Functional Studies On The Mitochondrial Outer Membrane Protein Porin Of Fusarium Graminearum In Growth And Development,Pathogenicity And Autophagy

Fusarium head blight is a worldwide disease caused by Fusarium graminearum,which occurs seriously in the main wheat production areas in China,not only causing loss of wheat yield,but also food safety problems caused by the residual mycotoxins in wheat grains,which is a serious threat to human and animal health.F.graminearum is the dominant pathogen of wheat scab.The secondary metabolite deoxynivalenol(DON)produced by F.graminearum is one of the most toxic and common mycotoxins,and also the key cause of Fusarium head blight.Therefore,studying the growth,development,and pathogenic mechanism of F.graminearum is very important for the prevention and control of Fusarium head blight.Mitochondria are essential organelle in eukaryotic cell,which mediate energy production,metabolism,signal pathways and programmed cell death in primary cells.Porin,also known as voltage dependent anion selective channel(VDAC),is the most abundant protein on the outer membrane of mitochondria,and its barrel like structure promotes the exchange of ions and small metabolites such as ATP between mitochondria and cytoplasm.In recent years,the function of Porin in biological activities has gradually been revealed,and indepth research has been conducted in mammals and even yeast.However,there is relatively little understanding of Porin function in filamentous fungi.In this study,a Porin homolog XP＿011318840.1,named Fg Porin with gene number FGSG＿09933,was identified from the F.graminearum PH-1 database by comparing the protein sequences of Saccharomyces cerevisiae Porin1 and Porin2.Fg Porin encodes 283 amino acids and has only one functional domain,Porin＿3.The amino acid sequence comparison and evolutionary tree analysis revealed that Fg Porin is closely related to the Porin genes of Neurospora crassa and Fusarium oxysporum,and has a more consistent species evolutionary trend.These could indicate that the Fg Porin found in this study is a direct homolog of the Porin genes of other filamentous fungi.The Fg Porin knockout fragment was constructed by Split-marker PCR and then the porin gene was knocked out using the principle of homologous recombination with the help of PEG-mediated protoplast transformation.After resistance screening,PCR and Southern blot validation,a Porin gene deletion mutant(Δporin)was obtained,and a Δporin-C revertant strain was further obtained on the basis of Δporin.In terms of the growth and development of F.graminearum,The colony growth rate of Δporin strain on PDA,CM,and 1.5% glycerol medium slowed down,the height of aerial hyphae decreased,and the ability to utilize non fermentable carbon sources decreased.The conidia production of Δporin strain decreased,the number of septum decreased,and it could not produce ascus and ascospore.In terms of pathogenicity of F.graminearum,The infection ability of Δporin strain on wheat ears and wheat germ sheaths significantly decreased,the production of DON toxin significantly decreased,and the expression level of TRI gene affecting DON toxin synthesis also significantly decreased.The defective formation of toxin bodies and lipid droplets in the Δporin strain suggests that Fg Porin plays an important role in the synthesis of toxin bodies and lipid droplets,affecting the pathogenicity of F.graminearum by influencing the formation of lipid droplets and toxin bodies and the synthesis of DON toxin.FgPorin is an outer mitochondrial membrane protein and we investigated the effect of Porin deletion,on mitochondrial morphology and function in F.graminearum.The results showed abnormal mitochondrial outcome,decreased mitochondrial membrane potential,and decreased production of both endogenous hydrogen peroxide and ATP in Δporin strain,indicating that Fg Porin is essential for normal mitochondrial morphology and function.In terms of autophagy in F.graminearum,the Δporin strain had a milder onset of autophagy.After nitrogen-deficient incubation,when autophagic vesicles appeared to accumulate in the cytoplasm of the mycelium of the wild-type PH-1 strain and severe autophagy had already occurred,the Δporin strain had not yet produced autophagic vesicles,suggesting that Fg Porin affects autophagy and that the absence of Fg Porin in the strain attenuates autophagy.In addition Porin acts as a barrel protein on the outer mitochondrial membrane and interacts with other free proteins to assist in the transport of small molecules.We have focused on Porin’s interactions with itself and with the lipid transport complex Ups1/2-Mdm35 in the mitochondrial membrane mesenchyme.Results from yeast two-hybrid and Bi FC experiments show that Porin can interact with itself and with Ups1/2 to assist in the smooth entry of substances from outside the mitochondrial membrane into the mitochondria.In F.graminearum,Fg Porin and Fg Ups1/2 together affect lipid transport on the strain’s mitochondria and,in addition,may play a role in other metabolic pathways.Knockouts of Fg Ups1/2 and Fg Mdm35 were attempted,but only the Δups2 strain was obtained,and comparative tests yielded that Fg Ups2 had no effect on the growth and pathogenicity of F.graminearum.the inability of Fg Ups1 and Fg Mdm35 to obtain mutants by protoplast transformation suggests that deletion of Fg Ups1 and Fg Mdm35 may be lethal.In summary,Fgporin plays an important role in the growth and development,asexual reproduction,sexual reproduction and pathogenicity of F.graminearum,affecting mitochondrial integrity and cellular autophagy.