Proteomics of the Honeydew from the Brown Planthopper and Green Rice Leafhopper Reveal They Are Rich in Proteins from Insects, Rice Plant and Bacteria

SIMPLE SUMMARY: Plant sap-sucking insects can secrete saliva into the plant and excrete honeydew on the plant surface, which both interfere with the plant’s immune system. However, knowledge about the composition of insect honeydew and the roles it plays in plant defenses is limited. In the present study, a diversity of proteins in the honeydew of two major rice pests, the brown planthopper and green rice leafhopper, were identified by way of proteomic analysis. Results revealed that the honeydew contains some insect proteins that originate from the saliva and guts, proteins that originate from plant sap via the insect digestion system as well as many bacterial proteins from insect or plant symbionts and from plant surfaces. These abundant proteins from insects, microbes and plants in the honeydew might be elicitors, effectors or self-recognized molecules for plant defenses, which provide further insights into their roles in the multitrophic interactions of plants–insects–microbes. ABSTRACT: Honeydew is a watery fluid excreted by plant sap-feeding insects. It is a waste product for the insect hosts. However, it plays important roles for other organisms, such as serving as a nutritional source for beneficial insects and bacteria, as well as elicitors and effectors modulating plant responses. In this study, shotgun LC–MS/MS analyses were used to identify the proteins in the honeydew from two important rice hemipteran pests, the brown planthopper (Nilaparvata lugens, BPH) and green rice leafhopper (Nephotettix cincticeps, GRH). A total of 277 and 210 proteins annotated to insect proteins were identified in the BPH and GRH honeydews, respectively. These included saliva proteins that may have similar functions as the saliva proteins, such as calcium-binding proteins and apolipophorin, involved in rice plant defenses. Additionally, a total of 52 and 32 Oryza proteins were identified in the BPH and GRH honeydews, respectively, some of which are involved in the plant immune system, such as Pathogen-Related Protein 10, ascorbate peroxidase, thioredoxin and glutaredoxin. Coincidently, 570 and 494 bacteria proteins were identified from the BPH and GRH honeydews, respectively, which included several well-known proteins involved in the plant immune system: elongation factor Tu, flagellin, GroEL and cold-shock proteins. The results of our study indicate that the insect honeydew is a complex fluid cocktail that contains abundant proteins from insects, plants and microbes, which may be involved in the multitrophic interactions of plants–insects–microbes.