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Heterotrimeric G-proteins are composed of Gα, Gβ and Gγ subunits and regulate many fundamental processes in plants. Nonetheless plants have a considerably simpler repertoire of G-protein signaling components than metazoans. In animals, ligand binding to 7 transmembrane (7TM) cell surface receptors designated GPCRs leads to G protein activation, however, activation of the plant G protein complex is constitutive, therefore the exact role of plant 7TM proteins is unclear. MLOs are the best characterized 7TM plant proteins. While genetic ablation of either MLO2 or G proteins alters resistance to pathogens, it is unknown if G proteins directly couple signaling through MLO2. Here we exploited two well-documented phenotypes of Arabidopsis mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition, to assess the relationship of MLO2 proteins to the G protein complex. Although our data reveal modulation of antifungal defence responses by Gβ and Gγ subunits, our findings are overall inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants defective in the Gβ subunit show delayed accumulation of a subset of defence-associated genes following exposure to the microbe-associated molecular pattern (MAMP), flg22. Moreover Gβ mutants were found to be hypersusceptible to spray-inoculation with the bacterial pathogen, Pseudomonas syringae. In sum, our data do not support a function for MLO proteins as GPCRs, but unravel a role for Gβ and Gγ subunits as modulators of basal defence against biotrophic and hemibiotrophic phytopathogens.

Heterotrimeric G-proteins are composed of Gα, Gβ and Gγ subunits and regulate many fundamental processes in plants. Nonetheless plants have a considerably simpler repertoire of G-protein signaling components than metazoans. In animals, ligand binding to 7 transmembrane (7TM) cell surface receptors designated GPCRs leads to G protein activation, however, activation of the plant G protein complex is constitutive, therefore the exact role of plant 7TM proteins is unclear. MLOs are the best characterized 7TM plant proteins. While genetic ablation of either MLO2 or G proteins alters resistance to pathogens, it is unknown if G proteins directly couple signaling through MLO2. Here we exploited two well-documented phenotypes of Arabidopsis mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition, to assess the relationship of MLO2 proteins to the G protein complex. Although our data reveal modulation of antifungal defence responses by Gβ and Gγ subunits, our findings are overall inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants defective in the Gβ subunit show delayed accumulation of a subset of defence-associated genes following exposure to the microbe-associated molecular pattern (MAMP), flg22. Moreover Gβ mutants were found to be hypersusceptible to spray-inoculation with the bacterial pathogen, Pseudomonas syringae. In sum, our data do not support a function for MLO proteins as GPCRs, but unravel a role for Gβ and Gγ subunits as modulators of basal defence against biotrophic and hemibiotrophic phytopathogens.

Powdery mildew is a common fungal disease of monocotyledonous and dicotyledonous plant species. Successful pathogenesis by the biotrophic fungus depends on the presence of plant-specific MLO (MILDEW RESISTANCE LOCUS O) proteins, as mutations in particular MLO genes confer durable powdery mildew resistance in barley, tomato and Arabidopsis. In the absence of MLO, fungal spores fail to invade the host epidermal cell, resulting in an early termination of fungal pathogenesis. MLO proteins define a family of heptahelical plasma membrane-localized proteins, reminiscent of G-protein coupled receptors (GPCRs) in metazoans that activate heterotrimeric G-protein signaling. A genetic approach was chosen in this study to assess the role of MLO proteins as putative plant GPCRs and results from these experiments demonstrate that powdery mildew susceptibility conferred by MLO is independent of the heterotrimeric G-protein complex. However, data from this analysis suggest a function of the heterotrimeric G-protein in basal defense mechanisms against powdery mildew fungi as well as in the integration of MAMP (microbe-associated molecular patterns) perception into downstream immune responses. Metabolomic analysis performed in this study indicate that the adapted powdery mildew fungus, Golovinomyces orontii, is able to suppress the accumulation of the defense-relevant indolic glucosinolate, 4MI3G (4-methoxyindol-3-ylmethylglucosinolate) in Arabidopsis, thereby inhibiting the PEN2-dependent glucosinolate defense pathway. This inhibition requires functional MLO, suggesting that successful defense suppression either operates through the MLO protein or that it requires the formation of post-invasive fungal infection structures. Other data obtained here demonstrate that MLO proteins negatively regulate transcriptional activation of defense-related genes in response to powdery mildew challenge as well as upon MAMP treatment, implicating MLO functions in MAMP-triggered defense signaling.