LAUSR

Although much is known about plant traits that function in nonhost resistance against pathogens, little is known about nonhost resistance against herbivores, despite its agricultural importance. Empoasca leafhoppers, serious agricultural pests, identify host plants by eavesdropping on unknown outputs of jasmonate (JA)–mediated signaling. Forward- and reverse-genetics lines of a native tobacco plant were screened in native habitats with native herbivores using high-throughput genomic, transcriptomic, and metabolomic tools to reveal an Empoasca-elicited JA-JAZi module. This module induces an uncharacterized caffeoylputrescine–green leaf volatile compound, catalyzed by a polyphenol oxidase in a Michael addition reaction, which we reconstitute in vitro; engineer in crop plants, where it requires a berberine bridge enzyme-like 2 (BBL2) for its synthesis; and show that it confers resistance to leafhoppers. Natural history–guided forward genetics reveals a conserved nonhost resistance mechanism useful for crop protection. Description A volatile defense against leafhoppers In established ecosystems, plants often fend off their insect attackers with the use of chemical defenses that are elicited by herbivore wounding. Some of these same insects are pests in the agricultural setting as well, attacking plants that have not benefited from chemical defenses evolved over the ages. Bai et al. leveraged genetic diversity in a population of Nicotiana attenuata plants that they grew in the plant’s native habitat in Arizona to study how their chemical defenses provide resistance to the herbivorous leafhoppers. A multi-omics approach led to the identification of a volatile compound from leaves that confers resistance to those leafhoppers. —PJH Polyphenol oxidase chemistry underlies nonhost resistance against herbivorous insect pests in a native tobacco plant. INTRODUCTION Although much is known about plant traits that function in nonhost resistance against pathogens, little is known about nonhost resistance against herbivores, despite its agricultural importance, because of the lack of fieldwork. Empoasca leafhoppers, serious agricultural pests, identify host plants by eavesdropping on unknown outputs of jasmonate (JA)–mediated signaling in a native tobacco plant that is naturally variable in its JA signaling. The known sectors of this tobacco plant’s specialized defense metabolism are not effective against this insect, which calls for an unbiased approach. RATIONALE An unbiased forward-genetics approach based on the screening of a 26-parent recombinant inbred line population in a natural habitat with native herbivores was wedded with unbiased transcriptomic and mass spectrometry–based metabolomic analyses of reverse-genetics lines to identify defense chemistries produced by this native tobacco when probed by leafhoppers. Synthetic biology approaches were used to reconstitute these chemistries in crop plants. RESULTS The analysis revealed an Empoasca-elicited JA-JAZi module that pointed to the phenolamide master transcription factor, MYB8, as a central genetic hub clustering with putrescine-derived phenolamides. Using tobacco plants silenced for components of JA signaling (JAZ and MYC2 genes) and phenolamide biosynthesis, the central role of a MYC2-MYB8-JAZi branch of JA signaling was confirmed; however, infiltration of MYC2-silenced plants with known putrescine-derived phenolamides did not alter Empoasca preference. Subsequent detailed structural analysis revealed an unknown metabolite whose abundance was regulated by the MYC2-MYB8-JAZi branch of JA signaling and was negatively correlated with Empoasca damage. Previous work on this unknown metabolite suggested a conjugate of caffeoylputrescine with a C-6 aldehyde produced during wound-induced lipid peroxidation—a process that leads to the formation of green leaf volatiles. Metabolite quantitative trait locus (mQTL) analysis and coexpression analysis pointed to two polyphenol oxidases (PPOs) and one berberine bridge enzyme-like 2 (BBL2) gene associated with the metabolite’s biosynthesis. The function of the proteins encoded by these genes was tested in both in vitro [Escherichia coli expression and enzymatic assays with (Z)-3-hexenal and caffeoylputrescine] and in vivo (transient expression in Solanum chilense and Vicia faba) systems. The structure of the unknown metabolite was identified by nuclear magnetic resonance (NMR) to be a caffeoylputrescine–green leaf volatile compound (CPH), catalyzed by a PPO in a Michael addition reaction and requiring BBL2 in planta. Synthetic biology approaches confirmed the function of CPH in nonhost resistance against Empoasca leafhoppers in Nicotiana attenuata lines silenced to be defective in CPH production; in V. faba, a bean crop host plant of the leafhoppers unable to produce caffeoylputrescine; and in S. chilense. CONCLUSION The natural history–driven multi-omics framework used for the discovery of CPH and its marriage with synthetic biology approaches highlight how readily the results of millions of years of innovation by natural selection can be amortized and transferred to crop plants to catalyze a greener and ecologically more nuanced revolution in plant protection. Crop plants face challenges not substantially different from those faced by native plants; they are constantly tested by hidden herbivore communities that challenge the host-nonhost distinction. In a world of climate change and globally homogenized herbivore communities, opportunistic associations will dominate natural and man-made ecosystems. CPH represents a chemical innovation that allows a native plant to cope with these opportunistic associations and is readily engineered in crop plants. Opportunistic leafhopper attack elicits caffeoylputrescine–green leaf volatile defenses. Attack elicits a JAZi-mediated sector of JA signaling to condense the products of three branches of specialized metabolism (green leaf volatile, phenylpropanoid, and polyamine pathways) in a native tobacco plant through a PPO-catalyzed and BBL2-mediated Michael addition reaction to produce previously unobserved defense chemistry (CPH) that was reconstituted in crop plants for durable nonhost resistance. CP, caffeoylputrescine; AT1, acyltransferase 1.