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

@entity851 @entity3261 @entity281 @entity375 @entity71836
UNASSIGNED: We used insertional mutagenesis by @entity3261 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 @entity281 , like switching to @entity851 . For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H+-ATPase @entity71836 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   Chku80 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 @entity281 of A. thaliana. @entity71836 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 @entity375 genes for plasma membrane pumps. We propose that expression of a specific proton pump early during @entity281 may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi.
