Magnaporthe oryzae Transcription Factor MoBZIP3 Regulates Appressorium Turgor Pressure Formation during Pathogenesis

The devastating fungus Magnaporthe oryzae (M. oryzae) forms a specialized infection structure known as appressorium, which generates enormous turgor, to penetrate the plant cells. However, how M. oryzae regulates the appressorium turgor formation, is not well understood. In this study, we identified MoBZIP3, a bZIP transcription factor that functioned in pathogenesis in M. oryzae. We found that the pathogenicity of the MoBZIP3 knockout strain (Δmobzip3) was significantly reduced, and the defect was restored after re-expression of MoBZIP3, indicating that MoBZIP3 is required for M. oryzae virulence. Further analysis showed that MoBZIP3 functions in utilization of glycogen and lipid droplets for generation of glycerol in appressorium. MoBZIP3 localized in the nucleus and could bind directly to the promoters of the glycerol synthesis-related genes, MoPTH2, MoTGL1 and MoPEX6, and regulate their expression which is critical for glycerol synthesis in the appressorium turgor pressure generation. Furthermore, the critical turgor sensor gene MoSln1 was also down regulated and its subcellular localization was aberrant in Δmobzip3, which leads to a disordered actin assembly in the Δmobzip3 appressorium. Taken together, these results revealed new regulatory functions of the bZIP transcription factor MoBZIP3, in regulating M. oryzae appressorium turgor formation and infection.

Enhancement of the hydrophilic feruloyl glycerol synthesis using A-35 as a catalyst and its function characteristics

Feruloyl glycerol (FG) is the hydrophilic ester of ferulic acid (FA), which has a high solubility in water and a strong ability to resist ultraviolet (UV) radiation. In this work,...

MAL62 overexpression enhances uridine diphosphoglucose-dependent trehalose synthesis and glycerol metabolism for cryoprotection of baker’s yeast in lean dough

Background In Saccharomyces cerevisiae, alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. In addition, the overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear. Results RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose. Moreover, in strains lacking trehalose synthesis, the cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in the intracellular glycerol concentration. This result was consistent with the elevated transcription of glycerol synthesis pathway members. Conclusions The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol also plays an important role. These findings shed new light on the mechanism of yeast response to freezing in lean bread dough and can help to improve industrial yeast strains.

MAL62 overexpression enhances uridine diphosphoglucose-dependent trehalose synthesis and glycerol metabolism for cryoprotection of baker’s yeast in lean dough

Background: In Saccharomyces cerevisiae, alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. In addition, the overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear.Results: RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose.Moreover, in strains lacking trehalose synthesis, the cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in the intracellular glycerol concentration. This result was consistent with the elevated transcription of glycerol synthesis pathway members.Conclusions: The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol also plays an important role. These findings shed new light on the mechanism of yeast response to freezing in lean bread dough and can help to improve industrial yeast strains.

MAL62 overexpression enhances uridine diphosphoglucose-dependent trehalose synthesis and glycerol metabolism for cryoprotection of baker’s yeast in lean dough

Background: In Saccharomyces cerevisiae, the alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. Overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear.Results: RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased transcription of uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose. Moreover, in strains lacking trehalose synthesis, cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in intracellular glycerol concentration which was consistent with the elevated transcription of glycerol synthesis pathways. Conclusions: The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol plays a secondary role. These findings shed new light to the mechanism of yeast response to freezing in lean bread dough and can help the improvement of industrial yeast strains.

Long-Term Adaption to High Osmotic Stress as a Tool for Improving Enological Characteristics in Industrial Wine Yeast

Industrial wine yeasts owe their adaptability in constantly changing environments to a long evolutionary history that combines naturally occurring evolutionary events with human-enforced domestication. Among the many stressors associated with winemaking processes that have potentially detrimental impacts on yeast viability, growth, and fermentation performance are hyperosmolarity, high glucose concentrations at the beginning of fermentation, followed by the depletion of nutrients at the end of this process. Therefore, in this study, we subjected three widely used industrial wine yeasts to adaptive laboratory evolution under potassium chloride (KCl)-induced osmotic stress. At the end of the evolutionary experiment, we evaluated the tolerance to high osmotic stress of the evolved strains. All of the analyzed strains improved their fitness under high osmotic stress without worsening their economic characteristics, such as growth rate and viability. The evolved derivatives of two strains also gained the ability to accumulate glycogen, a readily mobilized storage form of glucose conferring enhanced viability and vitality of cells during prolonged nutrient deprivation. Moreover, laboratory-scale fermentation in grape juice showed that some of the KCl-evolved strains significantly enhanced glycerol synthesis and production of resveratrol-enriched wines, which in turn greatly improved the wine sensory profile. Altogether, these findings showed that long-term adaptations to osmotic stress can be an attractive approach to develop industrial yeasts.

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Background: Dunaliella salina can produce glycerol under salt stress, and this production can quickly adapt to changes in external salt concentration. Notably, glycerol is an ideal energy source. In recent years, it has been reported that the mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is highly important to study the relationship between the MAPK cascade pathway and salt stress in D. salina and modify it to increase the prodution of glycerol. Results: In our study, we identified and analysed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1 and DsMEK1-X2 were both localized in the cytoplasm. qRT-PCR assays showed that DsMEK1-X2 was induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol production compared to the control and DsMEK1-X1-oe under salt stress. Under salt stress, the expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control. This finding indicated that DsMEK1-X2 was involved in the regulation of DsGPDH expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increased the proline content and reduced the MDA content under salt stress, and DsMEK1-X1 was able to regulate oxidative stress; thus, we hypothesized that DsMEK1-X1 could reduce oxidative damage under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 could interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1; however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines did not exhibit increased gene expression, except for DsMAPKKK9. Conclusion: Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can respond to salt stress by two different pathways. The DsMEK1-X1 response to salt stress reduces oxidative damage; however, the DsMAPKKK1/2/3/9/10/17- DsMEK1-X2-DsMAPK1 cascade is involved in the regulation of DsGPDH expression and thus glycerol synthesis under salt stress.

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) is involved different way in salt stress regulation in Dunaliella salina

Background：Dunaliella salina can produce a large amount of glycerol under salt stress, which can quickly adapt to the change of external salt concentration, and glycerol is one of the ideal energy sources. In recent years, it has been reported that Mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is urgent to study the relationship between MAPK cascade pathway and salt stress in D. salina, and help it to increase the content of glycerol. Results： In our study, we identified and analyzed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1, DsMEK1-X2 both localized in the cytoplasm. The qRT-PCR assays showed that DsMEK1-X2 induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol compared to the control and DsMEK1-X1-oe under salt stress. The expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control under salt stress. It means that DsMEK1-X2 is involved in the regulation of DsGPDHs expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increasing the proline content and reducing the MDA content under salt stress, and DsMEK1-X1 can regulate oxidative stress, thus we speculate that DsMEK1-X1 can reduce the damage of oxidative under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 can interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1, however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines do not increased genes expression, except for DsMAPKKK9. Conclusion： Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can response to salt stress in two different pathways, DsMEK1-X1 response to salt stress by reducing oxidative damage, however, DsMAPKKK1/2/3/9/10/17- DsMEK1-X2-DsMAPK1 cascade is involved in the regulated of DsGPDH expression and thus glycerol synthesis under salt stress.