Functional Analysis Of Laccase Gene ShLAC1 In Scleromitrula Shiraiana And Chitin Recognition Receptor In Mulberry

Mulberry is a nutritious and healthful fruit.However,mulberry sclerotial disease is the most serious fungal diseases for mulberry fruits.The main symptom of mulberry sclerotial disease is that the pathogens form one or several black sclerotia within the diseased drupelets.The cell death and pigment loss in the diseased drupelets,and finally the diseased mulberry fruits turned to mummified white fruits.Therefore,mulberry sclerotial disease is also known as mulberry albinism in China.The pathogens of mulberry sclerotial disease are necrotrophic fungi.Necrotrophic fungi have more complex infection mechanism than biotrophic and hemibiotrophic fungi.Even the model fungi of Necrotroph,Sclerotinia sclerotiorum and Botrytis cinerea,their pathogenesis is not very clear.Moreover,the pathogens of mulberry sclerotial disease may be specific parasitic mulberry,and the related reports on the pathogenesis of pathogens are rare.The study of pathogenic mechanism of pathogens has very important theoretical and practical significance.However,understanding the pathogenic mechanism of pathogens is only one aspect of solving the problem of diseases.The ultimate way to reduce the occurrence of diseases is to cultivate disease-resistant or disease-tolerant mulberry varieties.The study of the mulberry immune response triggered by pathogens of sclerotial disease is the theoretical basis for disease resistance breeding of mulberry.The innate immunity of plants can be divided into two layers: PAMP-triggered immunity(PTI)and effector-triggered immunity(ETI).PTI is a low-cost and broad-spectrum immune response that has less side-effect on the plants themselves.It is very important to study the PTI response of mulberry to the selection of broad-spectrum disease-resistant or disease-tolerant mulberry varieties.In this study,we cloned and identified a key gene ShLAC1 in melanin biosynthesis of S.shiraiana,and proved that the melanin and pathogenicity are closely related.Through the sequencing of the transcriptome of diseased fruits and the corresponding healthy fruits in three different diseased stages of mulberry sclerotial disease,the genes expression in defense-related metabolic pathways and resistance-related responses were analyzed.From the transcriptome data,receptor proteins that recognize fungal chitin were identified,and then the proteins that related to the recognition and transmission of chitin signaling were studied.The results of this study were as follows:1.ShLAC1 is a key gene for pathogenicity-related melanin biosynthesisS.shiraiana is the pathogen of mulberry shrunken fruit disease.S.shiraiana strain SX-001 was isolated from the diseased fruit in mulberry orchard in Southwest University.On the artificial medium,the growth of S.shiraiana is slow and the melanin is secreted.After the colony stops growing,S.shiraiana would secrete a circle of microspores in the middle of the colony,but could not form sclerotia.The sclerotium may only form in diseased fruit.Paraffin sections of diseased drupelets showed that black outer wall of sclerotium was formed by the fractured black hyphae.The sclerotium prosenchyma is composed of hypha and drupelets tissue.The fungal melanin not only has a defensive role,but also is associated with pathogenicity.The melanin of S.shiraiana is 1,8-DHN melanin that was identified by GC-MS.We have obtained ShLAC1,related to melanin biosynthesis,by molecular cloning technology.ShLAC1-encoded laccase catalyzes the polymerization of 1,8-DHN monomers to form 1,8-DHN melanin.In order to verify the important role of ShLAC1 in the melanin biosynthesis of S.shiraiana,we obtained two stable ShLAC1 silenced transformants by agrobacterium-mediated RNAi technology.ShLAC1 silenced transformants not only significantly reduced melanin synthesis,and mycelium vegetative growth is also inhibited.In vitro catalytic experiments showed that the cultured filtrate of the ShLAC1 silenced transformants catalyzed the melanin formation of 1,8-DHN monomers was significantly less than that of the wild type strain filtrate.This indicated that ShLAC1 is a key gene for the biosynthesis of 1,8-DHN melanin in S.shiraiana.Since ascospores are the only source of primary infection of mulberry sclerotial disease,and we are unable to obtain sclerotium of S.shiraiana on the artificial medium and cannot obtain ascospores.So,it is impossible to directly verify whether the pathogenicity of ShLAC1 silenced transformants is affected.Therefore,we have to indirectly verify the pathogenicity of ShLAC1 silenced transformants from other aspects.Oxalic acid is an important pathogenic factor for sclerotium-formed plant pathogenic fungi.Therefore,we regarded the concentration of oxalate produced by fungi as an important indicator of the pathogenicity of the strain.UPLC assay showed that the oxalic acid concentration of the filtrate of wild type strain was 2.13 mM,while the ShLAC1 silencing transformants could not detect oxalic acid.Similarly,ShSCD1,the other key gene in the melanin biosynthesis,silenced transformant iSCD's oxalic acid production was also significantly lower than that of the wild