Functional characterization of CgCTR2, a putative vacuole copper transporter that is involved in germination and pathogenicity in Colletotrichum gloeosporioides

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 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 ctr2Delta 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.

FIG. 1.

GFP expression in REMI mutant N159. (A) Strain N159 (bottom) was grown in different media, and GFP signal was compared with a strain expressing GFP from the constitutive GPDA promoter (top). The following media were used: REG medium (frames a and d), EMS medium (frames b and e), and PE medium (frames c and f). Scale bar, 5 μm. (B) The GFP signal in strain N159 is reduced in PE medium and recovered in REG medium. The addition of copper and EGTA (a copper chelator) did not affect GFP levels. Contrast and brightness of the lower part of the left picture (PE medium) were intensified to allow visualization of the very low level of GFP expression under these conditions. Scale bar, 30 μm.

FIG. 2.

Schematic diagram of the genomic sequence at the insertion site in strain N159. The insertion site of the transforming vector pAS1-GFP is 70 bp upstream from the start codon of a putative reading frame. Two transcripts are encoded with alternative splicing at 741 bp and 786 bp, corresponding to putative peptides of 183 aa and 168 aa, respectively. Both transcripts have the same reading frame at the 3′ end. Black bars are exons; lines are introns. Positions of the protein transmembrane domains are predicted to be between aa 32 to 54 and 146 to 169 in the larger peptide and between aa 32 to 54, 109 to 132, and 136 to 159 in the smaller peptide. GUS, glucuronidase; HPH, hygromycin B phosphotransferase.

FIG. 3.

Complementation of S. cerevisiae ctrΔ mutants with CgCTR2. An S. cerevisiae ctr1Δctr2Δctr3Δ triple mutant was transformed with the S. cerevisiae CTR2 gene or with CgCTR2. Strains were grown on ethanol-glycerol medium (YPEG) with various concentrations of copper. The triple mutant lacking all three copper transporters requires at least 20 μM CuCl₂ for growth on this medium (vector). Transformation of the triple ctr mutant with S. cerevisiae CTR2 (Sc CTR2) restores the ability of the strains to grow on YPEG medium with only 15 μM CuCl₂. Transformation of the yeast mutant strain with either the long or short CgCTR2 gene fully restored the ability of the complemented strains to grow on medium with 15 μM CuCl₂ (CgCTR2). Both transcripts had an identical effect (the result with the long transcript is presented). Growth of the various mutants on synthetic medium (Sc-ura) was unaffected.

FIG. 4.

CgCTR2 is localized in the vacuole membrane. Transformants expressing a CgCTR2-CFP fusion protein were visualized using confocal and fluorescent microscopes. (A) Confocal images of CFP and DMY-64 signals. Different samples are shown due to the overlap of the spectra between CFP and DMY-64. Frames a and b, CFP and DIC images; frames c and d, DMY-64 and DIC images. (B) Fluorescent microscopy of Hoechst-stained nuclei and DIC images. Frame e, Hoechst staining; frame f, DIC image (white arrow, vacuole; black arrow, nucleus); frame g, merge. (C) Fluorescent microscope images of CFP and Hoechst staining. Hyphae containing large (left) or small (right) vacuoles are shown. Frames a and b, DIC images; frames c and d, CFP (white arrows show large vacuoles; yellow arrows show small vacuoles); frames e and f, Hoechst staining; frames g and h, merged images of CFP and Hoechst staining. (D) Confocal images of FM4-64 and CFP signals. Frames a and b show the results of a short incubation with FM4-64. The FM4-64 signal (a) is detected on the outer cell membrane; CFP signal (b) is inside the cell; Frames c, d, and e show results of longer incubations with FM4-64: frame c, CFP; frame d, FM4-64; frame e, merged image. Membranes of large vacuoles are stained with FM4-64; CFP signal is localized inside these vacuoles as well as around nuclei. Scale bar, is 5 μm.

FIG. 5.

