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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.

plos one

A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration