Characterization And Bioinformatics Analysis Of Six Soybean Proteins Interacting With Soybean Mosaic Virus P3 Protein

Soybean mosaic virus(SMV)is one of the most prevalent and devastating viral pathogens in soybean-growing regions worldwide.It causes seed quality and yield loss deficiency seriously,and has been a great threat to soybean production.SMV is classified as the genus potyvirus in the family potyviridae with narrow hosts,limited primarily to the leguminosea.Potyvirus is the largest plant viruse in the world and responsible for severe economic losses to agriculture.Potyviruses encoded 11 proteins and interact with host proteins to complete their infection cycle.P3 protein is the most variable polyprotein in potyviruses,and not only plays a major role in virus replication and movement,but also is responsible for symptom development.Previously,our lab isolated several host proteins interacting with SMV P3 via a two-hybrid yeast system by screening a soybean cDNA library.In this study,we selected randomly six genes,which are GmTIM、GmHR、GmADF2、GmRTNLB2、GmeEF1A、GmSEC,and cloned full-length coding sequence of them from soybean susceptible cultivar’nannong 1138-2’.The main obtained results are as follows:1.The preliminary validation of interaction of six soybean candidate proteins with SMV P3 protein via yeast two hybrid(Y2H)methodMembrane protein yeast two hybrid system was performed according to the manufacturer’s protocols this study,SMV P3 and full-length coding regions of six soybean protein(GmTIM、GmHR、GmADF2、GmRTNLB2、GmeEF1A and GmSEC)respectively constructed into the yeast expression vector pBT3-STE and pPR3-N,Western blotting tests show that the genes can expressed with correct in the yeast cell.Co-transformation of yeast results show that the SMV P3 respectively with six soybean protein in SD/-Ade/-His/-Leu-Trp/X-a-Gal medium grew well,and blue colonies were considered as positive clones.The results show that the interaction of SMV P3 with the six soybean proteins.2.Further verification of interaction between six soybean proteins and SMV P3 protein in vivo and analysis of protein subcellular localizationBimolecular fluorescence complementation were performed to confirmed the interaction of the above in vivo.SMV P3 and six soybean protein(GmTIM、GmHR、GmADF2、GmRTNLB2、GmeEF1A and GmSEC)were inserted into the BiFC pEarleyGate201-YC and pEarleyGate202-YN.The fusion expression vectors were introduced into onion epiderma cells with transient expression by Agrobacterium-mediated method.The agro-infiltrated leaves were observed under a spectral confocal laser scanning microscope.The results show that the strong interactions SMV P3 with six soybean protein,emerging steady fluorescent.Interaction of P3 with soybean GmTIM、GmHR occurred in the cytomembrane of Nicotiana benthamiana cells.Interaction of P3 with soybean GmADF2 occurred in both the cytomembrane and cytoskeleton.Interactions of P3 with soybean GmRTNLB2、GmeEF1A、GmSEC occurred in the cytomembrane、the nucleus and cytoskeleton.Subcellular localization results were similar to BiFC results,GmTIM、GmeEF1A protein was located in the cell membrane,GmADF2 protein was located in the cell membrane and cytoskeleton,GmHR、GmRTNLB2、GmSEC and P3 proteins were located in the cell membrane and the nucleus.The above results show that the interactions SMV P3 with six soybean proteins.3.Expression patterns of six soybean genes under SMV stressThe study analyzed genes expression of soybean genes using RT-PCR after soybean mosaic virus(SMV)treatment at a different time.Results showed that different expression levels of GmTIM、GmHR、GmADF2、GmRTNLB2.GmeEF1A and GmSEC genes under SMV stress.GmTIM mRNA levels showed rising 2 h post-inoculation,showed the highest levels 24 h post-inoculation,had a rising 7d post-inoculation.GmHR mRNA levels showed no change in early stage of post-inoculation,the highest in 12 h post-inoculation,GmADF2 mRNA levels showed a slow upregulated in early stage of post-inoculation,the highest in 12 h post-inoculation.GmRTNLB2 mRNA levels had a obvious upregulated 14 d post-inoculation.GmeEF1A mRNA levels showed a slow upregulated in early stage of post-inoculation,the highest in 24 h post-inoculation,a downregulated after that.GmSEC mRNA levels showed a upregulated in early stage of post-inoculation,the highest in 24 h post-inoculation.4.Bioinformatic assay of Six soybean genesThe study analysised bioinformatics of six soybean genes,GmTIM belongs to transmembrane protein,there are four transmembrane region,amino acid sequence is relatively conservative,the phylogenetic tree analysis indicate that GmTIM is closely related to Phaseolus vulgaris,less similarity with other plant species.GmHR is a transmembrane protein,there are three transmembrane region,the amino acid sequence is relatively conservative,there are a lot differe.nt from amino acid sequence of other plant species,the phylogenetic tree analysis indicate that GmHR is closely related to Medicago truncatula and Phaseolus vulgaris,is a near related to Arabidopsis thaliana、Nicotiona sylvestris and Oryza sativa,less similarity with other plant species Theobroma cacao and Ricinus communis.Amino acid sequence alignment of GmADF2 results showed GmADF2 amino acid sequences is relatively conservative,the C-terminal is more conservative than N-end,the phylogenetic tree analysis showed GmADF2 is closely related to amino acid sequences of Cicer arietinum ADF2,is a near related to Nicotiona sylvestris and Solanum tuberosum,less similarity with other plant species.GmRTNLB2 results show that GmRTNLB2 belongs to transmembrane protein,there are four transmembrane region,the amino acid sequence alignment results show GmRTNLB2 amino acid sequence is not particularly conservative,the phylogenetic tree analysis showed GmRTNLB2 is closely related to amino acid sequences of Cicer arietinum,is a near related to Gossypium arboreum,less similarity with RTNLB2 of other plant species.Amino acid sequence alignment of GmeEF1A results show amino acid sequence of GmeEF1 A is very conservative,a little differences in sequence,the phylogenetic tree analysis showed GmeEF1 A is closely related to amino acid sequences of Cicer arietinum,is a near related to Solanum tuberosum and Nicotiona sylvestris,less similarity with other plant species.GmSEC results show GmSEC belongs to transmembrane protein,there are ten area across the membrane,Amino acid sequence alignment of GmSEC results showed that the amino acid sequence of GmSEC is very conservative,GmSEC、MtSEC and CaSEC have some unique amino acid sequence,it is noted that all of them belongs to Leguminosae,the phylogenetic tree analysis showed GmSEC is closely related to amino acid sequences of Medicago truncatula and Cicer arietinum,is a near related to Arabidopsis thaliana、Nicotiona sylvestris and Solanum lycopersicum,less similarity with other plant species.The results demonstrated the direct interactions between SMV P3 and each of six soybean proteins(GmTIM、GmHR、GmADF2、GmRTNLB2、GmeEF1A and GmSEC).The possible role of these soybean genes in SMV infection was also discussed in this study.