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The PKA pathway is a signaling pathway involved in virulence in Candida albicans. This mechanism can be activated via addition of glucose and activation involves at least two proteins, namely Cdc25 and Ras1. Both proteins are involved in specific virulence traits. However, it is not clear if Cdc25 and Ras1 also affect virulence independently of PKA. C. albicans holds a second, atypical, Ras protein, Ras2, but its function in PKA activation is still unclear. We investigated the role of Cdc25, Ras1, and Ras2 for different in vitro and ex vivo virulence characteristics. We show that deletion of CDC25 and RAS1 result in less toxicity towards oral epithelial cells, while deletion of RAS2 has no effect. However, toxicity towards cervical cells increases in both the ras2 and the cdc25 mutants while it decreases in a ras1 mutant compared to the WT. Toxicity assays using mutants of the transcription factors downstream of the PKA pathway (Efg1) or the MAPK pathway (Cph1) show that the ras1 mutant shows similar phenotypes as the efg1 mutant, whereas the ras2 mutant shows similar phenotypes as the cph1 mutant. These data show niche-specific roles for different upstream components in regulating virulence through both signal transduction pathways. [ABSTRACT FROM AUTHOR]

Abstract: Aim: The goal of the study was to investigate the cellular tolerance mechanism in response to honokiol exposure. Methods and Results: The broth microdilution method was employed to test the sensitivity of different Saccharomyces cerevisiae strains to honokiol. Intracellular levels of reactive oxygen species (ROSs) were determined by DCFH‐DA staining. The phosphorylation of Hog1 was evaluated by Western blot analysis. The mRNA expressions of genes involved in the Ras‐cyclic AMP (cAMP) pathway were analysed by real‐time reverse transcription polymerase chain reaction. We found that the sod1▵ mutant was hypersensitive to honokiol and produced more ROS compared with wild‐type and sod2▵ cells. Hog1 was phosphorylated in response to honokiol exposure and deletion of HOG1 increased the sensitivity to honokiol. The expressions of genes involved in the Ras‐cAMP pathway were down‐regulated after honokiol exposure; exogenous cAMP significantly reduced the phosphorylation of Hog1, although the level was higher than the control level. Conclusions: In addition to SOD1, the Ras‐cAMP cascade and Hog1 MAP kinase pathway is essential for protecting against honokiol‐induced oxidative stress. Significance and Impact of the Study: Our results provide insight into the understanding of the action mechanism of honokiol. [ABSTRACT FROM AUTHOR]

Previously, we reported that Rck1 up-regulates Ras2 and pseudohyphal growth of Saccharomyces cerevisiae . Here, we further investigate the involvement of Rck1 in the activation of pseudohyphal growth. Rck1 activated phosphorylation of the deubiquitinase Ubp3 through a direct protein interaction between Rck1 and Ubp3. The N-terminal Bre5 binding region of Ubp3 physically interacted with Rck1, and Ubp3 and Rck1 co-precipitated. Overexpression of UBP3 using a high-copy plasmid resulted in the upregulation of Ras2, and deletion of UBP3 blocked the upregulation of Ras2 by RCK1 overexpression. Treatment with the proteasome inhibitor MG132 resulted in accumulation of Ras2, indicating that Rck1 is involved in Ras2 degradation in a proteasome-dependent manner. Furthermore, deletion of UBP3 blocked the upregulation of FLO11 , a flocculin required for pseudohyphal and invasive growth induced by RCK1 overexpression in S. cerevisiae . Taken together, these results demonstrate that Rck1 promotes S. cerevisiae pseudohyphal growth via the activation of Ubp3 phosphorylation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Rck1 up-regulates phospho-Kss1 and phospho-Fus3. [•] Rck1 up-regulated Ras2 at the protein level. [•] Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Abstract: A high throughput screening assay was developed based on the yeast chronological aging model and applied in evaluating several factors that mediate lifespan, including inoculum size, cellular state in nutrient-rich medium, and calorie level. Using our assay, we confirmed the previously reported genetic mimics of calorie restriction, including deletion of TOR1, SCH9 or RAS2. In contrast, deletion of SIR2 had longevity effect but seemed to produce only small beneficial effect on the response to calorie restriction. Overall, this new high throughput screening assay may facilitate identification of calorie restriction mimetics with a rapid and simple protocol, uncomplicated data analysis, and high sensitivity. In addition, the assay also provides quantifiable data including lag-time, growth rate, doubling time, and survival percentage. [Copyright &y& Elsevier]

Reporter assay and Northern hybridization analysis revealed that the deletion of the RAS2 gene induced the expression of stress-responsive genes such as YAK1, CTT1, HSP12, and TSA2 in the laboratory yeast YNN27, but not in the sake yeast UT-1, suggesting that the Ras-cAMP-PKA signaling pathway does not play a very important part in the regulation of transcription of general stress-responsive genes in sake yeasts. However, these analyses showed that ethanol induces other stress-response element (STRE)-driven genes in the strain UT-1, with the exception of YAK1 which encodes a growth inhibitory protein, implying an ethanol-specific response. The good growth of the sake yeast in the presence of ethanol could be partially explained by YAK1 mRNA levels being unaffected by ethanol. Although the ras2 disruption of strain UT-1 did not potentiate ethanol tolerance, the disruptant could grow well in the presence of ethanol, and acquired ethanol tolerance, as is the case with the wild-type strain. These results suggest that specific stress responses of the sake yeast, which are different from those of the laboratory yeast, result in high ethanol tolerance and hence good growth in the presence of ethanol. [Copyright &y& Elsevier]

Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP-dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. R, Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing seventy based on the number of PKA-dependent traits affected [Cannon. J. F., Gitan. R. & Tatchell. K. (1990) J. Biol. Chem. 265, 11 897-11 904). In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total cataiytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild-type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1-6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit. [ABSTRACT FROM AUTHOR]

Simple Summary: Here, we studied two closely related medicinal and aromatic plants from Asia, called Russian sage or from their previously used Latin name–Perovskia. These plants contain various specialized metabolites called phenylpropanoids that contribute to their medicinal uses. In our experiments, several different specialized phytochemicals were traced down in the roots and leaves with the major metabolite called rosmarinic acid, known for health beneficial properties. In order to check if the composition of these plants is regulated by specific genes encoding proteins that assemble these phytochemicals, we analyzed their expression during the growth season (spring, summer and fall). Despite being the closest kin, the two species of Russian sage displayed different seasonal changes in the composition of bioactive metabolites and the activity of genes responsible for their production. The genes' activity was correlated with rosmarinic acid content in the roots but not in the green parts of the plants. Two genes pointed out were linked to the regulation of rosmarinic acid biosynthesis, called RAS (for Rosmarinic Acid-Synthase) and a newly reported version of an oxidizing enzyme called Cyp98A14. These discoveries broaden our understanding of relationships between the genes' activity and production of bioactive constituents in herbs such as the two studied species of Russian sages. Salvia abrotanoides Kar. and Salvia yangii B.T. Drew are medicinal and aromatic plants belonging to the subgenus Perovskia and used as herbal medicines in Asia. Derivatives of caffeic acid, mainly rosmarinic acid (RA), are the major phenolic compounds identified in these plants. Understanding the factors and molecular mechanisms regulating the accumulation of pharmacologically and ecologically relevant phenolic metabolites is essential for future biotechnological and medical applications. Up to date, no studies of phenylpropanoid biosynthetic pathway at the transcriptional level has been performed in the Perovskia subgenus. Using a combined qRT-PCR transcriptional activity analysis with LC-MS based metabolic profiling of roots and leaves at the beginning, in the middle and at the end of vegetation season, we have identified the following gene candidates with properties correlating to phenolic acid biosynthesis in S. abrotanoides and S. yangii: PAL, C4H, 4CL, TAT, HPPR, RAS1, RAS2 and Cyp98A14. A comparison of phenolic acid profiles with gene transcript levels revealed the transcriptional regulation of RA biosynthesis in the roots but not the leaves of the studied species. Additionally, RAS1 and Cyp98A14 were identified as rate-limiting steps regulating phenylpropanoid biosynthesis on a transcription level. In the future, this will facilitate the gene-based metabolic enhancement of phenolic compounds production in these promising medicinal herbs. [ABSTRACT FROM AUTHOR]

• The RAS2 gene was successfully knocked out and replaced in the genome of in Fusarium circinatum. • RAS2 is important for growth in Fusarium circinatum. • RAS2 is essential for pathogenicity in Fusarium circinatum. • RAS2 plays a role in conidial germination in Fusarium circinatum. In this study, we investigated to possible role of Ras2 in Fusarium circinatum - a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum. [ABSTRACT FROM AUTHOR]

The changing accessibility of nutrient resources induces the reprogramming of cellular metabolism in order to adapt the cell to the altered growth conditions. The nutrient-depending signaling depends on the kinases mTOR (mechanistic target of rapamycin), which is mainly activated by nitrogen-resources, and PKA (protein kinase A), which is mainly activated by glucose, as well as both of their associated factors. These systems promote protein synthesis and cell proliferation, while they inhibit degradation of cellular content by unselective bulk autophagy. Much less is known about their role in selective autophagy pathways, which have a more regulated cellular function. Especially, we were interested to analyse the central Ras2-module of the PKA-pathway in the context of peroxisome degradation. Yeast Ras2 is homologous to the mammalian Ras proteins, whose mutant forms are responsible for 33% of human cancers. In the present study, we were able to demonstrate a context-dependent role of Ras2 activity depending on the type of mTOR-inhibition and glucose-sensing situation. When mTOR was inhibited directly via the macrolide rapamycin, peroxisome degradation was still partially suppressed by Ras2, while inactivation of Ras2 resulted in an enhanced degradation of peroxisomes, suggesting a role of Ras2 in the inhibition of peroxisome degradation in glucose-grown cells. In contrast, the inhibition of mTOR by shifting cells from oleate-medium, which lacks glucose, to pexophagy-medium, which contains glucose and is limited in nitrogen, required Ras2-activity for efficient pexophagy, strongly suggesting that the role of Ras2 in glucose sensing-associated signaling is more important in this context than its co-function in mTOR-related autophagy-inhibition. [ABSTRACT FROM AUTHOR]