Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae

Abstract

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild-type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.

The entomopathogen Metarhizium anisopliae can modulate the secretion of lipolytic enzymes in response to different substrates including components of arthropod cuticle

“…The availability of the Agrobacteriummediated transformation system for constructing functional mutants of selected genes (Staats et al 2007) has made it possible to show that the interruption of a single chitinase gene results in the decreased efficiency of insect infection (Boldo et al 2009). On the other hand, little is known about the molecular mechanisms of Metarhizium host specificity.…”

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris

“…Significantly, T-DNA-tagged mutated genes or flanking sequences can be identified easily by thermal asymmetric interlaced (TAIL)-PCR, plasmid rescue, and IPCR (Inverse PCR) (Mullins et al 2001;Chen et al 2009;Sun et al 2009). Furthermore, T-DNA of A. tumefaciens can also integrate through homologous recombination in fungi, mainly for targeted gene disruption (Staats et al 2007). ATMT has been successfully employed in many entomopathogenic fungi (Leclerque et al 2004;Staats et al 2007) and medicinal fungus ).…”

Improving mycoinsecticides for insect biological control

“…Here, we will present a brief synopsis of the insect infection process and plant interactions mediated by these fungi, provide an overview of the major obstacles in practical applications of the fungi in the field and non-genetic engineering approaches at dealing with these issues, and then delve into the various design strategies used to genetically engineer the fungi for greater efficacy. Implicit in the ability to engineer entomopathogenic fungi has been the relatively recent development of robust tools for their genetic manipulation (Fan et al 2011;Fang et al 2004;Staats et al 2007;Ying and Feng 2006;Zhang et al 2010).…”