Transformation: a tool for studying fungal pathogens of plants

Abstract

Plant diseases caused by plant pathogenic fungi continuously threaten the sustainability of global crop production. An effective way to study the disease-causing mechanisms of these organisms is to disrupt their genes, in both a targeted and random manner, so as to isolate mutants exhibiting altered virulence. Although a number of techniques have been employed for such an analysis, those based on transformation are by far the most commonly used. In filamentous fungi, the introduction of DNA by transformation typically results in either the heterologous (illegitimate) integration or the homologous integration of the transforming DNA into the target genome. Homologous integration permits a targeted gene disruption by replacing the wild-type allele on the genome with a mutant allele on transforming DNA. This process has been widely used to determine the role of newly isolated fungal genes in pathogenicity. The heterologous integration of transforming DNA causes a random process of gene disruption (insertional mutagenesis) and has led to the isolation of many fungal mutants defective in pathogenicity. A big advantage of insertional mutagenesis over the more traditional chemical or radiation mutagenesis procedures is that the mutated gene is tagged by transforming DNA and can subsequently be cloned using the transforming DNA. The application of various transformation-based techniques for fungal gene manipulation and how they have increased our understanding and appreciation of some of the most serious plant pathogenic fungi are discussed.

Construction of pDESTR, a GATEWAY Vector for Gene Disruption in Filamentous Fungi

“…The gene disruption system using pDESTR, a novel Gateway destination vector reported in this paper gives an efficient way of gene disruption in filamentous fungi. The selection marker of hph cassette from pCB1004 was already known to work effectively in other filamentous fungi [5,11], indicating that pDESTR system will work also in many other fungal strains. A similar Gateway destination vector for gene disruption harboring phosphinothricin resistance gene (bar) cassette from pBARKS1 [7] is under construction.…”

“…These novel vectors may help the functional characterization of hypothetical genes of genome-sequenced fungi, by combination with non-homologous end joining (NHEJ) -deficient strains [6] and efficient transformation methods [5]. …”

Approaches to functional genomics in filamentous fungi

“…In recent years, efficient transformation systems have been developed for a wide range of filamentous fungi (reviewed in [1,4,5]). Successful transformation of fungi has been achieved by: CaCl 2 /polyethylene glycol [6][7][8], electroporation [9-15], particle bombardment [2,6,10,16,17], and Agrobacterium tumefaciens-mediated transformation (AMT) [1].…”

“…While our review covers some of the material in those papers, we have sought to give a broader, comparative overview of the approaches to functional genomics in filamentous fungi and to indicate the directions in which this research is heading. Other general reviews are also available [4,5] and this review updates that work with recent advances. …”

Promoter trapping in Magnaporthe grisea

“…Of these techniques, insertional mutation method including REMI (restriction enzyme-mediated insertional mutagenesis) (Balhadère et al, 1999) and ATMT (Agrobacterium tumefaciens-mediated transformation) (Rho et al, 2001) is a powerful way for functional genomic analysis. This technique has been successfully used to transform diverse filamentous fungi and isolate important genes (Mullins and Kang, 2001) relying on screening a large number of transformants. Although it is an efficient tool for tagging and cloning functional genes from fungi, many tagged genes that have no obvious phenotype cannot be detected.…”