“…In eukaryotes, there are two ways to activate the TORC1: (i) in cells, the amino acids and other nutrients stimulate their activity through Rag/Gtr GTPase, triggering the signal of Rag/Gtr dependent TORC1 activating amino acid uptake [ 67 ]; (ii) in yeast cells, TORC1 reacts to nitrogen sources through a potential mechanism and can sense and absorb several different nitrogen sources. The quality of nitrogen sources is defined by their ability to promote cell growth and glutamine accumulation, which is directly related to the ability to activate the TORC1 determined by Sch9 phosphorylation [ 57 , 58 ]. The preferred nitrogen source stimulates rapid and sustained Sch9 phosphorylation and glutamine accumulation.…”
Section: Discussionmentioning
confidence: 99%
“…The regulation of the chromatin state is an effective strategy to quickly and reversibly control cell growth in response to the fluctuational environmental conditions; the chromatin state globally stimulates protein expression by activating ribosome biogenesis and protein translation through the AGC family kinase Sch9 [ 57 ]. This Sch9 kinase is similar to the S6 kinase (S6K1/2) in mammals and is directly phosphorylated by the TORC1 on the vacuolar membrane [ 58 ]. On the contrary, TORC1 inhibits the degradation of large proteins through the phosphorylation of Atg13 to prevent its association with the Atg1, thereby inhibiting the induction of macrophages [ 59 ].…”
Leptographium qinlingensis is a fungal symbiont of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandii) that must overcome the terpenoid oleoresin defenses of host trees to invade and colonize. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include the decomposing and use of these compounds as a nitrogen source. Target of Rapamycin (TOR) is an evolutionarily conserved protein kinase that plays a central role in both plants and animals through integration of nutrients, energies, hormones, growth factors and environmental inputs to control proliferation, growth and metabolism in diverse multicellular organisms. In this study, in order to explore the relationship between TOR gene and carbon sources, nitrogen sources, host nutrients and host volatiles (monoterpenoids) in L. qinlingensis, we set up eight carbon source treatments, ten nitrogen source treatments, two host nutrients and six monoterpenoids (5%, 10% and 20%) treatments, and prepared different media conditions. By measuring the biomass and growth rate of mycelium, the results revealed that, on the whole, the response of L. qinlingensis to nitrogen sources was better than carbon sources, and the fungus grew well in maltose (carbon source), (NH4)2C2O4 (inorganic nitrogen source), asparagine (organic nitrogen source) and P. armandii (host nutrient) versus other treatments. Then, by analyzing the relationship between TOR expression and different nutrients, the data showed that: (i) TOR expression exhibited negative regulation in response to carbon sources and host nutrition. (ii) The treatments of nitrogen sources and terpenoids had positively regulatory effects on TOR gene; moreover, the fungus was most sensitive to β-pinene and 3-carene. In conclusion, our findings reveal that TOR in L. qinlingensis plays a key role in the utilization of host volatiles as nutrient intake, overcoming the physical and chemical host resistances and successful colonization.
