Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants.

Plant defenses induced by salicylic acid (SA) are vital for resistance against biotrophic pathogens. In basal and receptor-triggered immunity, SA accumulation is promoted by Enhanced Disease Susceptibility1 with its co-regulator Phytoalexin Deficient4 (EDS1/PAD4). Current models position EDS1/PAD4 upstream of SA but their functional relationship remains unclear. In a genetic and transcriptomic analysis of Arabidopsis autoimmunity caused by constitutive or conditional EDS1/PAD4 overexpression, intrinsic EDS1/PAD4 signaling properties and their relation to SA were uncovered. A core EDS1/PAD4 pathway works in parallel with SA in basal and effector-triggered bacterial immunity. It protects against disabled SA-regulated gene expression and pathogen resistance, and is distinct from a known SA-compensatory route involving MAPK signaling. Results help to explain previously identified EDS1/PAD4 regulated SA-dependent and SA-independent gene expression sectors. Plants have evolved an alternative route for preserving SA-regulated defenses against pathogen or genetic perturbations. In a proposed signaling framework, EDS1 with PAD4, besides promoting SA biosynthesis, maintains important SA-related resistance programs, thereby increasing robustness of the innate immune system.

Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.

The symbiotic association between plants and arbuscular mycorrhizal fungi is almost ubiquitous within the plant kingdom, and the early stages of the association are controlled by plant-derived strigolactones acting as a signal to the fungus in the rhizosphere and lipochito-oligosaccharides acting as fungal signals to the plant. Hyphopodia form at the root surface, allowing the initial invasion, and this is analogous to appressoria, infection structures of pathogenic fungi and oomycetes. Here, we characterize RAM2, a gene of Medicago truncatula required for colonization of the root by mycorrhizal fungi, which is necessary for appropriate hyphopodia and arbuscule formation. RAM2 encodes a glycerol-3-phosphate acyl transferase (GPAT) and is involved in the production of cutin monomers. Plants defective in RAM2 are unable to be colonized by arbuscular mycorrhizal fungi but also show defects in colonization by an oomycete pathogen, with the absence of appressoria formation. RAM2 defines a direct signaling function, because exogenous addition of the C16 aliphatic fatty acids associated with cutin are sufficient to promote hyphopodia/appressoria formation. Thus, cutin monomers act as plant signals that promote colonization by arbuscular mycorrhizal fungi, and this signaling function has been recruited by pathogenic oomycetes to facilitate their own invasion.