马铃薯Y病毒（PVY）突破寄主eif4e1基因抗性的机理解析

Potato virus Y (PVY) is an important disease on Solanaceae crops. Early studies of the applicant research group indicated that the PVY resistant tobacco cultivar (Va genotype) were eukaryotic initiation factor 4E1 (eIF4E1) gene deletion (hereafter as eif4e1 or va), spontaneous resistance-breaking PVY (hereafter as PVYvaB) mutants have detected. Through sequence analysis of PVYvaB and eIF4E family members, the applicant proposes the hypothesis that PVYvaB convert to utilize an unknown eIF4E family member to complete the infection cycle. This project intends to determine the resistance broken level by determining the key amino acid sites of PVYvaB VPg and constructing the infectious cloning of target mutation PVY wild type (PVYWT) VPg gene. Yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assay will be conducted to seek for PVYvaB interacting candidate gene(s) (refer as eIF4Ex) from eIF4E family. eif4ex/eif4e1 mutant, obtains by Crispr-Cas9 in va/va plant, will be used to verify molecular function of eIF4Ex in resistance against to PVYvaB and other potyviruses. The resistance durability of pyramiding eif4e1 and eif4ex genes together will be evaluated. This research will contribute to an understanding of the mechanism of PVY variation broken va resistance, providing guidance for disease management and developing durable resistance to PVY.

马铃薯Y病毒病（Potato virus Y, PVY）是茄科作物的重要病害，课题组前期研究表明抗PVY栽培烟草（va基因型）中va位点为eIF4E1基因缺失，并发现可克服va抗性的PVY分离物（命名为PVYvaB）。通过对PVYvaB和eIF4E家族成员的序列分析，申请人提出“PVYvaB转换利用某个eIF4E家族成员完成侵染循环”的假说。本项目拟确定PVYvaB VPg的关键氨基酸位点、并构建定点突变PVYWT VPg基因的侵染性克隆，明确抗性突破发生水平。同时利用酵母双杂交和双分子荧光互补实验确定与PVYvaB VPg互作的eIF4E（以eIF4Ex表示）。在va背景下，利用Crispr-Cas9技术获得eif4e1/eif4ex双突变材料，并鉴定其对PVYWT、PVYvaB和同属病毒的抗性，验证eIF4Ex的功能。阐明PVY变异克服va抗性的分子机理，可指导病害治理和持久抗性发掘。

为了研究PVY VPg与eIF4E家族基因在抗性获得和抗性突破中的机理，本项目利用野生型PVY坏死株系（PVYWT）摩擦接种va普通烟TN86，个别单株出现可克服va抗性的突变病毒（PVYvaB）。构建PVYvaB侵染性克隆，证明PVY VPg-K105Q突变可克服va抗性。为了寻找VPg-K105Q突变病毒在va烟草中利用的eIF4E家族基因，分别构建普通烟草中12个eIF4E家族基因和VPg-K105Q、VPg-WT的酵母双杂交载体，体外杂交表明VPg-K105Q与eIFiso4E-T蛋白互作，而VPg-WT不能互作。暗示VPg-K105Q在va烟草中转换利用eIFiso4E-T完成侵染循环。. 采用在va烟草中敲除eIFiso4E-T方法进行功能验证。苗期接种PVYWT和PVYvaB，结果表明单基因敲除va和eifiso4e-tKO/Va表现感PVYvaB，双基因敲除 eifiso4e-tKO/va表现抗PVYvaB。在接种PVYWT情况下，va烟草接种后14 d个别烟株抗性被突破、表现为非持久性，双基因敲除eifiso4e-tKO/va烟草接种后70 d抗性未被突破。表明在va背景下敲除eIFiso4E-T获得对PVYWT的持久抗性。苗期摩擦接种TVBMV和ChiVMV病叶汁液，症状调查和ELISA检测结果表明在va背景下敲除eIFiso4E-S获得对TVBMV和ChiVMV的抗性。其中eifiso4e-sKO/ eifiso4e-tKO /va三突材料持久抗PVY、广谱抗TVBMV和ChiVMV。本项目首次表明，异源四倍体普通烟草在va遗传背景下敲除eIFiao4E-T可获得对PVY的持久抗性、在va遗传背景下敲除eIFiso4E-S可获得对Potyviruses的广谱抗性。为了解Potyviruses与作物抗性的获得与突破提供了参考。