Identifying pathogenicity-related genes in the pathogen Colletotrichum magnum causing watermelon anthracnose disease via T-DNA insertion mutagenesis

Abstract

Fruit rot caused by Colletotrichum magnum is a crucial watermelon disease threatening the production and quality. To understand the pathogenic mechanism of C. magnum, we optimized the Agrobacterium tumefaciens-mediated transformation system (ATMT) for genetic transformation of C. magnum. The transformation efficiency of ATMT was an average of around 245 transformants per 100 million conidia. Southern blot analysis indicated that approximately 75% of the mutants contained a single copy of T-DNA. Pathogenicity test revealed that three mutants completely lost pathogenicity. The T-DNA integration sites (TISs) of three mutants were Identified. In mutant Cm699, the TISs were found in the intron region of the gene, which encoded a protein containing AP-2 complex subunit σ, and simultaneous gene deletions were observed. Two deleted genes encoded the transcription initiation protein SPT3 and a hypothetical protein, respectively. In mutant Cm854, the TISs were found in the 5'-flanking regions of a gene that was similar to the MYO5 encoding Myosin I of Pyricularia oryzae (78%). In mutant Cm1078, the T-DNA was integrated into the exon regions of two adjacent genes. One was 5'-3' exoribonuclease 1 encoding gene while the other encoded a WD-repeat protein retinoblastoma binding protein 4, the homolog of the MSl1 of Saccharomyces cerevisiae.

Keywords: ATMT; Colletotrichum magnum; T-DNA integration; fungal transformation; virulence gene.

Figure 1

Analysis of T-DNA copy number in C. magnum transformants generated by Agrobacterium tumefaciens-mediated transformation (ATMT). (A) Probe used for hybridization, HindIII and EcoRI restriction sites are indicated; (B) Genomic Southern blot analysis of 30 transformants of C. magnum (1–30) generated by ATMT. Genomic DNA was restricted with HindIII and EcoRI. One band (lanes marked with an asterisk) represents a single T-DNA integration event 20, 22, and 28 represent genomic Southern blot analysis of Cm699, Cm854, and Cm1078, respectively.

Figure 2

Phenotype and pathogenic development of C. magnum transformants produced by Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotype during axenic growth on PDA is shown in the left panel. Pathogenicity assays on detached watermelon leaves (4 days post-inoculation (dpi)). Structure of C. magnum transformants mycelia. Bars represent 50 μm.

Figure 3

Numbers of conidia and the conidia germination rates of the wild-type (WT) strain CAASZK4 and of mutant Cm854. (A) Numbers of conidia of the wild-type (WT) strain and of mutant Cm854; (B) Conidia germination rates of the wild-type (WT) strain CAASZK4 and of mutant Cm854. Data were analysed with GraphPad Prism 9.0 (https://www.graphpad.com/). Asterisks over the error bars indicate the significant difference at the p = 0.05 level.

Figure 4

The growth rate of the three pathogenicity mutants and WT strains obtained in this study. Data were analysed with SPSS Statistics 19.0 (WinWrap Basic; http://www.winwrap.com) by one-way analysis of variance, and means were compared using Duncan’s test at a significance level of p = 0.05. Letters over the error bars indicate the significant difference at the p = 0.05 level.

Figure 5

T-DNA integration sites patterns of T-DNA integration and in transformants Cm699, Cm854, and Cm1078. (A), (B), (C) Illustration of T-DNA integration into Cm699, Cm854, and Cm1078, respectively. The rectangle in gradient purple represents the ideal T-DNA between LB and RB. The rectangle in orange represents the T-DNA fragment coding the hygromycin resistance gene. In the blue arrow, the blue represents the exon as well as the white represents the intron. The integration sites were identified by whole genome re-sequencing in the text.