Effects of deletion of laccase gene Cglac10 on the asexual reproduction and infection ability of Colletotrichum gloeosporioides

Abstract:【Objective】Mango (Mangifera indica Linn.) is the second largest tropical fruit in China. Col- letotrichum gloeosporioides is an important plant pathogen with large host-range and wide distribution. Anthracnose mainly caused by C. gloeosporioides is one of the most serious diseases that reduce mango production. Identifying the pathogenesis-related genes and revealing their roles in fungal growth and in- fection, and further understanding the interaction between C. gloeosporioides and its host, will help to find new control targets and improve control effects. In previous work, we found out 13 laccase family members from C. gloeosporioides in a reference genome databases. Sequences alignment showed great difference. Therefore, we speculated that their function might be quite different. So far, only the func- tions of lac1 have been identified at the molecular level, and it affected mycelial growth and development, melanin deposition, conidia production, laccase activity, stress tolerance and virulence to the host. In order to further explore the role of Cglac10, another member of the laccase family, Cglac10 was dis- rupted and complemented, and its phenotype was analyzed in this paper.【Methods】The expression ofCglac10 gene in the process of infection of C. gloeosporioides on leaves was analyzed by qPCR. The knockout vector of the Cglac10 gene was constructed by inserting a hygB cassette into the coding re- gion, and transforming it into E. coli DH5. Hygromycin B was used as a marker to screen transformant, and the minimum inhibitory concentration of Hygromycin B on C. gloeosporioides was 400 μg · mL- 1. The knocked out mutants were obtained using PEG-mediated protoplast transformation. PCR amplifica- tion, Southern blot and qPCR were used to determine whether the transformants were Cglac10 deletion mutants. Then Cglac10 gene was complemented in mutants by a similar way. Basta resistance and PCR detection were used to determine whether the transformants were the complementary strains of mutantΔCglac10. The biological phenotypes including colony growth rate, sporulation, the formation of ap- pressorium, intracellular melanin content and laccase activity were tested. In addition, after inoculating the wild- type strain, ΔCglac10 and the complementary strain C- ΔCglac10H on the PDA plate with 0.04% guaiacol, the extracellular laccase activity was tested by the plate color reaction. The virulence was assessed on the mango fruits using unwounded and wounded inoculation method, respectively. The diameters of the lesions were observed and measured every day.【Results】The complete DNA and cDNA sequences of the laccase gene Cglac10 are 1989 bp and 1878 bp in length, respectively, encoding a putative protein with 625 amino acid. The Cglac10 gene was highly expressed during infection pro- cess, which was 8.02-13.66 times as high as that before infection. The phenotype indicated that there was no significant difference between the complementary strain C-ΔCglac10H and the wild type, but the deletion of Cglac10 affected the asexual reproduction and virulence of C. gloeosporioides. In con- trast to darkish color of the wild-type strain, the colony of mutant ΔCglac10H turned pale, and melanin content of mycelium decreased by 67.33% after 10 d of culture. The mycelium growth rate ofΔCglac10H was nearly the same as the wild-type strain. The sporulation was 57.61% less than that of wild-type strain. Observation showed that the wild-type strain formed appressorium on the wet glass 4 h after inoculation, then melanized and matured at 6 h. However, ΔCglac10H occasionally formed appres- sorium 24 h after incubation, and the formation rate was only 44.85% of that of the wild type at 36 h. On the PDA medium containing 0.04% guaiacol, the wild-type strain could produce red-brown oxida- tive band around the colony 4 d after incubation, while ΔCglac10H had no oxidative band during the whole observation period. The intracellular laccase activity of ΔCglac10H was 57.61% lower than that of the wild-type strain 6 d after incubation in PDB. After inoculation on the unwounded ripe fruits of the mango cultivar Tainong, ΔCglac10H did not cause necrosis, while the wild-type strain could cause typical anthracnose necrotic spots. Six days after inoculation on wounded mango, ΔCglac10H caused le- sion spot which was significantly smaller than that of the wild-type strain, and the lesion diameter ofΔCglac10H was 52.94% less than that of wild-type strain【. Conclusion】The laccase gene Cglac10 of C. gloeosporioides on mango was cloned and identified. This gene was highly expressed during the process of infection of C. gloeosporioides on mango leaves. Deletion of Cglac10 gene affected the colony pigmentation, mycelium melanin content, sporulation, appressorium formation, laccase secretion and ac- tivity as well as virulence, indicating that Cglac10 played an important role in the C. gloeosporioidesasexual reproduction and infection. This discovery would deepen our understanding of the roles of lac- case in fungal biology and virulence.