The Saccharomyces cerevisiae Ptc1 protein phosphatase attenuates G2-M cell cycle blockage caused by activation of the cell wall integrity pathway.

Lack of the yeast Ptc1 Ser/Thr protein phosphatase results in numerous phenotypic defects. A parallel search for high-copy number suppressors of three of these phenotypes (sensitivity to Calcofluor White, rapamycin and alkaline pH), allowed the isolation of 25 suppressor genes, which could be assigned to three main functional categories: maintenance of cell wall integrity (CWI), vacuolar function and protein sorting, and cell cycle regulation. The characterization of these genetic interactions strengthens the relevant role of Ptc1 in downregulating the Slt2-mediated CWI pathway. We show that under stress conditions activating the CWI pathway the ptc1 mutant displays hyperphosphorylated Cdc28 kinase and that these cells accumulate with duplicated DNA content, indicative of a G2-M arrest. Clb2-associated Cdc28 activity was also reduced in ptc1 cells. These alterations are attenuated by mutation of the MKK1 gene, encoding a MAP kinase kinase upstream Slt2. Therefore, our data show that Ptc1 is required for proper G2-M cell cycle transition after activation of the CWI pathway.

Author Correction: The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1.

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase-encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.