Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana. HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Plant innate immunity activation relies on detection of invasion pattern (IP) molecules that are perceived by plant cells (1, 2). Non–self-recognition IPs include essential components of whole classes of microorganisms, such as fragments of flagellin, peptidoglycans, mc-3-OH-FAs from bacteria or fragments of chitin, and β-glucans from fungi and oomycetes, respectively (3, 4). Apoplastic IPs are sensed by plant plasma membrane–localized receptor kinases (RKs) or receptor-like proteins (RLPs) that function as pattern recognition receptors (PRRs) (5, 6). Activation of the immune response requires the recruitment of regulatory receptor kinases and receptor-like cytoplasmic kinases (RLCKs) by PRRs (7). Early cellular immune signaling of pattern-triggered immunity (PTI) includes ion-flux changes at the plasma membrane, rise in cytosolic Ca²⁺ levels, production of extracellular reactive oxygen species (ROS), and activation of mitogen-activated protein kinases (MAPKs) and/or Ca²⁺-dependent protein kinases (3, 8–10). Biosynthesis and mobilization of plant hormones, including salicylic acid, jasmonic acid, ethylene, abscisic acid and brassinosteroids, ultimately modulate plant resistance to phytopathogens (11–14).Rhamnolipids (RLs) are extracellular amphiphilic metabolites produced by several bacteria, especially Pseudomonas and Burkholderia species (15–17). Acting as wetting agents, RLs are essential for bacterial surface dissemination called swarming motility and for normal biofilm development (18–20). These glycolipids are produced from l-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) precursors (15, 21). HAAs are synthesized by dimerization of (R)-3-hydroxyalkanoyl-CoA in Pseudomonas, forming congeners through the RhlA enzyme (21). The opportunistic plant pathogen Pseudomonas aeruginosa and the phytopathogen Pseudomonas syringae produce extracellular HAAs (16, 22–24). In P. syringae, HAA synthesis is coordinately regulated with the late-stage flagellar gene encoding flagellin (22). HAA and RL production is finely tuned and modulates the behavior of swarming migrating bacterial cells by acting as self-produced negative and positive chemotactic-like stimuli (25). RLs contribute to the alteration of the bacterial outer membrane composition, by shedding flagellin from the flagella (26) and by releasing lipopolysaccharides (LPS), resulting in an increased hydrophobicity of the bacterial cell surface (27). In mammalian cells, RLs produced by Burkholderia plantarii exhibit endotoxin-like properties similar to LPS, leading to the production of proinflammatory cytokines in human mononuclear cells (28, 29). They also subvert the host innate immune response through manipulation of the human beta-defensin-2 expression (30). Moreover, RLs from Burkholderia pseudomallei induce interferon gamma (IFN-γ)–dependent host immune response in goat (31).In plants, RLs induce defense responses and resistance to biotrophic and necrotrophic pathogens (32, 33). They also contribute to the biocontrol activity of the plant beneficial bacterium P. aeruginosa PNA1 against oomycetes (17). Recently, it was reported that the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29) mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) sensing in Arabidopsis thaliana (hereafter, Arabidopsis) and that bacterial compounds comprising mc-3-OH-acyl building blocks including LPS and RLs do not stimulate LORE-dependent responses (34).Here, we show that the lipidic secretome produced by P. aeruginosa (RL secretome), mostly composed of RLs and HAAs, induces Arabidopsis immunity. HAAs are perceived through the RK LORE. We demonstrate that, albeit not being sensed by LORE, RLs trigger an immune response characterized by an atypical defense signature. Altogether, our results demonstrate that RLs and their precursors produced by Pseudomonas bacteria stimulate the plant immune response by two distinct mechanisms.