Regulatory Roles Of GGA Family Protein FgGga1 On Fusarium Graminearum Ascospore Discharge And Pathogenicity

Wheat scab（Fusarium head blight,FHB）is a fungal disease caused by Fusarium graminearum,which seriously endangers the yield and quality of wheat and causes huge economic losses.In recent years,FHB have increased year by year due to fungicide resistance and cultivation methods.Ascospores produced by sexual reproduction of F.graminearum are an important part of the FHB disease cycle,and its infestation is spread mainly by the wind and rain in the field.Ascospores infect wheat panicles through germination and growth colonization.In this process,the pathogen produces a variety of mycotoxins,which seriously threaten the life health of human and animal,among them,DON toxin is the most toxic and stable.Therefore,it is significant to elucidate the pathogenic mechanism of F.graminearum for developing effective disease control strategies.Vesicular trafficking is a conserved material transport process in eukaryotic cells,which affects the vegetative growth,DON toxin synthesis and pathogenicity in F.graminearum.GGA family proteins are adaptors of clathrin vesicles in eukaryotes and regulate trans-Golgi associated vesicular trafficking.Studies in Saccharomyces cerevisiae have shown that the GGA family proteins affect the vesicular trafficking pathway from endosomes to Golgi,but the function of this family proteins in F.graminearum is still unclear.Previous studies in our laboratory have found that Fg Gga1 affected physiological phenotypes of F.graminearum,such as vegetative growth,asexual reproduction and pathogenicity.Based on this,this study further explored the regulatory mechanisms of Fg Gga1and its functional domains on vesicular trafficking,ascospore discharge and pathogenicity.The main results are as follows:1.Fg Gga1 and its functional domains regulate vesicular trafficking pathwaysPrevious studies have shown that vesicular trafficking plays an important role in the pathogenicity of F.graminearum.In this study,we studied which organelles vesicular trafficking pathway is regulated by Fg Gga1.The results show that:（1）To test whether vesicular trafficking in mycelium was normal,we observed the localization of v-SNARE protein GFP-Fg Snc1.It was found that compared with the wild-type PH-1,Fg Snc1 in theΔFggga1 mutant failed to locate on the plasma membrane and diaphragm,but remained in the endosome,further research revealed that Fg Gga1 regulated Fg Snc1 trafficking through three functional domains:VHS,GAT^167-248and hinge.（2）We observed the location of GFP-Fg Snc1-PEM to detect whether the late secretory transport pathway is normal.It showed that Fg Snc1-PEM was retained in the trans-Golgi inΔFggga1 mutant;we also found that the loss of a single functional domain of Fg Gga1 does not affect the late secretion process.（3）The endocytosis transport pathway of bacteria was detected by FM4-64 staining,and found that Fg Gga1 regulated the transport pathway from endosome to vacuole through GAT^167-248 and hinge domain.The above results indicate that Fg Gga1 is involved in the vesicular trafficking process from endosome to trans-Golgi,trans-Golgi to plasma membrane and endosome to vacuole.2.Fg Gga1 and its functional domains affect the pathogenicity and DON toxin synthesis of F.graminearumCompared with wild-type PH-1,the deletion of Fg GGA1 and its VHS、GAT^167-248and hinge functional domain resulted in 97%,75%,97%and 79%reductions in pathogenicity respectively.DON toxin is an important pathogenic factor of F.graminearum.Therefore,the production of DON toxin and the expression of genes related to DON biosynthesis were further detected.The results showed that the DON toxin production ofΔFggga1 mutant was 92%lower than that of the wild type.The expression of TRI gene was significantly decreased.By changing the promoter of TRI1,Tri1 inΔFggga1 mutant was found to be able to localize in the toxin normally,indicating that Fg Gga1 is not involved in DON biosynthesis by regulating the localization of Tri1.The above results indicate that Fg Gga1 affects the pathogenicity of F.graminearum and DON biosynthesis by participating in TRI gene expression.3.Fg Gga1 affects the ascospore discharge of F.graminearum by regulating the transport of Fg Neo1F.graminearum ascospores discharged from the asci are the primary source of FHB infection in wheat under natural conditions.In this study,it was found that the deletion of Fg Gga1 or GAT^167-248 and hinge domains did not affect the production of perithecia and ascospores,but blocked the release of ascospores.The ascospore discharge of F.graminearum depends on the normal Ca²⁺concentration.Further study found that exogenous application of Ca²⁺could restore the ability of ascospore discharge fromΔFggga1 mutant.Through protein interaction technology,it was found that Fg Gga1 interacted with Ca²⁺ATPase Fg Neo1 and regulated the normal localization of Fg Neo1 in vacuoles through GAT^167-248 and hinge functional domains.Therefore,Fg Gga1-mediated transport from endosome to vacuoles affects the localization of Fg Neo1,and consequently affecting the release of ascospores of F.graminearum.In summary,GGA family protein Fg Gga1 affects the pathogenicity of F.graminearum by regulating vesicular trafficking between different organelles,but only affects ascospore discharge by regulating the transport from endosome to vacuoles.The above results provide a foundation for elucidating how vesicular trafficking between different organelles affects physiological phenotype of F.graminearum.