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Eukaryotic Cell, July 2008, p. 1098-1108, Vol. 7, No. 7
1535-9778/08/$08.00+0     doi:10.1128/EC.00109-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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,¹ Department of Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel,² Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907³

Received 5 April 2007/ Accepted 10 April 2008

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 means of a promoterless green fluorescent protein (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 C. gloeosporioides CTR2 (CgCTR2) gene fully complemented growth defects of yeast ctr2 mutants, and a CgCTR2-cyan fluorescent protein (CFP) fusion protein accumulated 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 downregulated during "pathogenic" germination and the early stages of plant infection. CgCTR2 overexpression and silencing mutants were generated and characterized. The Cgctr2 mutants had markedly reduced Cu superoxide dismutase (SOD) activity, suggesting that CgCTR2 is important in providing copper 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 appressorium formation and penetration efficiency. An 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. Our results show that copper is necessary for optimal germination and pathogenicity and that CgCTR2 is involved in regulating cellular copper balance during these processes.

Published ahead of print on 2 May 2008.

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