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ARS Home » Midwest Area » Wooster, Ohio » Corn, Soybean and Wheat Quality Research » Research » Publications at this Location » Publication #307155
Title: Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference

Author
item CHEN, YUTING - The Ohio State University
item Redinbaugh, Margaret
item MICHEL, ANDREW - The Ohio State University

Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/28/2015
Publication Date: 6/1/2015
Publication URL: http://handle.nal.usda.gov/10113/61301
Citation: Chen, Y., Redinbaugh, M.G., Michel, A.P. 2015. Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference. Insect Molecular Biology. 24(3):391-401.

Interpretive Summary: The black-faced leafhopper (Graminella nigrifrons) is the only known vector for Maize fine streak virus (MFSV), an emerging plant pathogen within the Rhabdoviridae. Within laboratory G. nigrifrons populations, individuals can be experimentally separated into three types based on their capacity for viral transmission: transmitters, acquirers and non-acquirers. In this study, we used RT-qPCR to compare the expression profiles of ten transcripts that putatively function in the insect immune response among the three types of leafhoppers: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and -LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in leafhoppers fed with MFSV infected maize compared to those on healthy maize. The three RNAi pathway transcripts (Dcr-2, Ago-2, Ars-2) were significantly lower in acquirers and non-acquirers relative to transmitters. Injection of leafhoppers with dsRNA encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90% and 75% over 14 and 22 day treatment period, respectively. However, MFSV acquisition and transmission was not significantly affected by the injection of either dsRNA. Knock down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 dpi, and included a higher number of ‘abnormally molted’ leafhoppers relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G. nigrifrons transcripts will facilitate the study of gene function and pathogen transmission in leafhoppers, and may provide approaches for developing novel targets of RNAi-based pest control.

Technical Abstract: Maize fine streak virus (MFSV) is an emerging virus of maize that is transmitted by an insect vector, the leafhopper called Graminella nigrifrons. Virus transmission by the leafhopper requires that the virus enter into and multiply in insect cells, tissues and organs before being transmitted to a new plant host during insect feeding. Our previous studies on the transmission of MFSV by G. nigrifrons identified three classes of insects in our laboratory colony: leafhoppers that could host the virus and transmit it or 'transmitters'; leafhoppers that could host the virus but not transmit it or 'acquirers'; and, and leafhoppers that do not host the virus or 'non-acquirers'. We used this insect - virus - plant host system to begin to identify insect that might be involved in virus multiplication and transmission. We examined the expression of ten insect genes that are likely to be important in the insect's immune system, and found that expression for five of the immune system-associated genes was lower in insects fed on virus-infected plants than in insects feeding on healthy plants. This result suggests that either MFSV or another factor in the MFSV-infected plant reduces the leafhopper immune response. Three genes that function in a pathway important for degrading viral RNA were more highly expressed in transmitters than in acquirers and non-acquirers. Although leafhopper gene expression correlated the transmission class, we needed a new approach to directly investigate the roles of these genes in virus transmission. Therefore, we developed a method for artificially lowering expression of specific genes in the leafhoppers using a technique called RNAi. We used a microinjector to deliver double-stranded RNA (dsRNA) into leafhoppers, and demonstrated 75 to 90% reduction in expression for the PGRP-LC and Dcr-2 genes over 14 to 22 days after injection. MFSV acquisition and transmission was not affected by reducing expression of either gene, suggesting they do not play a direct role in virus transmission. However, we found that more leafhoppers injected with dsRNA from PGRP-LC died by 27 days after inoculation than did leafhoppers injected with dsRNA for other leafhopper genes, suggesting this gene could be a target for controlling G. nigrifrons populations. The development of RNAi as a tool to reduce gene expression in leafhopppers will facilitate the study of gene function and pathogen transmission in this important group of insects.