Functional Characterization Of Key Genes Involved In The TOR Signaling Pathway In Fusarium Graminearum

Target of Rapamycin signaling pathway （TOR signaling pathway） which is prevalent in eukaryotes, coordinates cell growth in response to nutrient availability and stresses. As yet, the study of this cascade has been mainly focused on yeasts, Arabidopsis and mammals, little is known about this pathway in filamentous fungi. Thus, we are interested to investigate the role of this pathway in Fusarium graminearum with the purpose of getting more insights into the mechanism of TOR signaling in filamentous fungi, which may lay scientific basis for gene quelling of key genes involved in TOR signaling and development of novel drug to the end of control of Fusarium head blight.Such techniques as gene disruption and complementation, yeast two hybrid （Y2H）, Co-immunoprecipitation （Co-IP）, affinity capture were adopted to characterize eight key genes involved in TOR signaling and analyze their interactions. Results of our study showed that:1. Rapamycin （0.25μg/ml） is more effective against F. graminearum on plate assays, as compared with other two fungicides cabendazim and tebuconazole at the same concentration. Twisted hyphae with more septa were observed under microscope when treated with rapamycin. What’s more, these hyphae also accumulated more lipid bodies. Besides, conidiation was also quantitatively affected when mung bean broth was ameded with serial concentrations of rapamycin （0ng/ml,0.025ng/ml,2.5ng/ml,250ng/ml）.2. We found eight key genes of TOR signaling pathway in F. graminearum, which were named FgFKBP12, FgTOR, FgTAP42, FgPP2A, FgSIT4, FgPPGl, FgTIP41, FgAREA, respectively. Of the eight genes studied, three were unable to obtain knockout mutants, indicating their essential roles in growth, but deletion of the left five genes,△FgFKBP12,△FgSIT4,△FgPPG1,△FgTIP41,△FgAREA shows detectable phenotypic changes.3. F. graminearum harbors three FKBPs homologs which were named FgFKBP12, FgFKBP20, FgFKBP54. Deletion of FgFKBP12showed no visible difference with the wild-type in growth and pathogenicity, but conferred resistance to rapamycin and FK506while deletion of FgFKBP20or FgFKBP54didn’t confer resistance to rapamycin and FK506. Deletion of FgTOR was lethal as revealed by failure to obtain disruptants from four independent experiments. However, through UV irradiation, we were able to get one mutant which showed resistance to rapamycin. Sequencing of FgFKBP12and FgTOR confirmed an point mutation was occurred （C5597T）, resulting in alteration of Serine at1866to Leucine in the Fkbp12-rapamycin binding domain （FRB domain） of FgTor. To justify that this mutation was responsible for rapamycin resistance, yeast two hybrid was conducted with FgFKBP12and FgFRB. In the presence of rapamycin, FgFRB was able to interact with FgFkbpl2, however once mutated （FgFRBs18766L）,FgFRBs18766L was unable to interact with FgFkbp12even in the presence of rapamycin.4. Deletion of FgTAP42was lethal, indicating its essential role in cell growth. Complementation of yeast temperature-sensitive mutant BY4741tap42-11with FgTAP42could partially restore its severe growth defect at37℃, suggesting that the functions of Tap42orthologues may be conserved in the budding yeast and filamentous fungi. Subcellular localization of FgTap42in the budding yeast and that in F. graminearum showed FgTap42was mainly distributed in cytoplasm in S. cerevisiae tap42-11strain. However, in F. graminearum, FgTap42was localized to the cytoplasm in mycelia and near the nucleus in conidia. Y2H assays showed that FgTap42interacts with FgPp2A, FgSit4, and FgPpgl, but not with FgTip41. The interaction of FgTap42with FgSit4and FgPp2A was further confirmed by affinity capture and Co-IP assays. Through affinity capture, it was also found that FgTap42interacted with FgKogl, one component of TOR complex which is composed of FgTor, FgKogl and FgLst8. In addition, FgPpgl also interacted with FgTip41in our Y2H and Co-IP assays.5. Disruption of FgPP2A was lethal while deletion of FgSIT4（AFgSIT4） or FgPPGl （△FgPPG1） led to severe growth defect in mutants, fewer septa in conidia and accumulated fewer lipid bodies when challenged by rapamycin. Both mutants were seriously impaired in pathogenicy and abolished in their ability to undergo sexual development on carrot media. Furthermore, AFgSIT4and AFgPPG1showed great sensitivity to cell-wall stresses like congo red and calcofluor white but not to other stresses. Through affinity capture, we were able to identify a dual-specificity phosphatase FgMsg5（FGSG₀6977） which was a negative regulator of cell wall integrity pathway. The interactions were further confirmed by yeast two hybrid. Western Blotting showed that the phosphorylation level of FgSlt2in△FgSIT4and△FgPPG1was lower than that of the wild-type while the phosphorylation level of FgSlt2in△FgMSG5was higher than that of the wild-type,thus it’s reasonable that FgSit4and FgPpg1positively regulate cell wall integrity signaling pathway via negatively regulate FgMsg5, a negative regulator of cell wall signaling pathway. Although AFgSIT4and AFgPPGl shared many phenotypic changes, they were different in conidiation and DON production:△FgSIT4produced enough amouts of conidia and DONcomparable to the wild-type while△FgPPG1showed significantly decreased conidia and DON production.6. Replacement of FgTIP41（△FgTIP41） resulted in retarded vegetative growth, decreased pathogenicity and DON production although it had no obvious effect on conidiation and perthecia formation. Deletion of FgAREA （△FgAREA） caused seriously-impaired growth on minimal medium or minimal medium containing nitrate as the sole nitrogen source, and showed severely reduced virulence on wheat head and DON production, however AFgAREA was not impaired in perithecia development and conidiation in Carboxy Methyl Cellulose broth （CMC）. Thus, FgTip41and FgAreA are concluded to contribute to conidiation and virulence in F. graminearum.7. A model was proposed for TOR signaling pathway in F. graminearum: Rapamycin forms a complex with FgFkbp12, and this complex can bind to and inhibit FgTor, the key component of TOR complex. Meanwhile, FgTap42, a downstream effector of FgTor complex, exhibits its function by interacting with three phosphatases FgPp2A, FgSit4, FgPpg1. FgSit4and FgPpgl are involved in the regulation of cell wall integrity via a negative regulator FgMsg5. In addition, FgPpgl also regulates DON biosynthesis and virulence via its downstream transcription factors FgAreA, FGSG₀9019and FGSG₀9709. The function of FgPp2A remains to be determined. The study of TOR pathway in F. graminearum provides novel insights for drug development.