An Efficient Homologous Recombination-Based In Situ Protein-Labeling Method in Verticillium dahliae

Author:

Yang Jie12,Liu Mengran12,Jiao Yue3,Guo Hui-Shan1,Shan Chun-Min1ORCID,Wang Haiting1

Affiliation:

1. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

2. University of Chinese Academy of Sciences, Beijing 100049, China

3. Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100176, China

Abstract

Accurate determination of protein localization, levels, or protein−protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.

Funder

Chinese Academy of Sciences

Publisher

MDPI AG

Subject

General Agricultural and Biological Sciences,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology

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