Abstract
We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H+-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi.
Highlights
Many aspects of fungal pathology have been discovered by forward genetic screens using different strategies for generating mutant alleles
A mutant screen for genes involved in pathogenicity in Colletotrichum higginsianum In order to obtain mutants affected in pathogenicity we used Agrobacterium tumefaciens mediated transformation (ATMT) of C. higginsianum conidia
Transformants were selected on Potato dextrose agar (PDA) plates containing hygromycin (Fig 1A), single colony purified on selective plates and further propagated on oatmeal plates (OMA) plates
Summary
Many aspects of fungal pathology have been discovered by forward genetic screens using different strategies for generating mutant alleles. Primary hyphae grow biotrophically between the plasma membrane of the host cell and the plant cell wall generating structures referred to as interfacial bodies which are thought to be critical areas for effector delivery [12, 13]. During this initial infection stage, the host cell remains alive and host defense mechanisms and / or pathogen recognition must be suppressed. Plant pathogenic fungi use a series of different mechanisms to accomplish this These include active suppression of defense by apoplastic and cytoplasmic effectors and mechanisms to reduce recognition of pathogen-associated molecular patterns (PAMPS) [13]. Carbohydrateactive enzymes, proteases and necrosis inducing peptides are upregulated [17]
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