Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioides

Abstract

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Keywords: Colletotrichum gloeosporioides; MAP kinase; appressorium; pathogenicity.

Figure 1

The amino acid sequence alignment of Cgl-Slt2 of C. gloeosporioides (JQ322774), Maf1 of C. orbiculare (AAL50116), Mps1 of M. oryzae (AAC63682) and Slt2 of S. cerevisiae (AAB68912). Sequence alignment was performed using CLUSTALW. Identical amino acids are indicated by white letters on a black background. Similar residues are indicated by a gray background. Gaps introduced for alignment are indicated by hyphens. The protein kinase subdomains are indicated by the Roman numerals above the sequences. The protein kinase activation sequence TEY is indicated by asterisks.

Figure 2

Copy number determination and the targeted gene disruption of Cgl-SLT2. (a) A Southern blot analysis shows the Cgl-SLT2 copy number in the C. gloeosporioides PeuB genome. Genomic DNA was digested with different restriction enzymes, including EcoRI, HindIII, XbaI and XhoI, and probed with the Cgl-SLT2 ORF. (b) A schematic representation of the targeted gene disruption of C. gloeosporioides Cgl-SLT2. A linearized plasmid harboring the disruption construct was transformed into the C. gloeosporioides spheroplasts. A portion of the Cgl-SLT2 ORF was replaced with an hph cassette via homologous recombination. A 1 kb fragment was used as a probe for Southern blot analysis. (c) A Southern blot analysis of the strain with disrupted Cgl-SLT2 and wild-type. Genomic DNA was digested with XhoI and probed with Cgl-SLT2. (d) Cgl-SLT2 expression analysis in wild-type and Cgl-slt2 strains via reverse transcription PCR using GPD as a control. Gene disruption resulted in the loss of Cgl-SLT2 transcripts. M: Molecular marker.

Figure 3

Light microscopy images of Cgl-slt2 (a) and wild-type strains (b) on minimal media agar. The Cgl-slt2 mutant exhibited increased hyphal branching compared to the wild-type. The image was captured using an Olympus light microscope under 100X magnification. The scale bar indicates 50 μm.

Figure 4

Appressorium formation in wild-type and Cgl-slt2 strains. Appressorium formation induced from conidia harvested from C. gloeosporioides grown on PDA supplemented with 1 M sorbitol in wild-type (a) and Cgl-slt2 strains (b) Appressorium formation induced from conidia harvested from C. gloeosporioides grown on PDA alone in wild-type (c) and Cgl-slt2 strains (d). The Cgl-slt2 mutant showed an impaired ability to form mature appressoria, and the melanization of appressoria was not observed. The image was captured using an Olympus light microscope under 200X magnification. (a: appressorium; gt: germ tube; c: conidium; sg: second germ tube). The scale bar indicates 10 μm.

Figure 5

A pathogenicity assay with wild-type and Cgl-slt2 strains on mangoes. (a) Mangoes were spray-inoculated with a 10⁵ conidia/mL suspension. Images were obtained 10-days post-inoculation. The picture in the left panel is the wild-type strain, the middle panel is the mutant strain (Cgl-slt2) and the right panel is the control (sterile dH₂O). (b) Wounded mangoes were inoculated with 25 μL of a 10⁵ conidia/mL suspension. The picture in the left panel is the wild-type strain, the middle panel is the mutant strain (Cgl-slt2) and the right panel is the control (sterile dH₂O). Cgl-slt2 mutants were unable to infect wounded or unwounded fruits compared to the wild-type.