ORCID IDs: 0000-0001-7137-8797 (J.L.); 0000-0002-3904-6984 (S.B.); 0000-0003-3192-978X (S.V.); 0000-0002-3550-5522 (V.J.); 0000-0001-9147-3871 (W.P.W.).Signaling networks among multiple phytohormones fine-tune plant defense responses to insect herbivore attack. Previously, it was reported that the synergistic combination of ethylene (ET) and jasmonic acid (JA) was required for accumulation of the maize insect resistance1 (mir1) gene product, a cysteine (Cys) proteinase that is a key defensive protein against chewing insect pests in maize (Zea mays). However, this study suggests that mir1-mediated resistance to corn leaf aphid (CLA; Rhopalosiphum maidis), a phloem sap-sucking insect pest, is independent of JA but regulated by the ET-signaling pathway. Feeding by CLA triggers the rapid accumulation of mir1 transcripts in the resistant maize genotype, Mp708. Furthermore, Mp708 provided elevated levels of antibiosis (limits aphid population)-and antixenosis (deters aphid settling)-mediated resistance to CLA compared with B73 and Tx601 maize susceptible inbred lines. Synthetic diet aphid feeding trial bioassays with recombinant Mir1-Cys Protease demonstrates that Mir1-Cys Protease provides direct toxicity to CLA. Furthermore, foliar feeding by CLA rapidly sends defensive signal(s) to the roots that trigger belowground accumulation of the mir1, signifying a potential role of long-distance signaling in maize defense against the phloem-feeding insects. Collectively, our data indicate that ET-regulated mir1 transcript accumulation, uncoupled from JA, contributed to heightened resistance to CLA in maize. In addition, our results underscore the significance of ET acting as a central node in regulating mir1 expression to different feeding guilds of insect herbivores.

SummarySome plant-derived anti-herbivore defensive proteins are induced by insect feeding, resist digestion in the caterpillar gut and are eliminated in the frass. We have identified several maize proteins in fall armyworm (Spodoptera frugiperda) frass that potentially play a role in herbivore defense. Furthermore, the toxicity of one of these proteins, ribosome-inactivating protein 2 (RIP2), was assessed and factors regulating its accumulation were determined.To understand factors regulating RIP2 protein accumulation, maize (Zea mays) plants were infested with fall armyworm larvae or treated with exogenous hormones. The toxicity of recombinant RIP2 protein against fall armyworm was tested.The results show that RIP2 protein is synthesized as an inactive proenzyme that can be processed in the caterpillar gut. Also, caterpillar feeding, but not mechanical wounding, induced foliar RIP2 protein accumulation. Quantitative real-time PCR indicated that RIP2 transcripts were rapidly induced (1 h) and immunoblot analysis indicated that RIP2 protein accumulated soon after attack and was present in the leaf for up to 4 d after caterpillar removal. Several phytohormones, including methyl jasmonate, ethylene, and abscisic acid, regulated RIP2 protein expression. Furthermore, bioassays of purified recombinant RIP2 protein against fall armyworm significantly retarded caterpillar growth.We conclude that the toxic protein RIP2 is induced by caterpillar feeding and is one of a potential suite of proteins that defend maize against chewing herbivores.