| The macro fungus Lentinula edodes is the most productive edible mushroom in China and all over the world that has excellent economic,edible and medicinal value.Virus disease is one of the most important infectious diseases in L.edodes.The disease symptoms,such as mycelial degeneration,abnormal browning and fruiting,fruiting body deformity and wilting,may be related to mycovirus infection.Studies have shown that virus in L.edodes is most likely to be latent infection or conditionally pathogenic,and the occurrence of symptoms may be related to high temperature induction.At present,the influence mechanism of virus on L.edodes,and the role of adverse environmental factors,such as high temperature,in the interaction process are still unclear.The purpose of this study is to clarify the virus diversity in L.edodes and further explore the interaction between L.edodes and virus affected by temperature.(1)Using r RNA-deppleted lnc RNA sequencing data,the virus diversity of 8representative strains of L.edodes was analyzed based on a local bioinformatics analysis platform,which was the first analysis of L.edodes virome.A total of 30 viruses composed of 37 contigs were found,including 6 ds RNA viruses,21(+)ss RNA viruses and 3(-)ss RNA viruses,of which 27 were found for the first time in this study.These viruses are distributed in 11 known families and 2 proposed families(genera).Multiple viruses co-infection in L.edodes strains was frequently occurred,and the abundance of virus expression is actually inconsistent.(2)The full-length genome of Lentinula edodes partitivirus 1(Le PV1)was sequenced.Its genome consists of two ds RNAs.SDS-PAGE analysis showed that Le PV1 encoded a 72 k Da coat protein and could form 34 nm spherical particles.RT-PCR analysis showed that the detection rate of Le PV1 in core collection was 48.2%(27/56),and the detection rate in wild collection was higher than that in cultivated collection.Le PV1 virions from the abnormal strain SX12 were transfected into Y3334 virus-free strain by PEG mediated protoplast transformation.The biological characteristics of Y3334virus-free strain and virus-infected strain were compared.Le PV1 did not affect the growth rate of MYG mycelium of strain Y3334,nor did it affect its thermotolerance.(3)Lentinula edodes mycovirus HKB(Le V)is another kind of ds RNA virus widely existing in L.edodes population.RT-PCR analysis showed that the detection rate of Le V in the core collection was 23.2%(13/56)with more higher in cultivated population.Le V in strain SY1 was eliminated with using ribavirin,and ISSR analysis showed that ribavirin had no significant effect on the genetic stability of L.edodes.Le V had no significant effect on mycelial growth rate,ripening aging and resistance to Trichoderma atroviride at 25 °C.However,Le V had negative effect on the above three characters after heat stress(37 °C).In other words,Le V affected the thermotolerance of SY1.Le V in core collections did not produce virus particles and had no obvious molecular variation.Low temperature(16 °C,19 °C)was beneficial to Le V replication,while high temperature(>31 °C)inhibited it.Otherwise,the relative expression of Le V replicase gene increased in matured mycelium exposed to high temperature(37 °C,40 °C,42 °C).(4)A total of 19 RNAi key genes including 3 Dicer-like(DCL),8 Argonaute(AGO)and 8 RNA-dependent RNA polymerase(RDR)genes were predicted in L.edodes genome.No heat responsive cis-acting elements were found in their promoter regions.At25 °C,there was no significant change in the expression of any of the 19 RNAi key genes after Le V removal.However,after heat treatment,the expression levels of one DCL gene,two AGO genes and four RDR genes were significantly decreased after Le V removal.Le AGO8 was almost not expressed in the virus-free strain after heat stress.(5)Le V-infected strain VI and Le V-free strain VF originated from strain SY1 was used as sequencing materials.Matured mycelia grown on the plate were treated at 25 °C and 37 °C for 48 h,respectively.After that,the mycelia were divided into two treatment:(1)T1 period,at the end of high treatment;(2)T2 period,followed by cultured under25 °C for 15 days.The m RNA and s RNA of all samples were sequenced and analyzed.At 25 °C,Le V had no significant effect on the transcriptome level of the host,but significantly reduced the thermotolerance of the host.The results showed that the heat shock protein of VI strain was more sensitive to heat stress,and the response of transcription factors was different from that of VF strain.VF was specifically enriched in the metabolic pathway related to repair.The cell membrane of VI was more damaged by high temperature than VF.The difference of gene response of VI and VF to heat stress was mainly in T1,and the total number of different genes in T2 was far less than T1.27 new mil RNAs were identified from L.edodes firstly.The abundance of most mil RNAs was very low.Only 11 mil RNAs were more than 100 reads,and led-mil R-21 was the highest one.Similar to the expression pattern of key RNAi genes such as Le AGO8,led-mil R-21 was significantly down regulated only after VF experienced heat stress,and was highly expressed in other treatments.Stem-loop RT-q PCR analysis confirmed this result.LE01Gene01783,one of the target genes of led-mil R-21,is a CBFB-NFYA type transcription factor that responsive to heat stress,was only up-regulated after heat stress in virus-free strain VF.Among the 2030 genes differentially expressed in VF after heat stress,613 had potential binding sites of transcription factor LE01Gene01783.KEGG enrichment analysis revealed multiple amino acid metabolic pathways including tryptophan metabolism.Heat stress induced Le DCL1 to degrade Le V genome,especially 5’-UTR terminal and ORF1 upstream.After heat stress,the total abundance of vsi RNA increased,but the abundance of single vsi RNA was relatively low.There was no significant difference in the target gene expression of Le V derived vsi RNA.It is very likely that Le V vsi RNA will not directly affect the gene expression of L.edodes.Based on the above results,L.edodes should has a mil RNA regulatory mechanism to cope with heat stress.The down-regulation of led-mil R-21 activates the downstream thermotolerance repair pathway,and the existence of Le V may interfere with this pathway,which reduces the thermotolerance of the Le V-infected strain.This study explained the diversity of L.edodes virus from the perspective of omics for the first time,analyzed the complete genome and molecular biological characteristics of Le PV1,identified the adverse effect of Le V on thermotolerance of L.edodes,and preliminarily revealed the effect of Le V on the regulatory pathway of led-mil R-21 coping with high temperature response.The results here have very important theoretical value and guiding significance for exploring the interaction mechanism between virus and L.edodes,as well as for the prevention of L.edodes virus diseases. |
PDF Full Text Request