Ergosterol-induced immune response in barley involves phosphorylation of phosphatidylinositol phosphate metabolic enzymes and activation of diterpene biosynthesis

Abstract

Introduction

Lipids are a diverse group of molecules that play crucial roles in plant nutrition, development, and plant–microbe interactions. As major constituents of the plasma and organelle membranes, they work in conjunction with the cell wall to establish the interface for environment interactions. Here, they act as structural elements, modulate physicochemical membrane properties, function as (stress) signaling molecules, and influence subcellular protein localization through lipid–protein interactions (Noack & Jaillais, 2020; Macabuhay et al., 2022; Zarreen et al., 2023).

Lipids are categorized into three main classes based on their chemical structures: sphingolipids, sterols, and (glycero)phospholipids (Moreau & Bayer, 2023). Among these, phosphoinositides stand out as crucial, low-abundance signaling molecules, which are derived from phosphatidylinositol (PI), a ubiquitous phospholipid containing myo-inositol in its head group. PI can be phosphorylated at various positions by phosphatidylinositol kinases (PIKs) to produce phosphatidylinositol phosphates (PIPs). In plants, phosphatidylinositol 4-phosphate (PI4P) is the most abundant phosphoinositide, although PI3P, PI5P, and diphosphorylated forms, such as PI(4,5)P₂ and PI(3,5)P₂ have also been detected (Munnik & Vermeer, 2010).

During abiotic and biotic stress, PIPs can be interconverted and hydrolyzed to produce the signaling lipid phosphatidic acid (PA). PIPs are also associated with disease resistance (Xing et al., 2019; Qin et al., 2020), cytoskeletal rearrangements (Sinha et al., 2024), endo- and exocytosis (Synek et al., 2021; Marković & Jaillais, 2022), and the formation of membrane nanodomains (Gronnier et al., 2017; Jaillais & Ott, 2020). Nanodomains, accommodating membrane-associated kinases, and receptor-like kinases can act as signaling hubs during plant–microbe interactions to enable perception of microbe- or damage-associated molecular patterns (MAMPs or DAMPs) and subsequent immune signaling (Couto & Zipfel, 2016; Jaillais & Ott, 2020).

Microbe-associated molecular pattern recognition initiates a signaling cascade often involving an increase in cytosolic calcium concentration ([Ca²⁺]_cyt), production of reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein (MAP) kinases (MAPKs). This cascade ultimately leads to altered gene expression and secretion of chemically diverse antimicrobial compounds, such as phytoalexins (Siebers et al., 2016; DeFalco & Zipfel, 2021). Altogether, this response is known as pattern-triggered immunity (PTI). While plant lipids are important signaling molecules, microbial lipids can also be detected by plants as MAMPs. Lipid MAMPs can be directly recognized by plant receptors, as in the case of the medium-chain-3-hydroxy fatty acids from Pseudomonas syringae lipopolysaccharides (Kutschera et al., 2019) or processed by secreted enzymes before recognition, such as ceramide D from Phytophora infestans (Kato et al., 2022).

The fungal sterol lipid ergosterol, a 5,7-diene oxysterol, has also been shown to be perceived as a MAMP by plants (Kasparovsky et al., 2003). Sterols are core membrane components that regulate membrane organization, stability, and permeability (Macabuhay et al., 2022; Der et al., 2024). Ergosterol is the main sterol in most fungal membranes (Jordá & Puig, 2020) and is absent from plant membranes. It has been shown to induce early immune responses in various plant species. These responses include ROS accumulation in Beta vulgaris (Rossard et al., 2010), increase in [Ca²⁺]_cyt in Nicotiana tabacum (Kasparovsky et al., 2003; Vatsa et al., 2011), medium alkalinization in B. vulgaris and N. tabacum (Rossard et al., 2010; Vatsa et al., 2011), and induced expression of immunity-related genes such as pathogenesis related (PR) genes and WRKY transcription factors in Vitis vinifera, N. tabacum, and Solanum lycopersicum (Laquitaine et al., 2006; Lochman & Mikes, 2006; Lindo et al., 2020). Despite being known as a MAMP for over two decades, the perception and signaling pathway of ergosterol remains largely unknown. This study investigates the molecular signaling mechanisms activated in barley (Hordeum vulgare) in response to lipid extracts, containing a mixture of lipids from the mycelium of the beneficial root endophytic fungus Serendipita indica (Sebacinales, Basidiomycota). Our data demonstrate that fungal lipids induce immunity in barley, with ergosterol identified as the primary immunogenic component of S. indica lipids and detected in the apoplast, the aqueous space between the host cells, of S. indica-colonized barley roots. By integrating transcriptomics, phosphoproteomics, and metabolomics, we provide evidence that PIP signaling and diterpene biosynthesis are activated upon exposure to fungal lipids. Furthermore, we demonstrate that PA enhances lipid-mediated apoplastic ROS production in barley. Notably, fungal colonization alters the host's phytosterol content and suppresses the ergosterol-induced ROS burst, suggesting a counterstrategy against lipid-mediated host immunity.