Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the Gβγ dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Gα or Gβ subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gβ-deficient mutants while Gα-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gβ-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gβ-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Gα-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gβγ functional subunit but not by Gα. We hypothesize that Gβγ acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Gα and Gβ subunit genes and two Gγ subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gγ genes revealed specialized roles for each Gγ subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gβγ1 and Gβγ2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gγ subunit in this root response differs, with Gβγ1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gβγ2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gβγ signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gγ subunits form functional Gβγ dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein–mediated signaling in plants.

Summary: Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1‐2 on pathogenesis‐related gene expression, as well as the genetic interaction between agb1‐2 and a number of mutants of established defense pathways. Gβ‐mediated signaling suppresses the induction of salicylic acid (SA)‐, jasmonic acid (JA)‐, ethylene (ET)‐ and abscisic acid (ABA)‐dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET‐dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ‐ and SA‐deficient mutants were more susceptible than wild‐type plants, whereas JA‐ and ET‐insensitive and ABA‐deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ‐mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1‐2 mutants was compensated by coi1‐21 and jin1‐9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling.

In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.