Analysis of CgCTR2 gene expression. (A) Copper effect. Mycelium was collected after growth in REG medium for 44 h and incubated in medium containing various amounts of copper. After 4 h RNA was extracted, and cDNA was produced. RT-PCR was performed on the cDNA with CgCTR2-specific primers, and samples were removed every 5 cycles starting at 15 cycles and up to 40 cycles. Picture shows samples collected after 35 cycles. Top, CgCTR2; bottom, CgCDC42. (B) Expression of CgCTR2 in different media and during pathogenic germination. Mycelium was grown for 48 h in different media, and then RNA was extracted, and transcript was detected by Northern blot analysis (left). Spores were collected from EMS plates and incubated in PE medium, which induces pathogenic germination (right). RNA was extracted at several time points starting at 2 h, which represents the onset of germ tube formation. Top, CgCTR2; bottom, rRNA. (C) Expression during early stages of pathogenic and saprophytic germination. Spores were collected from plates and incubated in PE or EMS shake cultures. (D) In planta CgCTR2 gene expression. Pea leaves were inoculated with fresh spores, and tissue samples were collected at time intervals beginning at 4 h and up to 72 h postinoculation. Quantitative PCR was performed as described in panel A. Shown are results after 40 cycles. Top, CgCTR2, bottom, CgCDC42. Lanes M, molecular weight ladders.

FIG. 6.

Analysis of CgCTR2-silenced and overexpression transgenic strains. (A) N159 and transgenic strains transformed with the CgCTR2 RNAi cassette (49 or 9). Strains were grown for 48 h in REG medium, conditions under which CgCTR2 gene expression is relatively strong. (B) CgCTR2-overexpressing isolates. Strains were grown for 48 h in PE medium, conditions under which CgCTR2 gene expression is low. RNA was extracted, and RT-PCR was performed with primers specific for CgCTR2 (upper panel) and CgCDC42 (lower panel). Lanes M, molecular weight ladders; wt, wild type.

FIG. 7.

Activity assay of Cu/Zn SOD in wild-type and Cgctr2 mutant strains. Strains were grown in CD medium without copper for 48 h. Mycelium was harvested, and SOD activity was determined with or without the addition of 5 μM copper. Without copper the wild type showed an additional band (arrow) that could not be detected in the RNAi and N159 mutant strains. This band was intensified and was present in all strains following the addition of 5 μM copper. The other two bands were unchanged under all conditions and might represent the activity of Mn SOD. WT, wild type.

FIG. 8.

Effect of spore density on germination rate. Spores of the wild-type (▪) and N159 (□) strains were collected from EMS plates, diluted, and germinated in PE medium. After 2.5 h the number of germinated spores was counted, and the percentage of germination was calculated. Germination in the mutant strains was markedly reduced compared to the wild type at spore densities of 10⁶/ml or higher but was similar to the wild type at or below 10⁵ spores/ml.

FIG. 9.

Pathogenicity of Cgctr2 mutant strains. (A) Comparison of infection by the CgCTR2-silenced and overexpression transgenic strains. A. virginica plants were inoculated with spore suspensions (5 × 10⁴ spores/ml) of wild type, N159, RNAi strains 9 and 49 (only strain 9 is shown), overexpression (OX) strains 29 and 35 (only strain OX29 is shown), and the N159 complemented strain C10. The picture shows plants at 6 days postinoculation. Numbers indicate the average fresh weight of six plants ± standard error. (B) Pea leaves were inoculated with spores of strain N159 (frames a to c) or with a GFP-expressing strain (frames d to f). After 24 h there is almost no external growth by the GFP-expressing strain (frame d), while N159 develops a significant amount of mycelium on the leaf surface (frame a). Leaves infected by strain N159 (frames b and c) develop necroses after 48 h, which are similar in size and number to the necroses that develop in leaves infected by the GFP-expressing strain (frames e and f). Images in frames a and d are from a light microscope; images in frames b to f are from a fluorescent stereoscope. (C) A. virginica plants were inoculated with optimal (5 × 10⁴), suboptimal (2 × 10⁴), and supraoptimal (8 × 10⁴) spore densities. Disease was recorded after 6 days. Full symptoms were developed by the inoculation of plants with 8 × 10⁴ spores/ml of strain N159. All the experiments were repeated several times with similar results. wt, wild type.