PDMP Induce Resistance Against TMV Via ABA Mediated-callose Accumulation

Tobacco mosaic virus(TMV)disease is the most common and serious virus disease in flue-cured tobacco planting.The prevention and control of TMV has always been a valuable and difficult project in agricultural production.Recent developments in fungiderived antiviral molecules provided a new strategy for the effective control of plant viruses,such as TMV Dry mycelium of Penicillium chrysogenum(DMP)has been reported to defend against plant virus infection by eliciting anti-virus-related gene expression in recent reports.Our previous studies also confirmed that DMP initiated a defensive response to TMV infection.Although DMP has been broadly applied to field prevention and has achieved considerable economic and ecological benefits,the mechanism of DMP-induced antiviral activities remains largely unknown.DMP-derived polysaccharide extract and polypeptide extract(PDMP)were obtained,and we found that both polysaccharide extract and PDMP activated the tobacco antiviral response to TMV infection.However,the molecular mechanism involved in the PDMPinduced anti-TMV response is still unclear.Thus,we investigated the molecular mechanism by using RNA-seq,qPCR,western blot,CRISPR/Cas9,callose staining and immunogold technology.The main results are as follows:(1)Results of RNA-seqOnly four differentially expressed genes(DEGs)in the TMV vs PT(PDMP+TMV)group were annotated as ABA2,IRAK4 and β-1,3-glucanase.However,these DEGs were not significantly different in the CK vs PDMP group.These results suggested that β-1,3glucanase and ABA2 have important relationships with the defense response induced by PDMP,moreover PDMP-induced TMV resistance is a state of priming.Directed acyclic graphs of GO enrichment showed that PDMP treatment alone could not directly induce a defensive response in plants,moreover PDMP pretreatment before TMV inoculation could induce the plant to produce a defense response,which indicated that PDMP-induced TMV resistance is a state of priming.Analysis of the KEGG database showed that many resistance-related signaling pathways were significantly enriched in the CK vs TMV and the CK vs PT groups;only the "carotenoid biosynthesis" significantly enriched in the TMV vs PT gourp,but not in the CK vs PDMP group.The "carotenoid biosynthesis" is closely correlated with precursors of abscisic acid(ABA),and previous data have also showed that large amounts of callose deposition were observed after TMV inoculation by PDMP;thus,we speculate that PDMP-induced callose deposition may be closely related to ABA biosynthesis.(2)Construction of N.benthamiana ABA biosynthesis pathway mutants.We successfully constructed ABA synthesis pathway mutants with NbZEP gene mutations using CRISPR/Cas9 technology.(3)PDMP promotes ABA biosynthesis-mediated callose deposition against TMV infection.The results showed that the infection speed of TMV was markedly inhibited,and callose accumulation around the plasmodesmata was significantly decreased in the PT group compared with the TMV group.In the mutant plants,there was no significant difference between the PT group and the TMV group,and callose accumulation was significantly reduced in the PT group.After treatment with ABA,TMV green fluorescence was decreased,deposition and expression of callose were increased,and the diameter of plasmodesmata was decreased in the PT group of the mutant plants.These results suggest that PDMP resistance to TMV by inducing callose deposition around the plasmodesmata is dependent on the ABA biosynthesis pathway.In this study,our results suggested that callose deposition around plasmodesmata in wild-type plants is mainly responsible for the restriction of TMV movement during the PDMP-induced defensive response to TMV infection and that ABA biosynthesis apparently plays a crucial role in PDMP-induced callose priming for enhancing defense against TMV.Our study provides new insights into the molecular mechanism of PDMPmediated resistance to virus in tobacco.