Airborne Immunity: A Bacterial Volatile Primes Plant Defenses by Unlocking a Phytoalexin Biosynthetic Checkpoint

Microbial volatile organic compounds (mVOCs) enable plants to perceive microbial activity prior to physical contact, yet the contribution of individual bacterial volatiles to immune signalling and disease resistance remains incompletely understood. Here, headspace GC-MS analysis demonstrates that the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) emits a distinct volatile blend containing the bacterium-specific compounds 2-methylbutanoic acid (2-MBA) and 3-methylbutanoic acid (3-MBA), with 2MBA as a dominant component. Exposure of Arabidopsis thaliana to the complete Pst DC3000 mVOC blend induced extensive transcriptional reprogramming activation of pattern-recognition receptor-associated genes, MAP kinase signalling components, WRKY transcription factors, camalexin biosynthetic genes and early defense responses. The latter included cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, K⁺ channel activation, hydrogen peroxide accumulation, and nitric oxide production, which culminated in camalexin accumulation in wild-type shoots and roots. In contrast, these early signaling and physiological responses were strongly attenuated in the camalexin-deficient pad3 mutant. Phenotypic priming assays confirmed that mVOC pre-exposure enhances resistance against subsequent Pst DC3000 infection largely through this PAD3-dependent mechanism, while exogenous camalexin administration proved independently sufficient to restore robust pathogen protection, indicating that PAD3-dependent camalexin biosynthesis contributes substantially, but not exclusively, to volatile-induced resistance. Application of synthetic 2-MBA, and to a lesser extent 3-MBA, was sufficient to recreate rapid [Ca²⁺]_cyt elevation and ROS production. Quantitative expression profiling revealed that synthetic 2-MBA modulates gene expression in an organ-specific manner, upregulating shoot volatile perception and salicylic acid pathways, inducing root calcium and immune responses, and systemically activating auxin signaling and tryptophan biosynthetic genes. Together, these findings identify 2-MBA as a primary active component of the Pst DC3000 volatile blend and indicate that full volatile-induced defence depends on PAD3-dependent camalexin accumulation together with integration of multiple volatile signals. Our results reveal how plants integrate distinct bacterial volatiles to trigger early signaling and coordinate localized and systemic camalexin-dependent immunity.