Resistance Evaluation Of Lentinula Edodes Germplasm Population To Trichoderma Spp. And The Molecular Mechanism Of Resistance To T.Atroviride Mediated By LeTLP1

Lentinula edodes is an artificially cultivated edible mushroom with the highest annual yield in China,occupying an important position in the edible mushroom industry.Infection of L.edodes bags or sticks by Trichoderma spp.is one of the main factors leading to the significant reduction of L.edodes production and the decline of industrial benefits.Except for contaminating the sterilized culture material and competing for nutrition and growth space with the mycelia of L.edodes,Trichoderma spp.can also infect the mycelia of L.edodes at various stages,such as the mycelium,color-turning and fruiting body stage,causing the rot of L.edodes rods.Mushroom rods are more vulnerable to Trichoderma infection,especially when mycelia of mushroom rods grow weakly due to high temperature damage.The rot of L.edodes rods spreads rapidly once it occurs,which often results in huge economic losses for mushroom production.The evaluation and analysis of L.edodes resistance to Trichoderma spp.and demonstrating the molecular mechanism of Trichoderma resistance provide important theoretical values and practical significances for the breeding of Trichoderma resistant varieties of L.edodes and reducing the adverse impact of Trichoderma on L.edodes production.In this study,the plate confrontation culture method was used to evaluate the resistance of L.edodes germplasm population to T.harzianum and T.atroviride.The resistance identification model of L.edodes against T.atroviride was established,and the strongly resistant strain Y3334 and highly susceptible strain Y55 of T.atroviride were selected from the Core Germplasm Bank of L.edodes.The key genes related to the difference of resistance between L.edodes and T.atroviride were mined using the comparative transcriptome method,and the gene cloning and bioinformatics analysis were performed.The gene function was investigated through gene overexpression and RNAi gene silencing transformation.The bioinformatics analysis and the function of the resistance gene were studied as follows:1.The identification model of L.edodes resistance to T.atroviride in L.edodes germplasm population was established,and the strongly resistant strain Y3334 and highly susceptible strain Y55 were selected with significant difference in the resistance of L.edodes to T.atroviride.The resistance differentiation of 56 L.edodes core collections to T.atroviride was studied with plate confrontation culture method.With the infection rate after contact(IRAC)of T.atroviride on L.edodes mycelia as the core index,the resistance identification method was established.The mechanism of the resistance difference between Y3334 and Y55 at the physiological and biochemical levels was demonstrated based on the experiments of mycelium growth on up-down dual culture,the interaction effect of water-soluble and volatile substances of mycelia and the effects of different glucose amounts on the resistance of L.edodes to Trichoderma spp..The results confirmed that there was a significant resistance difference between highly resistant strain Y3334 and highly susceptible strain Y55.The results of plate confrontation showed that T.harzianum had strong infection ability to the tested strains of L.edodes,and had obvious entanglement and re-parasitism to the mycelium of L.edodes.All core germplasms of L.edodes could not effectively prevent T.harzianum infection.The resistance of the tested strains to T.harzianum could be divided into weak resistance and high sensitivity type.2.The key gene Le TLP1 significantly related to the resistance of L.edodes to T.atroviride was mined by transcriptome analysis.Applying the highly resistant strain Y3334 and highly susceptible strain Y55 of L.edodes as research materials,the transcriptional level of the mycelial samples interacted with T.atroviride for 24 hours was analyzed by comparative transcriptomics.The results showed that more specific GO terms and KEGG pathways were enriched in Y3334 compared to Y55.Homology comparison was used for predicting the differentially expressed genes in pathogenesis in L.edodes.Among them,thaumatin-like proteins(TLP)gene Le01Gene05009(Le TLP1)was up-regulated for 32 folds in Y3334,but no significant difference in Y55.Ten differentially expressed genes in pathogenesis were selected and evaluated for the accuracy of transcriptome data with q RT PCR.q RT PCR results,which showed that the expression level of Le TLP1 in Y3334 was significantly affected by T.atroviride infection,and the expression increased significantly along with the infection.3.The overexpression and silencing transformants confirmed that Le TLP1 gene positively regulated the resistance of L.edodes to T.atroviride.Thaumatin-like proteins is a pathogenesis related protein,which has the functions of antifungal,response to biotic and abiotic stressors.The Le TLP1 gene was silenced by RNAi in the highly resistant L.edodes strain Y3334.q RT-PCR analysis showed that the expressions of Le TLP1 gene in the silencing transformants Le TLP1-Ri-3 and Le TLP1-Ri-8 were down-regulated to 65%and 57% of the wild-type strain Y3334,respectively.The infection rates of T.atroviride to the two silencing transformants increased by 70% and 50%,respectively.The Le TLP1 gene was over-expressed in the highly susceptible strain Y55.q RT-PCR analysis showed that the expression of Le TLP1 gene in the overexpressed transformants Le TLP1-OE-3 and Le TLP1-OE-10 increased about 5 and 7 folds,respectively.The infection rates of T.atroviride decreased to 40% and 10%,respectively.The results showed that the thaumatin-like protein gene Le TLP1 is a key gene of L.edodes against Trichoderm.4.The antifungal activity of Le TLP1 protein was verified by exogenous prokaryotic expression method,and it was clear that amino acid variation was crucial for the activity of L.edodes thaumatin-like protein.The molecular structure and physicochemical properties of Le TLP1 proteins(Y3334-S-TLP and Y55-W-TLP)were systematically analyzed in highly resistant L.edodes strain Y3334 and highly susceptible strain Y55.It was found that the two Le TLP1 proteins were composed of 255 amino acids,and only two amino acid residues were different.In addition,the signal peptide structure,transmembrane structure,tyrosine sulfation site,glycosylation site,theoretical isoelectric point,the hydrophilic coefficient and fat coefficient were very similar.Nevertheless,significant differences were found in the phosphorylation sites.Applying the Le TLP1 protein tlpx truncated signal peptide structure as the template,we constructed the expression vectors of p ET32a-S-Le TLPX and p ET32a-W-Le TLPX and Escherichia coli engineering bacteria,and the recombinant Le TLP1 proteins(Trx A-S-TLPX-His and Trx A-W-TLPX-His)were successfully induced and expressed.After the two recombinant proteins were purified,the accuracy of the induced protein sequences was verified by SDS-PAGE and LC-MS.The antibacterial experiment against T.atroviride was carried out by filter paper method.The filter papers containing recombinant Le TLP1 protein can significantly inhibit the mycelial growth of T.atroviride.The recombinant Le TLP1 protein of highly resistant strain Y3334 can more effectively inhibit the mycelial growth of T.atroviride compared with highly susceptible strain Y55,indicating that Le TLP1 has significant antifungal activity.The mutation of amino acid K-Q at position 245 can affect its activity,but it is not the only factor causing the resistance difference of Trichoderma in L.edodes mycelium,and the induced expression extent of Le TLP1 gene maight be more important.In this study,the resistance model of L.edodes against T.atroviride in L.edodes germplasm population was established,the thaumatin-like gene Le TLP1 significantly related to the resistance of L.edodes to T.atroviride was excavated,and the antifungal activity of thaumatin-like Le TLP1 was verified.These results lay an important foundation for the further study of the molecular mechanism of L.edodes resistance to Trichoderma spp.,and show obvious practical value for the L.edodes breeding with resistance to other microorganisms and diseases.