Cowpea chloroplastic ATP synthase is the source of multiple plant defense elicitors during insect herbivory

In cowpea ( Vigna unguiculata), fall armyworm ( Spodoptera frugiperda) herbivory and oral secretions ( OS) elicit phytohormone production and volatile emission due to inceptin [Vu-In; (+)ICDINGVCVDA(-)], a peptide derived from chloroplastic ATP synthase gamma-subunit (cATPC) proteins. Elicitor-induced plant volatiles can function as attractants for natural enemies of insect herbivores. We hypothesized that inceptins are gut proteolysis products and that larval OS should contain a mixture of related peptides. In this study, we identified three additional cATPC fragments, namely Vu-(GE+) In [(+)GEICDINGVCVDA(-)], Vu-(E+) In [(+)EICDINGVCVDA(-)], and Vu-In-A [(+)ICDINGVCVD(-)]. Leaf bioassays for induced ethylene (E) production demonstrated similar effective concentration(50) values of 68, 45, and 87 fmol leaf(-1) for Vu-In, Vu-(E+) In, and Vu-(GE+) In, respectively; however, Vu-In-A proved inactive. Shortly following ingestion of recombinant proteins harboring cATPC sequences, larval OS revealed similar concentrations of the three elicitors with 80% of the potential inceptin-related peptides recovered. Rapidly shifting peptide ratios over time were consistent with continued proteolysis and preferential stability of inceptin. Likewise, larvae ingesting host plants with inceptin precursors containing an internal trypsin cleavage site rapidly lost OS-based elicitor activity. OS containing inceptin elicited a rapid and sequential induction of defense-related phytohormones jasmonic acid, E, and salicylic acid at 30, 120, and 240 min, respectively, and also the volatile (E)-4,8-dimethyl-1,3,7-nonatriene. Similar to established peptide signals such as systemin and flg22, amino acid substitutions of Vu-In demonstrate an essential role for aspartic acid residues and an unaltered C terminus. In cowpea, insect gut proteolysis following herbivory generates inappropriate fragments of an essential metabolic enzyme enabling plant non-self-recognition.