Abstract
Coniothyrium minitans is a sclerotial parasite, which has been investigated for commercial control of crop diseases caused by Sclerotinia sclerotiorum. Previously, we obtained a T-DNA insertional mutant, ZS-1TN24363, which did not produce melanin during conidiation. To understand the function of melanin in C. minitans, we cloned the gene that was disrupted by the T-DNA insertion, and found that this gene, called CmMR1, encoded a putative protein of 1,011 amino acids, which is a homolog of the transcription factor MR. Full-length CmMR1 contains 3,167 bp, with three exons and two introns. To confirm that the disrupted gene is responsible for the melanin-deficiency of the mutant, CmMR1 was disrupted and three targeted knockout mutants were obtained. Biological assays showed that the phenotype of the targeted knockout mutants was similar to that of the T-DNA insertional mutant. Furthermore, gene complementation confirmed that CmMR1 is responsible for the mutant phenotype. CmMR1 disruption did not affect hyphal growth, conidiation, and parasitization of C. minitans, however, the ROS accumulation increased and tolerance to UV light decreased significantly in the mutants. Our result may enhance the understanding of melanin in the ecology of C. minitans on molecular level.
Highlights
Coniothyrium minitans is a mycoparasite of Sclerotinia spp. and certain species in other related genera (Campbell, 1947; Li et al, 2006), and it parasitizes both the hypha and sclerotia of Sclerotinia sclerotiorum
Complementary DNA was synthesized using a cDNA synthesis kit provided by TransGen Biotech (Beijing, China); DNA sequencing analysis was performed by the TianYiHuiYuan (Guangzhou, China) and the DNA sequence was analyzed using the Basic Local Alignment Search Tool for Nucleotides (BLASTN) against the local database of C. minitans to retrieve sequence information of the putative disrupted gene; the expression of the putative disrupted gene was determined using RT-PCR with primer pairs CmMR1-F/CmMR1-R
The reactive oxygen species (ROS) content in CmMR1 mutants was determined using NBT staining, and the results showed that transfer DNA (T-DNA) insertional mutant ZS-1TN24363 and the gene targeted knockout mutants had deeper color, whereas the wild-type strain ZS-1 and the gene complemented transformants were brown in color (Figure 5)
Summary
Coniothyrium minitans is a mycoparasite of Sclerotinia spp. and certain species in other related genera (Campbell, 1947; Li et al, 2006), and it parasitizes both the hypha and sclerotia of Sclerotinia sclerotiorum. C. minitans is a coelomycete, which grows on sclerotia, and produces pycnidia with numerous conidia on or in the sclerotia (Whipps et al, 2008). It grows well on media such as on potato dextrose agar (PDA) and wheat kernel and forms pycnidia. C. minitans synthesizes melanin, which accumulates on the pycnidia, conidia, and the aging hyphae, lending a dark appearance to the entire mature colony
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