The Effect Of The Rab-interacting Lysosomal Protein In Fusarium Graminearum On Biosynthesis Of DON Toxin

Wheat is one of the world's three major food crops.Wheat scab is an important disease worldwide,which not only causes the reduction of food crops,but also produces a large number of mycotoxins that threaten the safety of humans and animals.Prevention and control of wheat scab is of great significance.The main pathogen of the disease is Fusarium graminearum,and the DON toxin produced by it is regulated by many factors.Rab7 protein plays a regulatory role through upstream regulators and downstream effectors.RILP(Rab interaction lysosomal protein)is one of the effectors of Rab7.The Rab7 gene affects the biosynthesis of DON toxin in F.graminearum,but the the effect of RILP on DON toxins is unknown.The function of RILP was characterized in F.graminearum.Bioinformatics analysis revealed that FgRILP contains multiple conserved domains: NST1(negative salt tolerance),TolA,MIP-T3 and Atrophin-1.FgRILP of F.graminearum PH-1 was in the same branch as the putative protein of the other F.graminearum and the stress response protein of F.culmorum by phylogenetic analysis.It is supposed that the FgRILP is involved in salt stress.The FgRILP gene knockout and over-expression mutants were constructed by homologous recombination technique and phenotype of the mutants was analyzed under salt stress and non-stress.The result showed that there was no difference in the colony morphology between the mutant strain and the wild type under the condition of non-salt stress,but the growth of the FgRILP knockout mutant mycelium slowed down,and the hyphae of the over-expressed mutant grew faster(p<0.05).Compared with the wild type,the FgRILP over-expression mutants had raised sporulation,but the knockout mutants had reduced sporulation.Spore germination rate of FgRILP mutant and wild type is consistent,Both wild type and FgRILP mutants were invasive,but the knockout mutants had less infection power than PH-1 and FgRILP over-expression mutants.It indicated that the FgRILP gene was associated with the growth,sporulation and pathogenicity of F.graminearum,but had no effect on spore germination or morphology.Compared with non-salt stress,the wild type and mutant mycelia became sparse and the growth rate slowed down significantly(p<0.01)under the stress of1.0M NaCl,0.08 M LiCl or Congo red.Under salt stress,conidia of mutant and wild-type strains were significantly deformed,the sporulation rate and the spore germination rate decreased significantly,the sporulation of FgRILP mutants was lower than that of wild type.Both the overexpressing mutant and the knockout mutant had lower sporulation than the wild type.Under NaCl stress,the mycelial growth rate of FgRILP knockout mutants was slower(p<0.05)than that of wild-type and FgRILP overexpressing mutants,and the pathogenicity of FgRILP knockout mutants was weaker than that of wild type.It suggested that the FgRILP gene regulated the growth and pathogenicity of the strain under salt stress.This study further analyzed the effect of FgRILP on the synthesis of DON toxin.In wheat substrate medium,the DON synthesis of FgRILP knockout mutants was significantly lower than that of wild type under non-stress and NaCl stress(p<0.05);DON of FgRILP knockout mutant under 0.08 M LiCl threat The amount of synthesis is slightly higher than that of the wild type.In the toxic liquid medium,the DON synthesis of FgRILP knockout mutant was significantly lower than that of wild type under NaCl stress(p<0.05).Under the stress of0.08 MLiCl,the DON synthesis of FgRILP knockout mutant was slightly higher than that of wild type.And after analyzing the expression of Tri5 gene,it was found that the relative expression level was consistent with the determination of toxin under NaCl stress.It indicated that FgRILP affected the synthesis of F.graminearum DON toxin and had a regulatory effect under salt stress.In addition,yeast two-hybrid assay was used to analyze the interaction between FgRILP and FgRab7.The results showed that FgRILP was no interaction between FgRab7.But the relative expression of two genes was analyzed by real-time quantitative PCR.The relative expression level of FgRILP gene in the FgRab7 knockout mutant was significantly lower than that in the wild type,but that of FgRab7 in the FgRILP knockout mutant was higer than that in the wild type.The result indicated a certain correlation between the two gene.This study investigated the function of FgRILP and its effect on the synthesis of DON toxin in F.graminearum.The results laid a foundation for revealing the molecular regulatory mechanism of DON toxin biosynthesis,and provided a theoretical basis for developing new targeted microbicides,reducing the pollution level of mycotoxin,and guaranteeing the edible safety of agricultural products.