Isolation And Characterization Of Deoxynivalenol-Resistant Mutant(s)in Arabidopsis

Fusarium head blight(FHB)of wheat is a global crop disease caused mostly by the fungal pathogen Fusarium graminearum(Fg),which can reduce the yield of wheat and other crops significantly.During the infection process,Fg produces the mycotoxin deoxynivalenol(DON)as an effector to facilitate this process.In addition,DON has been considered as one of the key contaminants of cereals and their derivatives.Over the last decade,the biodegradation of DON has become a hot topic.However,there are only very few reports on DON-resistant genes in plants.Most available researches are mainly focused on a set of genes encoding glucosyltransferase,which can glycosylate the C3 hydroxyl of DON,thus reducing the toxicity of DON.To uncover more DON-resistant genes in plants,we screened the ethyl methanesulfonate(EMS)mutant library of Arabidopsis to identify DON-resistant mutants,which may provide important genetic materials for future clarifying the molecular mechanism of DON resistance in plants.To evaluate the effect of DON on plant growth,firstly we phenotypically analyzed the Arabidopsis grown on DON-containing media in detail,and found that DON can remarkably inhibit the root growth of Arabidopsis.It increased the ROS level and decreased the number of meristematic cells in root tips.In addition,DON reduced the expression of several auxin transporters,including PIN1,PIN2 and PIN3,and the auxin signaling components AUX1 and DR5,which indicates that DON alters the microenvironment at the root tips,thus inhibiting Arabidopsis root growth.Four DON-resistant mutants were then isolated,designated as dor1-4,by screening an EMS mutagenized population of Arabidopsis for seedlings with roots exhibiting better growth in 5 ppm DON.These mutants also displayed enhanced resistance to Fg.We further analyzed one of the mutants dor1,of which a mutation was found in AT1G05680 through standard map-based cloning combined with Mutmap analysis.A single G to A transition mutation caused a Glycine to Arginine change.AT1G05680 encodes a glucosyltransferase(UGT74E2)which glycosylates auxin IBA.The heterologous expression in yeast demonstrated that the enzyme could also glycosylate the C3 hydroxyl of DON.However,further in vivo analysis of DON and its derivate D3 G showed that the mutation did not significantly change their composition in Arabidopsis,indicating that although UGT74E2 can glycosylate DON,the mutation is not the cause of DON resistance of dor1.Through phenotypic analysis of dor1 during early seedling development,we found that the mutant is smaller and has shorter roots than wild type under normal growth conditions.Upon auxin treatment,the auxin-dependent lateral root growth is reduced in dor1,which produced fewer lateral roots than wild type,indicating that the mutation in UGT74E2 reduces auxin sensitivity.In conclusion,the mutation of UGT74E2 gene in dor1 damped its sensitivity to auxin,which in turn may improve its resistance to DON and Fg.Therefore,the identification of this mutant provides a very good genetic material for clarifying the molecular mechanism of how DON interferes auxin signaling pathway and the role of auxin in Fg infection.