenic rice OE-P10 and SRBSDV infected rice were changed compared with that ofwildtype plants,indicating that the P10 protein may play important roles by affecting the function and structure of the chloroplast during SRBSDV infection.