Interaction Between the Coat Protein of Apple Chlorotic Leaf Spot Virus and Two E3 Ubiquitin Ligases of Pear and Their Subcellular Localization
Apple chlorotic leaf spot virus(ACLSV)possesses a positive single stranded RNA and widely infects pear and other fruit trees.The coat protein(CP)of the virus functions in the viral pathogenesis.In order to gain a deep understanding of the interaction characteristics between the virus and its host plants,a yeast two hybrid(Y2H)assay was carried out by using pGBKT7-CP(expressing ACLSV CP)as a bait vector to screen pear proteins in a cDNA library of pear'Hongxiangsu',and 33 host factors potentially interacting with ACLSV CP were identified.Furthermore,the interaction relationships between CP and two RING type E3 ubiquitin ligases(named MIEL and BOI)from pear were confirmed by both Y2H and bimolecular fluorescence complementation(BiFC)assays.In BiFC assays,the interaction signal of CP with BOI located at plasmodesma and nucleus,which was similar to the subcellular locations of the two proteins.The interaction signal of CP with MIEL located at endoplasmic reticulum and nucleus,which was similar to the subcellular locations of MIEL.The truncated mutants of the three proteins were constructed.In Y2H assays,only MIEL mutant containing the zf CHY domain(1-99 aa)interacted with CP,and other MIEL mutants and all BOI mutants failed to interact with CP,and all CP mutants cannot interact with the two host proteins.
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