Characterization Of The Function Of The Transcription Regulator YlSfl1 In Dimorphic Transition In The Yeast Yarrowia Lipolytica

Some fungal species can switch between oval-shaped cell morphology and hypha or pseudohypha under induction by environmental factors.This switch is called dimorphic transition.Dimorphic transition is a suitable model for the study of cell differentiation.In addition,dimorphic transition is also implicated in the pathogenesis in some pathogenic fungi.Therefore,the study on the regulatory mechanisms that control dimorphic transition is scientifically significant and valuable in medicare.Yarrowia lipolytica is a dimorphic yeast species.It forms hyphae or pseudohyphae under induction by certain environmental factors.At present,not much is known on the regulation of dimorphic transition in this yeast species than in yeasts Saccharomyces cerevisiae and Candida albians.Sfl1 is an important transcription regulator in yeast.It plays critical roles in the regulation of dimorphic transition in S.cerevisiae and C.albicans.In this thesis,we investigated the role of Y.lipolytica YISfl1 in filamentation,the signal transduction pathway to which YISfll belongs and the mechanism by which YISfl1 regulates filamentation.Firstly,we explored the role of YISfl1 in filamentation by the characterization of Ylsfl1?mutant,YlSFL1 overexpression and the examination of YlSfl1's subcellular localization.We found that Ylsfl1? mutant displays defect in filamentous growth.This mutant failed to form filaments when grown in culture media containing glycerol or glucose as the sole carbon source.When grown in media containing sodium acetate as carbon source,the mutant showed a delay in filamentous growth.On the other hand,YlSFL1 overexpression enhanced filamentation.These results indicate that YISfl1 promotes filamentous growth and is a positive regulator of dimorphic transition.This is very different from the roles of S.cerevisiae Sfll and C.albicans CaSfll but similar to the role of C.albicans CaSfl2.The Ylsfl1? mutant was also defective in invasive growth,suggesting that YlSfl1 may also regulate invasive growth.By comparing growth on solid culture media containing various carbon sources,wefound that Ylsfl1? mutant showed difference in the utilization of different carbon source.Ylsfl1? mutant displayed severe growth defect on oleic acid,suggesting that YlSfl1 may have a role in the expression of genes required for oleic acid metabolism.We observed that the YlSfl1-GFP fusion protein is concentrated in the nucleus.This finding supports the idea that YlSfl1 functions inside the nucleus as a transcription regulator.Next,we performed a preliminary investigation on the signaling pathway to which YISfl1 may belong.It is known that S.cerevisiae Sfll is controlled by Tpk2,a catalytic subunit of protein kinase A in the cAMP-PKA pathway.We deleted YITPK1,the gene coding for the catalytic subunit of protein kinase A in Y.lipolytica and found that Yltpk1? mutant did not show any defect in filamentation,which is different from S.cerevisiae tpk2? mutant.As shown earlier,YISfl1 overexpression can enhance filamentation in wild-type cells.We found that YlSfl1 overexpression in Yltpk1? mutant can still enhance filementation.We speculate that YISfll may not be controlled by the cAMP-PKA signaling pathway,which is different from S.cerevisiae Sfll.Ras controls filamentous growth via the MAPK signaling pathway and the cAMP-PKA signaling pathway in S.cerevisiae and C.albicans.We overexpressed YlSFL1 in Ylras2? mutant and overexpressed YlRAS2 in Ylsfl1? mutant.In both cases,we observed a partial enhancement in filamentous growth,suggesting that YISfll may not function downstream of Ras.Lastly,we randomly overexpressed genes in the filamentation-deficient YlsfllA mutant and screened for genes that when overexpressed can rescue the filamentation defect of this mutant.We randomly inserted the strong promoter hp4d into the genome of Y1sfl1? mutant and isolated six insertional mutants that regained the ability to undergo filamentous growth.The hp4d insertion sites in these mutants were determined by TAIL-PCR.We identified four mutants that may undergo hp4d-driven gene overexpression,one of which is YISOK2.YlSOK2's encoded protein product is a homolog of S.cerevisiae Sok2,a transcription regulator.We confirmed that YISOK2 overexpression can indeed partially rescue filamentous growth to Ylsfl1? mutant.In addition,YISOK2 overexpression can also enhance filamentation in wild-type cells.These findings indicate that YlSok2 is a positive regulator of dimorphic transition and it may function downstream of YlSfl1.We deleted YISOK2 in the wild-type strain but failed to identify any defects in filament formation the Ylsok2A mutant.This result suggests that although YlSok2 positively regulates filamentous growth,it is dispensable for this process.Our results indicate that the role of Y.lipolytica YISfll in dimorphic transition is clearly different from S cerevisiae Sfll and C.albicans CaSfll.The regulatory mechanism of Y1Sfl1 is also different from S.cerevisiae Sfl1.Additionally,we identified YlSok2,a transcription regulator that may act downstream of YlSfl1.Our findings provided new insight into the understanding of the regulation on dimorphic transition in Y.lipolytica.