Functional characterization of cgCTR2, a putative vacuole copper transporter that is involved in germination and pathogenicity in Colletotrichum gloeosporioides

Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel; Department of Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel; Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907

^* To whom correspondence should be addressed. Email: amirsh{at}ex.tau.ac.il.

	Abstract

Copper is a cofactor and transition metal involved in redox reactions that are essential in all eukaryotes. Here we report that a vacuolar copper transporter that is highly expressed in resting spores is involved in germination and pathogenicity in the plant pathogen Colletotrichum gloeosporioides. A screen of C. gloeosporioides transformants obtained by transforming with a promoter-less GFP construct led to the identification of transformant N159 in which GFP signal was observed in spores. The transforming vector was inserted 70 bp upstream of a putative gene with homology to the Saccharomyces cerevisiae vacuolar copper transporter gene CTR2. The CgCTR2 gene fully complemented growth defects of yeast ctr2 mutants, and a CgCTR2-CFP fusion protein localized in vacuole membranes confirming the function of the protein as a vacuolar copper transporter. Expression analysis indicated that CgCTR2 transcript is abundant in resting conidia and during germination in rich medium and down-regulated during "pathogenic" germination and early stages of plant infection. CgCTR2 overexpression and silencing mutants were generated and characterized. The cgctr-2 mutants had markedly reduced Cu SOD activity, suggesting that CgCTR-2 is important in providing cupper to copper-dependent cytosolic activities. The cgctr2 silenced mutants had increased sensitivity to H₂O₂ and reduced germination rates. The mutants were also less virulent to plants, but they did not display any defects in appressoria formation and penetration efficiency. External copper supply compensated for the hypersensitivity to H₂O₂ but not for the germination and pathogenicity defects of the mutants. Similarly, overexpression of CgCTR2 enhanced resistance to H₂O₂ but had no effect on germination or pathogenicity. The vacuolar copper transporter CgCTR2 is probably involved in this process, possibly by exporting copper from the vacuole to the cytoplasm, thereby providing the copper to cytosolic enzymes such as SOD.