Aphid Probing and Phloem Feeding Trigger Distinct Plant Immune Programs, Identifying a Broad‑Spectrum Trypsin Protease Inhibitor

Aphid feeding begins with brief exploratory probing and transitions to prolonged phloem ingestion, yet whether these distinct phases trigger different plant immune programs remains unknown. We show that probing and phloem feeding are associated with distinct plant immune cascades. Under probing-enriched conditions, we observed selective activation of jasmonate (JA) signaling, accompanied by significant ROS bursts and vascular callose deposition, whereas phloem feeding elicits only marginal salicylic acid accumulation. Probing-enriched conditions imposes severe fitness penalties on aphids, a phenotype recapitulated by exogenous methyl jasmonate. Consistent with this, aphids exhibit high feeding‑site fidelity, which likely minimizes probing events and helps evade these penalties. Transcriptome analysis identified the squash‑type trypsin protease inhibitor CsTrypPI1, characterized by six conserved cysteines, as a JA-dependent component upregulated under probing-enriched conditions. Recombinant CsTrypPI1 directly impairs aphid survival and fecundity in artificial diet assays. Transgenic cucumber lines constitutively over‑expressing CsTrypPI1 displayed broad‑spectrum resistance against the aphid Aphis gossypii, the whitefly Bemisia tabaci, and the lepidopteran Spodoptera litura. Our findings indicate that the probing versus phloem feeding comparison defines two distinct immunological trajectories, which helps reconcile conflicting evidence on JA and SA roles in plant and phloem‑feeder interactions. The identification of CsTrypPI1 as a broad‑spectrum resistance candidate highlights this behavioral-immunological interface as a promising intervention point for sustainable pest management.