EDS1 in tomato is required for resistance mediated by TIR-class R genes and the receptor-like R gene Ve.

In tobacco and other Solanaceae species, the tobacco N gene confers resistance to tobacco mosaic virus (TMV), and leads to induction of standard defense and resistance responses. Here, we report the use of N-transgenic tomato to identify a fast-neutron mutant, sun1-1 (suppressor of N), that is defective in N-mediated resistance. Induction of salicylic acid (SA) and expression of pathogenesis-related (PR) genes, each signatures of systemic acquired resistance, are both dramatically suppressed in sun1-1 plants after TMV treatment compared to wild-type plants. Application of exogenous SA restores PR gene expression, indicating that SUN1 acts upstream of SA. Upon challenge with additional pathogens, we found that the sun1-1 mutation impairs resistance mediated by certain resistance (R) genes, (Bs4, I, and Ve), but not others (Mi-1). In addition, sun1-1 plants exhibit enhanced susceptibility to TMV, as well as to virulent pathogens. sun1-1 has been identified as an EDS1 homolog present on chromosome 6 of tomato. The discovery of enhanced susceptibility in the sun1-1 (Le_eds1-1) mutant plant, which contrasts to reports in Nicotiana benthamiana using virus-induced gene silencing, provides evidence that the intersection of R gene-mediated pathways with general resistance pathways is conserved in a Solanaceous species. In tomato, EDS1 is important for mediating resistance to a broad range of pathogens (viral, bacterial, and fungal pathogens), yet shows specificity in the class of R genes that it affects (TIR-NBS-LRR as opposed to CC-NBS-LRR). In addition, a requirement for EDS1 for Ve-mediated resistance in tomato exposes that the receptor-like R gene class may also require EDS1.

Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway.

Efficient internalization of proteins from the cell surface is essential for regulating cell growth and differentiation. In a screen for yeast mutants defective in ligand-stimulated internalization of the alpha-factor receptor, we identified a mutant allele of TOR2, tor2G2128R. Tor proteins are known to function in translation initiation and nutrient sensing and are required for cell cycle progression through G1. Yeast Tor2 has an additional role in regulating the integrity of the cell wall by activating the Rho1 guanine nucleotide exchange factor Rom2. The endocytic defect in tor2G2128R cells is due to disruption of this Tor2 unique function. Other proteins important for cell integrity, Rom2 and the cell integrity sensor Wsc1, are also required for efficient endocytosis. A rho1 mutant specifically defective in activation of the glucan synthase Fks1/2 does not internalize alpha-factor efficiently, and fks1Delta cells exhibit a similar phenotype. Removal of the cell wall does not inhibit internalization, suggesting that the function of Rho1 and Fks1 in endocytosis is not through cell wall synthesis or structural integrity. These findings reveal a novel function for the Tor2-Rho1 pathway in controlling endocytosis in yeast, a function that is mediated in part through the plasma membrane protein Fks1.

The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.