The Different Expressions Of Ras1,Cdc42,Rac1 And Rho1 Under Different Yeast-hyphal Phases Of Trichosporon Asahii

Part I The different expressions of Rasl, Cdc42, Racl and Rhol between the yeast and hyphal phases of Trichosporon asahiiOBJECTIVEIn fungi, Ras1, Cdc42, Racl and Rhol play an important role in cell polarization and morphogenetic transition. Our study was designed to obtain yeast and hyphal phases of Trichosporon asahii (T.asahii) by comparative study. Then we aimed to measure the different expressions of Ras1, Cdc42, Racl and Rhol in both yeast and hyphal phases of T.asahii by real-time fluorescence quantitative PCR. Finally we sought to preliminary discuss the roles they play in the yeast-hyphal transition of T.asahii.METHODS1. Cornmal agar medium (CMA) was chosen to induce the yeast phase at 35℃. The morphology was observed and the hyphal formation rate was calculated at 1d,2d, 5d and 10d respectively. RPMI-1640 liquid medium was used to induce the hyphal phase at 37 ℃,150r/min. The morphology was observed and the hyphal formation rate was calculated at 4h,8h,12h and 24h respectively.2. The total RNA of yeast or hyphal phase was extracted, and the first chain cDNA was synthesized by reverse transcription.3. The different expressions of Rasl, Cdc42, Rac1 and Rho1 between the yeast and hyphal phases were detected by real-time fluorescence quantitative PCR.4. Statistics:all data were processed by SPSS13.0 statistical software, and the data were expressed as mean ± standard deviation. Data by real-time fluorescence quantitative PCR were 2-ΔΔCt levels calculated relative to the form with the control group set to 1. Two sample student’s t-test and analysis of variance tests were used for statistical analyses and P<0.05 was considered significantRESULTS1. The results of the induction of yeast and hyphal phases were as follows. The mean hyphal formation rate and standard deviation in CMA for 5d at 35℃ was (0.40±0.53)%, which would obtain enough yeast phase. The mean hyphal formation rate and standard deviation in RPMI-1640 medium for 24h at 37 ℃ was (99.33±0.57)%, which would obtain enough hyphal phase. T test was conducted and there was significance between yeast and hyphal phases (t=13.93, P<0.05).2. Dissociation curves showed that melting temperatures were homogeneous and the amplification specificity was obtained. No non-specific double stranded DNA products or primer dimers were observed. Therefore, SYBR Green RT PCR analysis was conducted. Amplification plots showed that the shape of S curves were complete and the curves were all smooth and stable, which was consistent with the quantitative detection criteria.3. The expressions of Cdc42、Rac1、Rho1 and Ras1 in yeast or hyphal phases were as follows. For Ras1, with the control group (yeast phase) set to 1, the expression in the hyphal phase was significantly higher (25.17-fold). Compared with the control group, there was statistically significant difference between yeast phase and hyphal phase (t=3.81, P<0.05). For Cdc42, with the control group (yeast phase) set to 1, the expression in the hyphal phase was significantly higher (14.68-fold). Compared with the control group, there was statistically significant difference between yeast phase and hyphal phase (t=3.63, P<0.05). For Racl, with the control group (yeast phase) set to 1, the expression in the hyphal phase was significantly higher (16.81-fold). Compared with the control group, there was statistically significant difference between yeast phase and hyphal phase (t=3.51, P<0.05). For Rhol, with the control group (yeast phase) set to 1, the expression in the hyphal phase was significantly higher (42.61-fold). Compared with the control group, there was statistically significant difference between yeast phase and hyphal phase (t =3.90, P<0.05).Part Ⅱ The different expressions of Cdc42 and Rho1 under different yeast-hyphal phases of Trichosporon asahiiOBJECTIVEThere are significantly different expressions of both Cdc42 and Rho1 in the yeast and hyphal phases of T. asahii. Firstly, our study was designed to obtain different yeast-hyphal phases by incubating T.asahii at different times respectively. Besides,4 strains of T.asahii isolated from different sources were introduced. Then we aimed to measure the different expressions of Cdc42 and Rhol of varied hyphal developments of T.asahii from different incubation times as well as 4 different strains by real-time fluorescence quantitative PCR. Finally we sought to further verify the roles they play in the yeast-hyphal transition of T.asahii.METHODS1. YPD liquid medium was chosen to induce the different hyphal developments of T.asahii type strain at 37℃,150r/min at 3h,6h,12h and 20h respectively. The morphology was observed and the hyphal formation rate was calculated. Besides,4 strains of T.asahii isolated from different sources were incubated for 24h by YPD liquid medium, at 37℃,150r/min. The morphology was observed and the hyphal formation rate was calculated respectively.2. The total RNA of varied hyphal developments was extracted, and the first chain cDNA was synthesized by reverse transcription.3. The different expressions of Cdc42 and Rho1 under different yeast-hyphal phases were detected by real-time fluorescence quantitative PCR.4. Statistics:all data were processed by SPSS 13.0 statistical software, and the data were expressed as mean ± standard deviation of the mean. Data by real-time fluorescence quantitative PCR were 2ΔΔCt levels calculated relative to the form with the control group set to 1. One way ANOVA was used for statistical analyses and P< 0.05 was considered significant.RESULTS1. The induction results under different incubation time showed that the mean hyphal formation rate and standard deviation for 3h was (9.00±3.61)%, for 6h was (36.00±5.29)%, for 12h was (77.67±6.81)% and