Genetic transformation of the tomato pathogen Pyrenochaeta lycopersici allowed gene knockout using a split-marker approach

Abstract

Pyrenochaeta lycopersici, as other soil-transmitted fungal pathogens, generally received little attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and important fruit yield reduction of agronomic relevant crops. The scope of this study was to develop a system allowing to investigate the functional role of P. lycopersici genes putatively involved in the corky root rot of tomato. A genetic transformation system based on a split-marker approach was developed and tested to knock out a P. lycopersici gene encoding for a lytic polysaccharide monooxygenase (Plegl1) induced during the disease development. The regions flanking Plegl1 gene were fused with the overlapping parts of hygromycin marker gene, to favour homologous recombination. We were able to obtain four mutants not expressing the Plegl1 gene though, when tested on a susceptible tomato cultivar, Plegl1 mutants showed unaltered virulence, compared with the wild-type strain. The strategy illustrated in the present work demonstrated for the first time that homologous recombination occurs in P. lycopersici. Moreover, a transformation system mediated by Agrobacterium tumefaciens was established and stable genetic transformants have been obtained. The transformation systems developed represent important tools for investigating both the role of genes putatively involved in P. lycopersici interaction with host plant and the function of other physiological traits which emerged to be genetically expanded from the recent genome sequencing of this fungus.

Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain

“…1a (Fu et al 2006;Aragona and Valente 2015). The split-marker constructs ku80-A and ku80-B which contain a 597-bp length overlap region located in the coding sequence of the ptrA marker were transformed into A. terreus CICC40205.…”

A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus

“…Indeed, work has focused on improving the frequency of gene targeting by homologous recombination, such as using split-marker-or deficient-NHEJ DNA repair pathway-strategies. However, split-marker technology is based on three crossover events, significantly reducing the transformation rate compared with classical recombination strategies based on a single crossover event (Aragona and Valente 2015;Wilson et al 2010).…”

Transcriptome Dynamics Underlying Chlamydospore Formation in Trichoderma virens GV29-8

“…The hygromycin resistance gene ( hyg ), which was amplified from the plasmid pKH-KO, conferred hygromycin resistance on the fungus. The protoplast transformation was used to generate the Chs Δ by transforming two fragments containing homologous region sequences of the Chs gene and partial hygromycin gene fragment into the protoplasts of wild-type T. virens GV29-8 ( Aragona and Valente, 2015 ; Li et al, 2017 ). The genotypes of Chs Δ mutants were confirmed by amplifying internal fragment of Chs (no PCR product generated), and the hyg fragment (PCR product was 719 bp in size).…”