RFP and GFP Detection in Planthoppers with LUYOR-3415RG

Researchers developed a cytorhabdovirus-based expression vector for gene functional studies in rice planthoppers and used fluorescent protein lamp LUYOR-3415RG to screen planthoppers infected with recombinant BYSMV expressing RFP and GFP reporter proteins.

Abstract

The brown planthopper (BPH, Nilaparvata lugens), the small brown planthopper (SBPH, Laodelphax striatellus), and the white-backed planthopper (WBPH, Sogatella furcifera) are problematic insect pests and cause severe yield losses through phloem sap-sucking and virus transmission. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, has been developed as versatile expression platforms in SBPHs and cereal plants. However, biosafe overexpression vectors based on recombinant BYSMV (rBYSMV) remain to be developed and applied to the three kinds of planthoppers. Here, we found that rBYSMV was able to infect SBPHs, BPHs and WBPHs through microinjection with crude extracts from rBYSMV-infected barley leaves. To ensure bio-safety of the rBYSMV vectors, we generated an rBYSMV mutant by deleting the accessory protein P3, a putative viral movement protein. As expected, the resulting mutant abolished viral systemic infection in barley plants but had no effects on BYSMV infectivity in insect vectors. Subsequently, we used the modified rBYSMV vector to overexpress iron transport peptide (ITP) in the three kinds of planthoppers and revealed the potential functions of ITP. Overall, our results provide bio-safe overexpression platforms to facilitate functional genomics studies of planthoppers.

RFP and GFP Fluorescence Observed Using LUYOR-3415RG Fluorescent Protein Excitation Lamp

Planthoppers infected with recombinant BYSMV viruses were selected with LUYOR-3415RG fluorescent protein excitation lamp (Luyor Instrument). The viruliferous planthoppers exhibiting RFP fluorescence were selected for phenotype analyses. Cuticle color and mortalities during eclosion were recorded. Planthoppers were anesthetized by carbon dioxide for about 1–3 min and photographed by Olympus stereomicroscope SEX 16 with equivalent light conditions. RFP/GFP fluorescence signals were acquired with RFP/GFP filter.

Fig. 1. RFP fluorescence in SBPHs, WBPHs and BPHs

Fig. 2. Distribution of BYR-GUS in different tissues of SBPHs, WBPHs and BPHs.

Fig. 3. GFP fluorescence in barley leaves

Fig. 4. GFP and RFP fluorescence in SBPHs and barley plants

Fig. 5. RFP fluorescence in planthoppers

Reference

Xu, W.-Y., Fang, X.-D., Cao, Q., Gao, Q., Gao, D.-M., Qiao, J.-H., Zang, Y., Xie, L., Ding, Z.-H., Yang, Y.-Z., Wang, Y., & Wang, X.-B. A cytorhabdovirus-based expression vector in Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. Insect Biochem. Mol. Biol. 142, 103703 (2022).