“…In eukaryotes, there are two ways to activate the TORC1: (i) in cells, the amino acids and other nutrients stimulate their activity through Rag/Gtr GTPase, triggering the signal of Rag/Gtr dependent TORC1 activating amino acid uptake [ 67 ]; (ii) in yeast cells, TORC1 reacts to nitrogen sources through a potential mechanism and can sense and absorb several different nitrogen sources. The quality of nitrogen sources is defined by their ability to promote cell growth and glutamine accumulation, which is directly related to the ability to activate the TORC1 determined by Sch9 phosphorylation [ 57 , 58 ]. The preferred nitrogen source stimulates rapid and sustained Sch9 phosphorylation and glutamine accumulation.…”
Section: Discussionmentioning
confidence: 99%
“…The regulation of the chromatin state is an effective strategy to quickly and reversibly control cell growth in response to the fluctuational environmental conditions; the chromatin state globally stimulates protein expression by activating ribosome biogenesis and protein translation through the AGC family kinase Sch9 [ 57 ]. This Sch9 kinase is similar to the S6 kinase (S6K1/2) in mammals and is directly phosphorylated by the TORC1 on the vacuolar membrane [ 58 ]. On the contrary, TORC1 inhibits the degradation of large proteins through the phosphorylation of Atg13 to prevent its association with the Atg1, thereby inhibiting the induction of macrophages [ 59 ].…”
Leptographium qinlingensis is a fungal symbiont of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandii) that must overcome the terpenoid oleoresin defenses of host trees to invade and colonize. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include the decomposing and use of these compounds as a nitrogen source. Target of Rapamycin (TOR) is an evolutionarily conserved protein kinase that plays a central role in both plants and animals through integration of nutrients, energies, hormones, growth factors and environmental inputs to control proliferation, growth and metabolism in diverse multicellular organisms. In this study, in order to explore the relationship between TOR gene and carbon sources, nitrogen sources, host nutrients and host volatiles (monoterpenoids) in L. qinlingensis, we set up eight carbon source treatments, ten nitrogen source treatments, two host nutrients and six monoterpenoids (5%, 10% and 20%) treatments, and prepared different media conditions. By measuring the biomass and growth rate of mycelium, the results revealed that, on the whole, the response of L. qinlingensis to nitrogen sources was better than carbon sources, and the fungus grew well in maltose (carbon source), (NH4)2C2O4 (inorganic nitrogen source), asparagine (organic nitrogen source) and P. armandii (host nutrient) versus other treatments. Then, by analyzing the relationship between TOR expression and different nutrients, the data showed that: (i) TOR expression exhibited negative regulation in response to carbon sources and host nutrition. (ii) The treatments of nitrogen sources and terpenoids had positively regulatory effects on TOR gene; moreover, the fungus was most sensitive to β-pinene and 3-carene. In conclusion, our findings reveal that TOR in L. qinlingensis plays a key role in the utilization of host volatiles as nutrient intake, overcoming the physical and chemical host resistances and successful colonization.
“…After verification of the genome editing event(s) by PCR and/or Sanger sequencing, the Cas9+sgRNA(s) multi-gene plasmid is removed from yeast cells. In our lab, we have successfully used this procedure to generate several mutations at the SCH9 locus, encompassing full gene knockout, deletion of specific gene regions, and domain replacement ( Novarina et al., 2021 ), as well as to perform gene knockouts and to introduce point mutations in several yeast genes ( Guerra et al., 2021 ). Figure 1 Schematic of the sgRNA(s) and Cas9 cloning through Golden Gate assembly The sgRNA and the Cas9 gene are cloned in a yeast expression vector through three consecutive Golden Gate assembly reactions.…”
Section: Before You Beginmentioning
confidence: 99%
“…This step-by-step protocol guides the reader from sgRNA design to verification of the desired genome editing event and provides preassembled plasmids for cloning the sgRNA(s), making this technology easily accessible to any yeast research group. For complete details on the use and execution of this protocol, please refer to Novarina et al. (2021) .…”
mentioning
confidence: 99%
“…For complete details on the use and execution of this protocol, please refer to Novarina et al. (2021) .…”
Overexpression of Saccharomyces cerevisiae protein phosphatase Ppz1 strongly impairs cell growth. Ppz1 is negatively regulated by its subunit Hal3, and Hal3 overexpression fully counteracts the toxic effects derived from high levels of the phosphatase. We show that Ppz1 localizes at the plasma membrane, and that co‐expression of Hal3 recruits Ppz1 to internal membranes (mostly vacuolar). This effect is not observed in a catalytically impaired mutant of Ppz1. Disruption of intracellular trafficking by deletion of the ESCRT‐0 component VPS27 abolishes both Hal3‐mediated relocalization of Ppz1 and normalization of cell growth, without affecting Ppz1 protein levels. We propose that Hal3 counteracts the toxic effects caused by excess of Ppz1 not only by inhibiting its enzymatic activity but also by recruiting the phosphatase to internal structures.
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