for 20h was (44.00±5.29)%, obtaining different yeast-hyphal phases. One way ANOVA was conducted and significant difference was existed between every 2 groups (F=83.26, P<0.05). Multiple comparison methods showed that significant difference was not existed between 6h and 20h groups (P>0.05) while significant difference were existed between the rest paired groups (P<0.05).2. The induction results of 4 strains of T.asahii isolated from different sources illustrated that the mean hyphal formation rate and standard deviation for CBS8904 was (1.75±0.43)%, for BZP09001 was (57.20±7.52)%, for CBS2479 was (72.26±5.73)% and for CBS8520 was (97.00±4.36)%, obtaining different yeast-hyphal phases. One way ANOVA was conducted and significant diffference was existed between every 2 groups (F=235.64, P<0.05). Multiple comparison methods showed that significant difference was existed between every 2 paired groups (P<0.05).3. Dissociation curves showed that melting temperatures were homogeneous and the amplification specificity was obtained. No non-specific double stranded DNA products or primer dimers were observed. Therefore, SYBR Green RT PCR analysis was conducted. Amplification plots showed that the shape of S curves were complete and the curves were all smooth and stable, which was consistent with the quantitative detection criteria.4. The expressions of Cdc42 and Rho1 in different yeast-hyphal phases were as follows.For different expressions of Cdc42 under different incubation times, with the control group (3h) set to 1, the expression for 6h was 10.16-fold, the expression for 12h was 20.10-fold and the expression for 20h was 5.89-fold. One way ANOVA was conducted and significant difference was existed between every 2 groups (F=13.15, P<0.05). Multiple comparison methods showed that significant difference were existed between 3h and 12h groups (P<0.05) while significant difference was not existed between the rest paired groups (P>0.05).For different expressions of Cdc42 of 4 strains of T.asahii isolated from different sources, with the control group (BZP09001) set to 1, the expression for CBS8904 was 0.30-fold, the expression for CBS2479 was 1.73-fold and the expression for CBS8520 was 4.07-fold. One way ANOVA was conducted and significant difference was existed between every 2 groups (F=12.11, P<0.05). Multiple comparison methods showed that the expression of CBS8520 was significantly higher than any .other groups (P<0.05) while there were no significant differences between the rest paired groups (P>0.05).For different expressions of Rhol under different incubation times, with the control group (3h) set to 1, the expression for 6h was 2.79-fold, the expression for 12h was 5.10-fold and the expression for 20h was 2.10-fold. One way ANOVA was conducted and significant difference was existed between every 2 groups (F=39.98, P<0.05). Multiple comparison methods showed that significant difference was not existed between 6h and 20h groups (P>0.05) while significant difference was existed between the rest paired groups (P<0.05).For different expressions of Rhol of 4 strains of T.asahii isolated from different sources, with the control group (BZP09001) set to 1, the expression for CBS8904 was 0.40-fold, the expression for CBS2479 was 1.24-fold and the expression for CBS8520 was 2.75-fold. One way ANOVA was conducted and significant difference was existed between every 2 groups (F=30.16, P<0.05). Multiple comparison methods showed that the expression of CBS8520 was significantly higher than any other groups (P<0.05) and the expression of CBS2479 was significantly higher than the CBS8904 (P<0.05) while there were no significant differences between the rest paired groups (P>0.05).CONCLUSION1.Our research has shown that the yeast phase of T.asahii could be easily obtained through CMA for 5d at 35 ℃ while the hyphal phase of T.asahii could be quickly acquired by RPMI-1640 medium for 24h at 37℃.2. Different incubation conditions resulted in different hyphal formation rates of T.asahii, which confirms that both the medium composition and the incubation time have influenced the formation of hyphae. On the other hand, even under the same incubation condition, different sources of T.asahii resulted in different hyphal formation rates, suggesting that the source of strain also plays an important role in hyphae formation.3. Our results showed that Ras1, Cdc42, Rac1 and Rho1 expressed in both yeast and hyphal phases of T.asahii. However, in yeast phase, there was only a small amount of gene expression while in the hyphal phase the expression was significantly higher. The findings suggest that Ras1, Cdc42, Rac1 and Rho1 are not hypha-specific genes, however, they may play an important role in cell polarization and hyphal formation.4. Different incubation times resulted in different hyphal formation rates of T.asahii. With the increase of hyphal formation rate, gene expression also rose, which further shows that both Cdc42 and Rhol are regulation factors in yeast to hyphal transition of T.asahii type strain, and they may play an important role in yeast-hyphal transition of T.asahii.5. Different sources of T.asahii resulted in different hyphal formation rates of T.asahii. With the increase of hyphal formation rate, gene expression still rose. What’s more, the cells of the strain CBS8904 were almost yeast like conidia while the cells of the strain CBS8520 were almost hyphae. Besides, The expressions of Cdc42 and Rhol between the two groups were significantly different. Taken together, these results suggest that both Cdc42 and Rho1 contribute to the hyphal formation of T.asahii. Because Racl, Ras1, Cdc42 and Rho1 proteins belong to the same Ras superfamily proteins, we may speculate that all of Ras1, Cdc42, Rac1 and Rho1 may play an important role in the yeast-hyphal transition of T.asahii.