Extra-large G proteins regulate disease resistance by directly coupling to immune receptors in Nicotiana benthamiana

Abstract

Heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are key regulators of eukaryotic intracellular signaling. Extra-large G (XLG) proteins are a subfamily of plant-specific Gα proteins interacting with plasma membrane-localized receptors to regulate multiple biological processes. The Nicotiana benthamiana genome encodes seven XLG proteins, NbXLG1–7, whose functions in disease resistance and underlying mechanisms are unknown. In this study, we silenced all the seven genes and found that disease susceptibility was enhanced when both NbXLG3 and NbXLG5 or NbXLG4 was silenced. Then, we generated N. benthamiana xlg3xlg5 double- and xlg4 single-mutant lines using the CRISPR-Cas9 approach. All the mutants showed reduced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, the fungal pathogen Sclerotinia sclerotiorum, and a series of oomycete pathogens, including Phytophthora capsici, Phytophthora infestans, and Phytophthora parasitica. We further demonstrated that NbXLG3/4/5 positively regulated microbial pattern-induced reactive oxygen species burst and defense gene expression by directly coupling to the tested plant immune receptors. In addition, we examined the role of NbXLG3/4/5 in abiotic stress tolerance and observed that NbXLG3 and NbXLG5 negatively regulated plant resistance to high-salt, mannitol, and PEG. Our study demonstrates the possible role of NbXLG3/4/5 in response to biotic and abiotic stresses and provides insights for the improvement of plant resistance to environmental changes.