Combined Transcriptome and Proteome Analysis of the Protein Composition of the Brochosomes of the Leafhopper Nephotettix cincticeps

SIMPLE SUMMARY: The Cicadellidae family comprises over 22,000 described species, renowned for the presence of brochosome coatings on their integuments. Brochosomes are protein–lipid particles, hollow spheres with a honeycomb-like surface, synthesized and secreted by a specialized segment of leafhopper Malpighian tubules. Brochosomes are believed to assist leafhoppers in resisting various threats. However, the exact protein composition of brochosomes remains poorly understood. In this study, we conducted a combined transcriptome and proteome analysis of the protein composition of brochosomes in the leafhopper Nephotettix cincticeps. Ultimately, we identified 22 candidate brochosome proteins. These proteins were divided into two groups: brochosomins (BSM) and brochosome-associated proteins (BSAP). Examination of conserved motifs and functional predictions unveiled potential roles for these proteins. Our findings indicated that BSM, along with some BSAP, are exclusive to the Cicadellidae family of leafhoppers. These findings provide insights into brochosome synthesis, function, and evolutionary origins in leafhoppers, highlighting the existence of species-specific orphan genes encoding BSM and some BSAP. Furthermore, this research underscores the complexity of brochosome assembly and its potential roles in leafhopper biology. ABSTRACT: Brochosomes, unique coatings on the integuments of Cicadellidae, are synthesized in specialized glandular sections of Malpighian tubules. However, limited knowledge exists regarding the protein composition of brochosomes. In this study, we conducted transcriptomic and proteomic profiling to characterize the brochosome protein composition in the rice green leafhopper Nephotettix cincticeps. Brochosomes were collected from the forewings of leafhoppers using ultrasonic treatment, allowing for more effective brochosome collection and shaking treatment, resulting in purer brochosomes. Transcriptome sequencing analysis identified 106 genes specifically expressed in the Malpighian tubules; combined with proteomic data, we identified 22 candidate brochosome proteins. These proteins were classified into 12 brochosomins (BSM) and 10 brochosome-associated proteins (BSAP) based on previous research. Conserved motif analysis and functional predictions unveiled unique motifs in each BSM, while BSAP appeared to play a crucial role in BSM folding and pathogen resistance. Comparative analysis of other Hemiptera species demonstrated that all BSM and some BSAP are specific to the Cicadellidae family. Our findings could contribute to understanding the mechanism of brochosome synthesis, its function, and evolutionary genesis.