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

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.

ChMob2 binds to ChCbk1 and promotes virulence and conidiation of the fungal pathogen Colletotrichum higginsianum

“…Attempts to amplify the junction between the two T-DNA fragments by PCR were not successful, presumably due to the inverted arrangement of the sequences (not shown). The presence of two T-DNAs at this genomic context is consistent with the results of previous Southern Blot analysis probed with a hygromycin resistance DNA fragment [25]. CH063_12012 encodes a predicted 333 aa protein closely related to yeast Mob2.…”

“…vir-88 was isolated from a pool of T-DNA insertion mutants with defects in virulence on Arabidopsis thaliana [25]. In contrast to Arabidopsis infected with wild-type C. higginsianum where all leaves showed strong necrotic lesions after 4 days, infection with vir-88 resulted only in small and much fewer lesions (Fig.…”

“…In order to identify the mutation responsible for the phenotype of vir-88 , we initially analyzed a T-DNA insertion site found in the original mutant screen which suggested the presence of two linked T-DNA insertions on supercontig5277 [26] close to CH063_12012 encoding a homolog to yeast Mob2 [25, 27]. By PCR analyses using a series of primers specific to that locus, both regions flanking the insertion could be isolated and were sequenced (Additional file 3: Text S1).…”

“…In order to obtain ΔChmob2 null mutants, we attempted to perform targeted gene knockout of ChMOB2 by transformation of the non-homologous end-joining defective mutant ΔChku80 [25] with the ChMOB2 deletion plasmid pCK3712. This plasmid harbors the hygromycin resistance cassette (hph) flanked by ChMOB2 upstream (-1015 to -198) and downstream sequence (+704 to +1513) on the T-DNA.…”

“…Previously, we described the identification of 75 C. higginsianum Agrobacterium tumefaciens -mediated transformation (ATMT) mutants, which are impaired in virulence on Arabidopsis thaliana [25]. Here, we show that for one of these mutants ( vir-88), a hypomorphic allele of ChMOB2 is responsible for the virulence phenotype and we analyzed the function of the protein encoded by this locus in C. higginsianum .…”