Plant Virology and Molecular Plant Pathology Laboratory, Division of Plant Pathology, SKUAST-K, Shalimar, Srinagar 190025, India.
Plant Virology and Molecular Plant Pathology Laboratory, Division of Plant Pathology, SKUAST-K, Shalimar, Srinagar 190025, India.
J Microbiol Methods. 2020 Apr;171:105885. doi: 10.1016/j.mimet.2020.105885. Epub 2020 Mar 5.
Thyrostroma carpophilum, a causal agent of shot hole disease of stone fruits, cause severe loss in economically important fruit crops of Kashmir. Understanding its pathogenesis at molecular level will aid in devising a better management strategy. In this study, we optimized Agrobacterium tumefaciens mediated transformation (ATMT) conditions for T. carpophilum using PBIF2-EGFP construct. Using this protocol, we obtained 328 positive transformants per 10 spores and subsequent sub-culturing of transformants on selective and non-selective media resulted in stable T-DNA integration. Southern blot analysis revealed that most of the transformants embodied single T-DNA integration. Using this method, we obtained a small-scale transformant library (2050 transformants). Among this pool, we tested 1005 transformants for their pathogenicity; out of which 185 showed complete pathogenicity loss, 35 displayed reduced virulence and 785 were pathogenically similar to wild type. Out of this experimental stock, three transformants from each category were randomly selected to dissect the infection assay. The findings deciphered that transformants with complete pathogenicity loss failed to penetrate the host tissue and a few transformants failed to sporulate in laboratory. Transformants from reduced category could not form appressorium and occasionally sporulated. Transformants similar to wild type were morphologically and pathogenically similar to wild type because of un-alteration in their modus operandi. Our work provides a new platform to understand the pathogenicity mechanism of T. carpophilum. The optimized ATMT protocol will help in developing large transformant library that can help to identify the virulence arsenals necessary for the pathogen to cause disease.
石果树穿孔病的病原菌枝孢菌，会给克什米尔地区有重要经济价值的水果作物造成严重损失。在分子水平上了解其致病机制将有助于制定更好的管理策略。本研究利用 PBIF2-EGFP 构建体优化了枝孢菌的农杆菌介导转化（ATMT）条件。利用该方案，我们从每 10 个孢子中获得了 328 个阳性转化体，随后在选择性和非选择性培养基上对转化体进行继代培养，导致 T-DNA 稳定整合。Southern blot 分析表明，大多数转化体都含有单个 T-DNA 整合。利用该方法，我们获得了一个小规模的转化体文库（2050 个转化体）。在这个文库中，我们测试了 1005 个转化体的致病性；其中 185 个转化体完全丧失了致病性，35 个转化体表现出毒力降低，785 个转化体与野生型相似。在这个实验种群中，从每个类别中随机选择三个转化体进行感染分析。研究结果表明，完全丧失致病性的转化体无法穿透宿主组织，少数转化体无法在实验室中形成孢子。毒力降低类别的转化体无法形成附着胞，偶尔会形成孢子。形态和致病性与野生型相似的转化体与野生型相似，因为它们的操作模式没有改变。我们的工作为理解枝孢菌的致病性机制提供了一个新的平台。优化的 ATMT 方案将有助于开发大型转化体文库，有助于鉴定病原菌致病所需的毒力武器。
