Identification And Subcellular Localization Of Host Factors From Malus Sylvestris Cv. R12740-7A Interacting With ACLSV

Apple chlorotic leaf spot virus(ACLSV) is one of the most economic important latent viruses on apple, which distributes worldwide. The virus can cause remarkable economic losses in both yield and quality when infection alone or in combination with other viruses. In this study, on the basis of obtaining the host proteins interacting with coat protein(CP) and movement protein(MP) of ACLSV by yeast two-hybrid system, the significant host factors were selected by bioinformatics analysis. Then the further study was made by using the yeast two-hybrid system and pull-down technology. After that, CP, MP and Malus15-1 were also subcellular localized in Nicotiana benthamiana. The results of this study will provide theoretical basis to reveal the biological function of host genes in ACLSV infection process and interacting significance between host and virus. The main results are as follows:1. The interaction between Malus15-1, Malus40-5 and CP, MP of ACLSV was verified by the method of yeast two-hybrid system. Firstly, yeast expression vectors of Malus4-1,Malus15-1, Malus40-5, Malus72-3, Malus85-1 and Malus86-1 were constructed. Then, the bait vector of p GBKT7-CP, p GBKT7-MP and yeast expression vector of host gene were co-transformed respectively into yeast strain Y2 H gold, and the transformants were cultured on the SD/-Ade/-His/-Leu/-Trp/X-α-Gal media. The growth and the color of yeast were observed after cultured. The results showed that Malus15-1 and Malus40-5 presented clear blue on selective media, which proved that CP and MP interacted with host factors Malus15-1 and Malus40-5.2. The interection between CP, MP and Malus15-1 was verified by the method of pull-down. Firstly, the prokaryotic expression vectors of p ET28a-CP, p ET28a-MP, and p ET28a-15-1 and p ET28a-40-5 were constructed and transformed into E.coli Rosetta(DE3) and induced to express. SDS-PAGE and Western blotting analysis of products from pull-down experiment showed that, there were 21.5 k Da and 16 k Da two bands, 50.8 k Da and 16 k Da two bands in the eluting products respectively, using CP and MP as bait to capture Malus15-1 protein. It showed that both CP and MP interacted with host factors Malus15-1. In the same way, there was only one band in eluting products using CP and MP as bait to capture Malus40-5. It showed that both CP and MP could not interact with host factors Malus40-5.3. The subcellular localization of CP, MP and Malus15-1were revealed. Transient expression vectors of p Cam35s-gfp-CP, p Cam35s-gfp-MP and p Cam35s-gfp-15 were constructed, subsequently transformed to agrobacterium tumefaciend strain EHA105 by frozen thawing method, and then injected into the tobacco leaf epidermis. Nicotiana.benthamiana leaves, expressed the fused protein containing target genes and fluorescent derived from the vector, were visualized by laser confocal scanning microscopy. The results showed that CP may be located in the nucleus and cytoplasm, MP may be located in cytoplasm and endoplasmic reticulum and Malus15-1 may be located in cytoplasm.