漆酶基因Cglac10的缺失对杧果炭疽病菌无性繁殖和侵染能力的影响

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

【目的】揭示漆酶基因Cglac10在杧果炭疽病菌(Colletotrichum gloeosporioides)生长及侵染致病过程中的功能。【方法】利用qPCR技术对Cglac10在该菌侵染过程中的表达进行分析,并通过同源重组和PEG介导的原生质体转化法获得敲除突变体ΔCglac10H和回复菌株C-ΔCglac10H。【结果】Cglac10基因在侵染过程中的表达量是侵染前的8.02~13.66倍。回复菌株C-ΔCglac10H的表型与野生型菌株的无显著差异;突变体ΔCglac10H除在菌丝生长速率方面与野生型菌株差异不明显外,菌落颜色变为白色,培养10 d时菌丝中黑色素含量下降了67.33%,产孢量显著下降;在水膜中培养至24 h时,ΔCglac10H才偶见形成附着胞,36 h时的形成率仅为野生型菌株的44.85%;在PDB中培养6 d后,ΔCglac10H的胞内漆酶活性较野生型菌株下降了57.61%;不刺伤时,ΔCglac10H几乎不致病,刺伤接种6 d后,观察到ΔCglac10H的致病力比野生型菌株下降了52.94%。【结论】Cglac10在C.gloeosporioides无性繁殖及侵染致病过程中起着重要作用。

【Objective】Mango(Mangifera indica Linn.)is the second largest tropical fruit in China.Colletotrichum 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 infection,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 functions 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 disrupted and complemented,and its phenotype was analyzed in this paper.【Methods】The expression of Cglac10 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 region,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 400μg·mL-1.The knocked out mutants were obtained using PEG-mediated protoplast transformation.PCR amplification,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 appressorium,intracellular melanin content and laccase activity were tested.In addition,after inoculating the wild-type strain,ΔCglac10 and the complementary strain C-ΔCglac10 H on the PDA plate with0.04%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 process,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-ΔCglac10 H and the wild type,but the deletion of Cglac10 affected the asexual reproduction and virulence of C.gloeosporioides.In contrast to darkish color of the wild-type strain,the colony of mutantΔCglac10 H turned pale,and melanin content of mycelium decreased by 67.33%after 10 d of culture.The mycelium growth rate ofΔCglac10 H 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,ΔCglac10 H occasionally formed appressorium 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 oxidative band around the colony 4 d after incubation,whileΔCglac10 H had no oxidative band during the whole observation period.The intracellular laccase activity ofΔCglac10 H 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,ΔCglac10 H did not cause necrosis,while the wild-type strain could cause typical anthracnose necrotic spots.Six days after inoculation on wounded mango,ΔCglac10 H caused lesion spot which was significantly smaller than that of the wild-type strain,and the lesion diameter ofΔCglac10 H 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 activity as well as virulence,indicating that Cglac10 played an important role in the C.gloeosporioides asexual reproduction and infection.This discovery would deepen our understanding of the roles of laccase in fungal biology and virulence.