Mechanisms Of Validamycin Inducing Plant Resistance And Controlling Fusarium Graminearum

Fusarium graminearum Schwabe[teleomorph Gibberella zeae（Schweinitz）Petch]is the most common causal agent of a destructive disease Fusarium head blight（FHB）in many parts of the world.It can also infect other crops,such as barley,rice,corn and oats,causing significant yield losses and grain quality reduction.In infected grains,F.graminearum produces trichothecene mycotoxins that are deadly hazardous to human and animals.At present,due to the lack of wheat varieties with high resistance to FHB,the main strategy to control FHB is chemical control.The commonly used fungicides include benzimidazoles,triazoles,phenamacrils,succinate dehydrogenase inhibitors,methoxyacrylates.However,with the long-term use of single fungicides in large quantities alone,the problem of pathogen resistance has become increasingly serious.Therefore,using antagonistic microorganisms and their secondary metabolites to biologically control plant disease,or combined with chemical fungicides,can not only reduce the use of chemical pesticides,but also slow down the generation of drug resistance of pathogens,which is beneficial to the ecological environment and human health.Validamycin A（VMA）is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus,and has been used to control rice sheath blight caused by Rhizoctonia solani for about 50 years in China,and no resistance has been found.In recent years,our previous study suggested that VMA can not only improve the disease resistance of wheat,but also can be used to control FHB and reduce the production of DON of F.graminearum.Although the control effect of VMA when used alone is not ideal,the combination of VMA and triazoles has significant synergistic effect.Although many studies have reported that VMA can enhance plant disease resistance and induce plant defense response,the optimal conditions for VMA to induce plant defense response and the signal transduction pathway needed to activate plant defense response have not been reported.Since the hyphae of R.solani are multinuclear,it is very difficult to study its gene function by the method of genetic transformation.So far the research on the mode of action of VMA still remains on the results of physiological and biochemical studies in the 1990s that VMA can be hydrolyzed to Validoxylamine（VAA）by glycosidase.VAA has similar structure with trehalose,and can inhibits trehalase activity,thus affecting the energy metabolism and cell wall structure of pathogens.However,there are many glucosidase genes in organisms,as well as neutral and acid trehalase.It is not known which glycosidase can hydrolyzed VMA and which trehalase can be inhibited by VMA.In this study,reactive oxygen species（ROS）bursts and callose deposition in Arabidopsis thaliana,rice（Oryza sativa L.）and wheat（Triticum aestivum L.）were induced by VMA and were most intense with VMA at 10 μg mL-1 at 24 h.Moreover,VMA induced resistance against Pseudomonas syringae,Botrytis cinerea,and Fusarium graminearum in Arabidopsis leaves,indicating that VMA induces broad-spectrum disease resistance in both dicots and monocots.In addition,VMA-mediated resistance against P.syringae was not induced in NahG transgenic plants,partially decreased in nprl mutants,and VMA-mediated resistance to B.cinerea was not induced in npr1,jar1,and ein2 mutants.These results strongly indicated that VMA triggers plant defense responses to both biotrophic and necrotrophic pathogens involving in salicylic acid（SA）,jasmonic acid/ethylene（JA/ET）signaling pathway and is dependent on NPR1.In addition,transcriptome analysis further revealed that VMA regulated the expressions of genes involved in SA,JA/ET,abscisic acid（ABA）and Auxin signal pathways.Taken together,VMA induces systemic resistance involving in SA,and JA/ET signaling pathways,and also exerts a positive influence on ABA and Auxin signaling pathways.In this study,the differentially expressed genes of F.graminearum and R.solani treated with VMA were analyzed,founding that VMA reduced the expression level of cellulose degrading enzymes:glucanase Novel00337 and glucosidase Novel00880 of R.solani,glucanase FGSG₁1777 and glucosidase FGSG₀7351 of F.graminearum.Interestingly,another glucosidase FGSG₀6605 was up-regulated by VMA in F.graminearum.In addition,VMA can also up regulated the expression of trehalose synthase genes AG1IA₀5577 and FGSG₀7926,and chitinase genes AG1IA₀1900 and FGSG₀0952 in R.solani and F.graminearum.These results suggested that the metabolism of trehalose,cellulose and chitin may be closely related to the action mode of VMA.In this study,the single deletion,double deletion and overexpression of neutral trehalase FgNTH（FGSG₀9895）and acid trehalase FgATH（FGSG₀5622）in F.graminearum were conducted by genetical