A Preliminary Study On The Involvement Of Phosphatidic Acid Synthase LPAT In Soybean Mosaic Virus Infection

Soybean(Glycine max)is the most important source of edible oil and animal feed in the world.The viral disease caused by Soybean mosaic virus(SMV)is one of the main diseases that affect soybean production in the world,and it is also one of the most important soybean diseases in the northeast,Huanghuaihai,Yangtze River valleys and southern soybean production areas in China.Because of its large damage area,which seriously affects soybean yield and quality,the research on SMV has always been valued by scholars at home and abroad.Plant viruses are obligate parasites and can only replicate and package their genomes in infected cells.In order to effectively use host cell resources for genome replication,plant viruses must interact with host cells,manipulate the host cell pathways,and ultimately transform host cells into "virus factories." Therefore,studying the functions of various replication-related or replication-regulating host factors in plant virus replication is one of the main frontiers of current virus research.In recent years,many studies have shown that cell lipids play an important role in the virus life cycle.Viruses can target lipid signals and lipid synthesis and metabolic pathways,thereby transforming host cells into the most suitable environment for virus replication.Currently,we recognize that lipids play a key role in viral replication,but our understanding of the lipid synthesis pathway involved in viral replication remains limited.How lipids or lipid synthesis pathways are involved in infection of Potyvirus,a virus that causes important agricultural diseases,is unclear.In this paper,we used the SMV Chinese isolate SC7 as a model,based on the SMV reverse genetics system,through exploring the interaction between SMV and membrane lipid synthetase,to find the key pathways and key factors of membrane lipid synthesis involved in virus replication.In order to explore whether there is an interaction between SMV and lipid synthetase,we cloned the genes related to the key enzymes in the lipid synthesis pathway in Arabidopsis and constructed a yeast two-hybrid screening library.The results showed that: Most of the phospholipid synthase in the plasma reticulum and chloroplast can interact with SMV replication protein 6K2,suggesting that the interaction between SMV replication and lipids may be carried out in the endoplasmic reticulum and chloroplast.We suggest that LPAT,a key protease that catalyses de initio PA synthesis,may be a key host factor in SMV replication.There are two main subtypes of LPATs in plants,which correspond to chloroplast localization or endoplasmic reticulum localization,respectively.We constructed Arabidopsis thaliana and Glycine max LPAT gene clones and constructed yeast two-hybrid vectors.Yeast two-hybrid experiments showed that 6K2 can interact with At LPAT1/2/3/4/5,and NIb can interact with At LPAT1;6K2 and NIb Both can interact with Gm LPAT1.In addition,6K2 can also interact with Gm LPAT2/3/4/5.Observation of subcellular localization of Nb/At/Gm LPAT1 showed that the LPAT1 of the three plants are all localized on the chloroplast.After virus infection,LPAT1 is recruited at the replication site of the virus.Next,we successfully silenced Nb LPAT1 using the TNV-p82 C mediated gene silencing system that has been successfully constructed in the laboratory.WB results show that silencing of Nb LPAT1 can significantly reduce the accumulation of SMV CP.These preliminary experimental results show that virus multiplication requires chloroplast-localized LPAT1.We speculate that LPAT1 is used by the virus to synthesize a large amount of membrane lipid precursor PA at the viral replication site with a membrane structure.By observing the subcellular localization of Nb/At/Gm LPAT2 and Nb/At/Gm LPAT4 showed that,the LPAT2/4 of the three plants present obvious endoplasmic reticulum localization Positioning.And LPAT2/4 co-located with the virus replication site after virus infection.After using the TNV-p82 C mediated gene silencing system to silence Nb LPAT2+4,WB results showed that SMV CP accumulation increased.We constructed the transient expression vectors of Nb/At/Gm LPATs and overexpressed these LPATs under SMV infection.The results showed the accumulation of SMV CP is significantly inhibited when Flag-At LPAT4,Flag-Gm LPAT2 b,and Flag-Gm LPAT2 d was overexpressed,respectively.The overexpression of Flag-At LPAT4-m AT,which has lost the acyltransferase activity,does not affect the accumulation of SMV CP.The results indicate that At LPAT4 relies on its acyltransferase activity to inhibit virus replication.Overexpression of At LPAT2-Flag and At LPAT2-Flag-m AT does not affect the accumulation of viral CP,but the expression size of At LPAT2-Flag and At LPAT2-Flag-m AT is larger than our predicted size,which is predicted to be a ubiquitinated protein.Based on these experimental results,we conclude that the non-ubiquitinated LPAT2/4 located in the endoplasmic reticulum inhibits viral replication,and its acyltransferase activity plays an important role in viral replication.It is speculated that its acyltransferase activity caused autophagy to degrade the virus.In addition,we tried to establish a replication system of SMV in Saccharomyces cerevisiae,but current experiments show that this yeast clone can only background translation of SMV CP protein but no replication.In the future,we will design and optimize this replication system from more perspectives,so that SMV can replicate in yeast,and lay the foundation for the identification of host factors that interact with SMV.In summary,this study identified LPAT subtypes that play a key role in SMV replication through the interaction between different subtypes of phosphatidic acid synthase LPAT and SMV and the genetic analysis of different subtypes of LPAT.Clarify the role of LPAT as a key host factor in virus replication.