Functional Analysis Of The Lentinula Edodes Laccase Gene (Lelcc4) Involved In Mycelia Browning And The Resistance To Trichoderma Atroviride

Lentinula edodes,also called Xianggu or shiitake,which is one of the edible and medicinal mushrooms from white rot species,has been widely cultured in China and the East Asian region by sawdust-based cultivation.The sawdust-based cultivation process of L.edodes can be generally divided into several stages including mycelia cultivation,brown film(BF)formation,primordium initiation and fruiting body development.Temperature and light are the main environmental factors affecting the growth and development of L,edodes.It has been reported that laccase genes are involved in the fruiting body development of L.edodes.14 laccase genes have been identified in the genome of L.edodes and their expression patterns are found to be different at different developmental stages.However,the specific functions of laccase genes during the growth and development of L.edodes has rarely been studied.The present study firstly optimized the genetic transformation system of L.edodes,and the function research of this gene in the BF formation and the resistance of L.edodes to Trichoderma atroviride was carried out by overexpression and RNAi of Lelcc4.At the same time,the melanin synthesis pathway involving laccase gene Lelcc4 was analyzed.The main findings are as follows:1.The pCAMBIA-1300-GFP vector containing the reporter gene expression element PLegpd::eGFP was used for Agrobacterium-mediated genetic transformation of L.edodes.The transformation efficiency of ATMT was evaluated under different Agrobacterium strains,acetylsyringone(AS)concentration,mediums for recipient incubation and genotypes.The results showed that the transformation efficiency of Agrobacterium EHA105 was higher than that of AGL1.Adding 200 mmol/L AS in induction medium and co-culture medium could obtain more resistant transformants.In dikaryon strain W1,the positive frequency of selected transformants was 30%when using 1/4M medium(1.95%oak sawdust,2%bran,0.2%gypsum,and 2%agar).While in the monokaryon strain W126,only the millet medium group obtained positive transformants with a positive frequency of 75.48%.Comparing the transformation efficiency of the five L.edodes strains YS55,YS3334,YS3357,S606 and W1,YS55 showed the highest transformation efficiency,with 85.12%of positive frequency for hygromycin-resistant transformants;W1 showed the lowest transformation efficiency,with 2.79%of positive frequency for hygromycinresistant transformants.The above results indicated that the genotype of recipients as well as the medium for mycelial incubation were suggested to play key roles in determining the transformation efficiency of L.edodes.2.Based on the draft genome sequence of L.edodes strain W1-26,the protein characteristics of 14 L.edodes laccase genes were analyzed.Bioinformatics analysis of the promoters in each gene revealed the variances in the number and type of stress-related Ciselements among different laccase genes.The qRT-PCR was used to analyze the expression profile of laccase gene family in L.edodes.The results demonstrated that the expression profile of the laccase gene family in L.edodes were significantly affected by different environmental stresses,such as photoperiods,carbon sources,and temperature.It is worth noting that the expression levels of genes such as Lelcc4 were significantly increased after low temperature treatment at 10℃ for 24 h;tyrosinase gene(Letry)was clustered with the same group as Lelcc4 and Lelcc7 in all the different photoperiod treatments.When the interspecies were interacted with T.atroviride 92-1,The relative expression levels of Lelccl,Lelcc2,and Lelcc4 was continuously increased and reached the highest value at 48 h,which was increased by 38.58,213.75 and 49.84 folds,respectively as compared with CK.3.The gene overexpression and RNAi were applied for the functional research of laccase gene Lelcc4.More than two stable transformants with overexpression or interference of Lelcc4 were selected,respectively.The phenotypic analysis for the transformants including mycelial growth rate,brown film(BF)formation and the resistance of L.edodes to T.atroviride 92-1 was then carried out.The results showed that after 10 days of light treatment,the browning bag of Lelcc4 overexpression transformants showed deeper color and bigger browning area than that in wild strain W1 and Lelcc4 RNAi transformants.Further observation of the mycelia by transmission electron microscopy demonstrated that the cell wall of the Lelcc4 overexpression transformants was thicker than that in the wild strain W1 and Lelcc4 RNAi transformants after light treatment.Hydrochloric acid cleavage method was used to extract melanin-like substances from brown mycelia,and the substances were identified as fungal melanin.The melanin content in the surface mycelia of Lelcc4 overexpression transformant,Lelcc4 RNAi transformants and wild-type W1 was calculated and the Lelcc4 overexpression transformant has the highest melanin content.Furthermore,the content of element C,N,S,H,the morphological structure,the redox potential and anti-ultraviolet radiation in the melanins of Lelcc4 overexpression transformant,Lelcc4 RNAi transformants and wild-type W1 were significant differences.Based on the draft genome sequence of L.edodes strain W1-26,according to the reported melanin biosynthetic pathway,combined with comparative transcriptome analysis,LC-MS analysis and The intermediate metabolite or inhibitors of melanin biosynthetic,it was speculated that eumelanin,DHN-melanin,pyo-melanin,GHB-melanin,catechol melanin and PAP-melanin biosynthetic pathways was existed in L.edodes mycelia.Meanwhile,Lelcc4 may envolved in the synthesis of eu-melanin,DHN-melanin and GHB-melanin.4.The resistance of Lelcc4 RNAi transformant to Trichoderma atroviride mycelia and spore suspension was significantly decreased.Besides,the hydrophobicity of Lelcc4 RNAi transformant was also decreased.The mycelia of Lelcc4 RNAi transformant was sensitive to different cell wall synthesis inhibitors and lysing enzyme.The relative content of melanin in the extracellular culture fluid of Lelcc4 overexpression transformant was increased,and the mycelia growth and conidia germination of T.atroviride was was inhibited by adding the extracellular culture fluid of Lelcc4 overexpression transformant.The browning mycelium produced during the confrontation culture between L.edodes and T.atroviride 92-1 was collected and the melanin was successfully extracted from it.the germination of Trichoderma conidia was inhibited and the resistance of L.edodes to T.atroviride 92-1 was enhanced when L.edodes melanin was added.In addition,the exogenous application of melanin synthesis intermediate metabolite or inhibitors further confirmed that when the DHN melanin synthesis was blocked,the resistance of L.edodes to T.atroviride 92-1 was reduced.In summary,it was revealed that the laccase gene Lelcc4 may involve in the mycelial browning and the resistance of L.edodes to T.atroviride by participating in cell wall synthesis and melanin biosynthesis.