transformation technology,founding that the deficiency of FgNTH and FgATH respectively reduces the sensitivity to VMA by 2.12-and 1.79-fold,indicating that FgNTH is the main target of VMA.FgNTH is responsible for vegetative growth,FgATH is critical to sexual reproduction,and both of them play an important role in conidiation and virulence in F.graminearum.Moreover,FgNTH resides in cytoplasm,affects the localization of FgATH,and positively regulates DON biosynthesis,however,FgATH resides in vacuole and negatively regulates DON biosynthesis.Furthermore,this study found that FgNTH interacts with FgPK（Pyruvate kinase）,a key enzyme in glycolysis,and the interaction can be reduced by VMA,and the deficiency of FgNTH affects the localization of FgPK under DON induction condition.Thus,this study concluded that VMA disturbs glycolysis by reducing the interaction between neutral trehalase and pyruvate kinase to decreases DON biosynthesis in F.graminearum.In addition,this study also found that the deletion mutant of FgNTH was more sensitive to triazoles tebuconazole.The relative expression levels and subcellular locations of FgCYP51A,FgCYP51B and FgCYP51C were determined in the mutants ΔFgNTH andΔFgATH,showing that FgNTH positively regulated the expression of FgCYP51A and FgCYP51B,and FgATH negatively regulated the expression of FgCYP51A,FgCYP51B and FgCYP51C.Furthermore,the protein affinity capture mass spectrometry analysis and CO-IP assay showed that FgNTH can interact with FgCYP51B.Therefore,this study concluded that FgNTH can regulate the expression levels of FgCYP51A and FgCYP51B via interacting with FgCYP51B,which makes VMA have synergistic effect with triazoles tebuconazole.In this study,the upstream gene of trehalose synthesis phosphoglucomutase FgPGM（FGSG₀0387）also was deleted and overexpressed by genetical transformation technology to determine whether it affects the sensitivity to VMA.This study found that FgPGM resided in cytoplasm of F.graminearum,and played an important role in vegetative growth,formation of aerial hyphae,branching at the tip of hyphae,conidiation,conidia morphology,conidia germination and virulence,but did not affect the penetration ability and sexual reproduction of mycelia,and the deletion of FgPGM was more sensitivity to VMA in F.graminearum.In addition,the deficiency of FgPGM reduced the pyruvate acid and DON production,indicating that FgPGM can regulate the glycolysis and the DON biosynthesis of F.graminearum.Therefore,this study thought that FgPGM has very important biological functions,and can regulated the sensitivity of VMA,and can regulate the biosynthesis of DON of F.graminearum,which makes it has the potential to develop as a new pesticides target to inhibit the DON biosynthesis and control FHB.In this study,the beta-glucosidase gene FgBGL1（FGSG₀6605）and FgBGLIB（FGSG₀7351）effected by VMA in F.graminearum were also deleted to study their regulatory role for the sensitivity to VMA.This result showed that the deficiency of FgBGLl decreased the sensitivity of F.graminearum to VMA,and made F.graminearum has similar sensitivity to different concentrations of VMA;the sensitivity of deletion mutantΔFgBGLIB was not changed when has 1 μg mL-1 VMA,and was increased to 10 μg mL-1 VMA,but was decreased to 100 μg mL-1 VMA.This result may be due to the fact that because of the deficiency of FgBGLl,VMA cannot be hydrolyzed to VAA,which has a better inhibitory effect on trehalase;FgBGL1B also is the target of VMA.Therefore,this study presumed that FgBGL1 may be a glycosidase for hydrolyzing VMA,FgBGLIB is another target of VMA expect for FgNTH and FgATH.In conclusion,this study found that VMA induces plant disease resistance via activating SA and ET/JA signal transduction pathways;FgNTH is the main target of VMA;FgBGL1 may be hydrolyze VMA;FgATH and FgBGL1B are also the targets of VMA;FgNTH and FgPGM can regulation DON biosynthesis by interacting with FgPK;VMA decreases the DON biosynthesis through reduce the interaction between FgNTH and FgPK;FgNTH can regulate the expression levels of FgCYP51A and FgCYP51B via interacting with FgCYP51B,which makes VMA has synergistic effect with triazoles fungicides.Therefore,this study illuminated the mechanism that VMA triggers plant defense response for the first time,and clarified its targets in F.graminearum,and revealed the mechanism of VMA that inhibits the DON biosynthesis of F.graminearum,and the synergistic effect of VMA with triazoles fungicides.This study will provide a theoretical basis for the creative application of VMA on inducing plant defense response to increase plant disease resistance,and for the combination of VMA and triazole fungicides to decrease the DON contamination,control FHB,reduce the use of chemical pesticides,even govern the resistance of pathogens to fungicides.