Tobacco Hornworm (Manduca sexta) Oral Secretion Elicits Reactive Oxygen Species in Isolated Tomato Protoplasts.

Plants are under constant attack by a suite of insect herbivores. Over millions of years of coexistence, plants have evolved the ability to sense insect feeding via herbivore-associated elicitors in oral secretions, which can mobilize defense responses. However, herbivore-associated elicitors and the intrinsic downstream modulator of such interactions remain less understood. In this study, we show that tobacco hornworm caterpillar (Manduca sexta) oral secretion (OS) induces reactive oxygen species (ROS) in tomato (Solanum lycopersicum) protoplasts. By using a dye-based ROS imaging approach, our study shows that application of plant-fed (PF) M. sexta OS generates significantly higher ROS while artificial diet-fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. Elevation in ROS generation was saturated after ~140 s of PF OS application. ROS production was also suppressed in the presence of an antioxidant NAC (N-acetyl-L-cysteine). Interestingly, PF OS-induced ROS increase was abolished in the presence of a Ca2+ chelator, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). These results indicate a potential signaling cascade involving herbivore-associated elicitors, Ca2+, and ROS in plants during insect feeding. In summary, our results demonstrate that plants incorporate a variety of independent signals connected with their herbivores to regulate and mount their defense responses.