Functional Characterization Of Thioredoxin Reductase In Fusarium Graminearum

Wheat scab is an important disease which has attracted worldwide attention due to the serious threat to the stable development of wheat production.Fusarium graminearum is one of the important pathogens that not only causes wheat scab,but also endangers the health of humans and animals because of consumption of Deoxynivalenol.Many enzymatic or non-enzymatic mechanisms can effectively scavenge reactive oxygen species.The most important mechanism is thioredoxin system which has strong antioxidant and free radical scavenging effects.Thioredoxin reductase?TRR?plays a key role in the cycling of redox and reductive thioredoxins,but its function in plant pathogenic fungi remains unclear.In this study,we used split-marker PCR to knockout TRR gene in F.graminearum.Seven individual mutants were identified by PCR and Southern blot.We found that FgTRR was localized in the cytoplasm.The domain analysis showed that FgTRR was predicted to harbor all domains typical for the TRR family,including a FAD-binding domain??GXGXX,where X is any amino acid?,a FAD-binding domain??GXFAXGDV?,and a NADPH-binding domain?GGGXXA?.Phylogenetic analysis by the neighbor-joining method revealed that TRR is highly conserved among three Fusarium species.The mycelial growth rate of the?TRR mutants were significantly slower than that of the WT strain.Although it had no effect on morphology,the?TRR mutants showed delayed conidial germination,indicating that FgTRR had defects in mycelial growth and conidial germination.The?TRR mutants showed reduced resistance in 0.7 M KCl,0.7 M NaCl,1 M sorbitol and 0.2g/L congo red,and increased resistance in 0.05%SDS,indicating that FgTRR was involved in the salt stress,cell wall and cell membrane integrity of F.graminearum.Moreover,the?TRR mutants showed increased sensitivity and decreased resistance in 5 mM H₂O₂ and 15?M menadione,suggesting that FgTRR affected the process of oxygen stress.Different from the the WT strain that produced normal perithecia and ascospores,the perithecia produced by the?TRR mutants were smaller in shape and could not produce ascospores,the?TRR mutants also exhibited female sterility,these results indicated that FgTRR was not necessary for sexual reproduction,but seriously affected the process of sexual reproduction.Compared with the WT,the pathogenicity of the?TRR mutants decreased by 94%,the production of DON and genes related to the biosynthesis of DON were significantly decreased,indicating that FgTRR was of great importance in the pathogenicity.Moreover,the?TRR mutants showed defects in pigmentation,and the expression level of genes related to pigment biosynthesis was significantly reduced.We further showed that the proportion of Annexin V-FITC-positive protoplasts was approximately 18%and 33%in the WT and the?TRR mutants treated with H₂O₂ for 1 h,and the accumulation of reactive oxygen species in the?TRR mutant were more than that in the WT strain,the above results showed that FgTRR was involved in the apoptotic process.Taken together,these results demonstrate that FgTRR plays an important role in growth and development,sexual reproduction,pigment synthesis,pathogenicity and apoptosis-like cell death.