MS/MS similarity scoring, biclustering, and
molecular networking were used to iden-
tify the unknownm/z347.19 metabolite. OS-
induced volatile emissions were collected
using polydimethylsiloxane (PDMS) tubing
from 650 MAGIC RILs planted in the main
MPI-CE glasshouse and analyzed by thermal
desorption–gas chromatography–mass spec-
trometry (TD-GC-MS).NaPPO1/2andNaBBL2
genes were elucidated by combining mQTL
analysis for herbivory-induced unknown
m/z347.19 and transcriptomics analysis of
the microarray and RNA-seq datasets of
OS-induced kinetics of WT and irMYC2and
irMYB8lines. The candidate genes were func-
tionally validated by VIGS and in vitro enzy-
matic assays usingE. coli–expressedNaPPO1,
NaPPO2, andNaBBL2with CP and (Z)-3-
hexenal. The CPH [CP-5–(Z)-3-hexenal] chem-
ical structure was characterized by NMR. CPH’s
resistance function againstEmpoascawas
tested by in vitro nonchoice assays with syn-
thesized CPH or by in planta choice assays
conducted with VIGS plants of EV,NaPPO1,
NaPPO2, andNaAT1. The biosynthetic path-
way of CPH was reconstituted inV. fabaand
S. chilenseby transiently coexpressingNaPPO1,
NaPPO2, andNaBBL2with CP and (Z)-3-
hexenal leaf infiltrations. The nonhost resist-
ance function of CPH againstEmpoascawas
further evaluated with the CPH-engineered
V. fabaandS. chilenseplants.
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ACKNOWLEDGMENTS
We thank B. Hong for technical support and fruitful discussions
regarding CPH’s chemistry; J. Li for insightful discussions regarding
insect treatments; V. Grabe and X. Jiang for microscopic support;
Prescott College, Northern Arizona University, the Arboretum at
Flagstaff, Prescott National Forest, and the Southwest Experimental
Garden Array for the use of a field plot at the Walnut Creek Center for
Education and Research; Brigham Young University for the use of
its Lytle Ranch Preserve, where this work started; R. Carlson and
N. Carlson for logistics support of the 2019 field team; G. Baldwin for
support in sowing the MAGIC population and plant transformations;
K. Gase for preparations of transformation constructs; the 2019
MPI-CE field team (E. McGale, H. Valim, R. Ray, J. Li, P. Harary,
C. Mahadevan, and M. Smith) for planting, screening, and oral
secretion–eliciting the MAGIC population; R. Ray for help in preparing
the eQTL dataset; C. Rocha for help with metabolite extractions
and analyses; and J. Gershenzon and S. O’Connor for comments
on an early draft of the manuscript.Funding:This work was
supported by the Max Planck Society, advanced grant no. 293926
of the European Research Council to I.T.B., and the Deutsche
Forschungsgemeinschaft - SFB 1127/2 ChemBioSys–239748522 to
I.T.B.Author contributions:Y.B., R.H., I.T.B., and D.L. conceived
and designed the project. Y.B., C.Y., R.H., C.P., D.K., K.B., I.T.B., and
D.L. performed experiments. Y.B., C.Y., C.P., E.G., I.T.B., and D.L.
analyzed data. Y.B., I.T.B., and D.L. wrote the manuscript, with
contributions from all authors.Competing interests:The authors
have filed a patent (EP 21 217 268.8 and US 63/293,193) on CPH
production and function. They declare no other competing interests.
Data and materials availability:All data are available in the
manuscript or the supplementary materials. Transformed lines are
available from I.T.B.
SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abm2948
Materials and Methods
Figs. S1 to S29
Tables S1 to S3
References ( 44 – 73 )
MDAR Reproducibility Checklist
Data S1 to S3
7 September 2021; accepted 22 December 2021
10.1126/science.abm2948Baiet al.,Science 375 , eabm2948 (2022) 4 February 2022 9of9
RESEARCH | RESEARCH ARTICLE