An efficient gene disruption method using a positive–negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi

Abstract

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0-4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.

New Downstream Signaling Branches of the Mitogen-Activated Protein Kinase Cascades Identified in the Insect Pathogenic and Plant Symbiotic Fungus Metarhizium robertsii

“…In the first dozen years of this century, robust genetic manipulation technologies including Agrobacterium tumefaciens -mediated fungal transformation and gene deletion based on homologous recombination had been developed for the Metarhizium fungi (Fang et al, 2006 ; Wang and St Leger, 2006 ; Xu et al, 2014 ; Su et al, 2018 ), and the genomes of Metarhizium species, M. robertsii , and M. acridum , were sequenced (Gao et al, 2011 ; Hu et al, 2014 ). This has been significantly speeding up the research on molecular mechanisms underlying insect infection and development by the Metarhizium fungi.…”