Thyrostroma carpophilum insertional mutagenesis: A step towards understanding its pathogenicity mechanism

• Published in: Journal of microbiological methods Vol. 171; p. 105885
• Main Authors: Rasool, Rovidha S., Padder, Bilal A., Wani, Ashraf Alam, Shah, Mehraj D., Masoodi, Khalid Z., Khan, Nisar A., Banoo, Aqleema, Khan, Imran
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-04-2020
• Subjects: Agrobacterium tumefaciens-mediated transformation; Ascomycota - genetics; Ascomycota - isolation & purification; Ascomycota - pathogenicity; DNA, Fungal - genetics; Fruit - microbiology; Insertional mutagenesis; Modus operandi; Mutagenesis, Insertional - methods; Pathogenicity genes; Plant Diseases - microbiology; Spores, Fungal - genetics; Thyrostroma carpophilum; Transformation, Genetic - genetics; Thyrostroma carpophilum; Agrobacterium tumefaciens-mediated transformation; Modus operandi; Pathogenicity genes; Insertional mutagenesis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2020.105885

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 104 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.
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•We optimized the conditions for Agrobacterium tumefaciens mediated transformation for T. carpophilum.•Our standardized ATMT conditions resulted in 328 transformants per 104 spores•Phenotypic evaluation of mutant library showed inability of hundreds of mutants to cause infection•Transformants with complete pathogenicity loss failed to penetrate the host tissue•This is first such in study T. carpophilum and can be explored to understand the pathogenicity mechanism of fungus