A highly efficient Agrobacterium tumefaciens-mediated transformation system for the postharvest pathogen Penicillium digitatum using DsRed and GFP to visualize citrus host colonization

Penicillium digitatum is a major postharvest pathogen of citrus crops. This fungus broadly spreads worldwide and causes green mold disease, which results in severe losses for citrus production. Understanding of the citrus infection by P. digitatum may help develop effective strategies for controllin...

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Bibliographic Details
Published in:	Journal of microbiological methods Vol. 144; pp. 134 - 144
Main Authors:	Vu, Tao Xuan, Ngo, Tho Tien, Mai, Linh Thi Dam, Bui, Tri-Thuc, Le, Diep Hong, Bui, Ha Thi Viet, Nguyen, Huy Quang, Ngo, Binh Xuan, Tran, Van-Tuan
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-01-2018
Subjects:	Agrobacterium tumefaciens-mediated transformation Citrus host colonization Fluorescent reporter genes Insertional mutagenesis Penicillium digitatum Postharvest pathogen Penicillium digitatum Citrus host colonization Fluorescent reporter genes Agrobacterium tumefaciens-mediated transformation Insertional mutagenesis Postharvest pathogen
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Summary:	Penicillium digitatum is a major postharvest pathogen of citrus crops. This fungus broadly spreads worldwide and causes green mold disease, which results in severe losses for citrus production. Understanding of the citrus infection by P. digitatum may help develop effective strategies for controlling this pathogen. In this study, we have characterized a virulent strain of P. digitatum isolated in Vietnam and established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for this fungal strain with two newly constructed binary vectors. These binary vectors harbor dominant selectable markers for hygromycin or nourseothricin resistance, and expression cassettes for the red fluorescent protein (DsRed) or the green fluorescent protein (GFP), respectively. Using the established ATMT system, the transformation efficiency of the Vietnamese strain could reach a very high yield of 1240±165 transformants per 106 spores. Interestingly, we found that GFP is much better than DsRed for in situ visualization of citrus fruit colonization by the fungus. Additionally, we showed that the transformation system can also be used to generate T-DNA insertion mutants for screening non-pathogenic or less virulent strains. Our work provides a new platform including a virulent tropical strain of P. digitatum, an optimized ATMT method and two newly constructed binary vectors for investigation of the postharvest pathogen. This platform will help develop strategies to dissect molecular mechanisms of host-pathogen interactions in more detail as well as to identify potential genes of pathogenicity by either insertional mutagenesis or gene disruption in this important pathogenic fungus. •Characterization of a virulent strain of Penicillium digitatum from Vietnam (PdVN1)•Optimization of the ATMT method using two newly constructed binary vectors•Successful DsRed and GFP expression using hygromycin and nourseothricin resistance•GFP is much better than DsRed for fungal visualization of citrus host colonization.•Generation of a non-pathogenic mutant by T-DNA insertion using the ATMT system
ISSN:	0167-7012 1872-8359
DOI:	10.1016/j.mimet.2017.11.019