type.The production of oxalic acid was also significantly inhibited after treatment of the wild-type strain with the agricultural agent tricyclazole that specifically inhibits the activity of two reductase tetrahydroxylnaphthalene reductase and trihydroxynaphthalene reductase in the melanin biosynthesis.This indicated that the inhibition of melanin biosynthesis,oxalic acid production is also inhibited.In addition,we also found that the adhesion of ShLAC1 silenced transformants on PDA was reduced.In summary,ShLAC1 silencing reduced melanin production and attenuated the pathogenicity of S.shiraiana.2.Transcriptome sequencing of mulberry fruits that infected mulberry sclerotial diseaseWe selected the diseased fruits that infected sclerotial disease at early stage(Stage 1),mid stage(Stage 2)and middle-late stage(Stage 3),and the corresponding period of healthy mulberry for transcriptome sequencing.Statistical analysis of differentially expressed genes revealed that the differentially expressed genes mainly appeared at the mid stage(Stage 2)and middle-late stage(Stage 3),but there were almost no differentially expressed genes at the early stage.First of all,we analyze the defense-related metabolic pathways.The results showed that a large number of differentially expressed genes were enriched in flavonoid biosynthesis pathway and phenylpropanoid biosynthesis pathway,such as chalcone isomerase(CHI)gene,leucoanthocyanidin dioxygenases(LDOX)gene,leucoanthocyanidin reductase(LAR)gene,bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase(DFR)gene,chalcone synthases(CHS)genes and peroxidases(POD)genes,cinnamyl-alcohol dehydrogenase(CAD)gene,phenylalanine ammonia-lyase(PAL)gene,caffeic acid 3-O-methyltransferase(COMT)gene were upregulated at the stage 2 or/and stage 3.However,in the plant hormone signal transduction pathway that closely related to plant immunity,most of the differentially expressed genes were down-regulated.In addition,from the two-layered immune response of plants,PTI and ETI,we identified 19 genes involved in calcium signal transduction,of which over half were up-regulated,and many of the plasma membrane-localized receptor protein differentially expressed genes.Among these Unigenes of plasma membrane-localized receptor protein,we also identified 13 LysM-containing Unigenes.LysM-containing proteins may be involved in the immune response triggered by the fungal chitin signalling.This is the research we will conduct next.In addition,we also identified 66 up-regulated R genes belonging to the second layer of the immune response ETI.3.LysM1 in MmLYK2 is a critical motif for interaction between MmLYK2 and MmLYP1Chitin signal recognition proteins are the LysM-containing plasma membranelocalized proteins.Based on the mulberry fruit transcriptome and mulberry genome data,we identified 11 LysM receptor-like proteins,including 9 receptor kinases(LYKs)and 2 receptor proteins(LYPs).The expression of MmLYP1 is induced by fungi and chitin,and MmLYP1 is a homolog of CEBiP and is a GPI-anchored protein.MmLYP1 has a 23 amino acid residue signal peptide at the N-terminus,two LysMs in the middle and a hydrophobic structure at the C-terminus.The predicted glycosylphosphatidylinositol modified site ? is at 334 N.Therefore,MmLYP1 is likely to be a chitin recognition receptor.In order to verify that MmLYP1 has chitin affinity,we incubated mulberry total protein with insoluble polysaccharide chitin magnetic beads,chitin,chitosan,cellulose and xylan,respectively,and then used polyclonal antibody specific for MmLYP1 to detect the target protein.The results of western blot show that MmLYP1 can be pulled down specifically by insoluble chitin(chitin magnetic beads and chitin).Furthermore,the GFP-tagged MmLYP1 fusion protein that transiently expressed in Nicotiana benthamiana leaves was pulled down by chitin.Since MmLYP1 lacks the intracellular structure,the chitin signal recognized by MmLYP1 require another protein with intracellular kinase to transmit.Therefore,we screened MmLYK2 from 9 receptor kinases,which is most likely to be the downstream protein of MmLYP1.Subcellular localization showed that both MmLYP1 and MmLYK2 were located on the plasma membrane.The interaction of MmLYP1 and MmLYK2 was demonstrated by Y2 H,BiFC and Co-IP.The interaction of MmLYP1 and MmLYK2 is an important step for chitin signaling.Because MmLYP1 only contains extracellular structure,the interaction between MmLYP1 and MmLYK2 occurs in the extracellular domain.LysM is the only predicted extracellular domain of MmLYP1 and MmLYK2,so the interaction between them may be related to LysM motif.Secondary structure analysis showed that the structure of MmLYP1 was very similar to that of CEBiP and AtCEBiP,both of which contained two LysM motifs,and the corresponding LysM sequences were similar.However,MmLYK2 has two LysM motifs,while OsCERK1 and AtCERK1 have one and three LysM motifs,respectively.The LysM1 of MmLYK2 interacted with all four LysM motifs in MmLYP1 and MmLYK2 in yeast.The chimera lacking the LysM1 of MmLYK2 did not interact with MmLYP1 and MmLYK2 in yeast and Nicotiana benthamiana cells.This indicated that the LysM1 of MmLYK2 is the key motif of the interaction between MmLYK2 and MmLYP1 and itself.