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Background: Most plant-infecting rhabdoviruses are transmitted by one or a few closely related insect species. Additionally, intraspecific differences in transmission efficacy often exist among races/biotypes within vector species and among strains within a virus species. The black-faced leafhopper, Graminella nigrifrons, is the only known vector of the persistent propagative rhabdovirus Maize fine streak virus (MFSV). Only a small percentage of leafhoppers are capable of transmitting the virus, although the mechanisms underlying vector competence are not well understood. Methodology: RNA-Seq was carried out to explore transcript expression changes and sequence variation in G. nigrifrons and MFSV that may be associated with the ability of the vector to acquire and transmit the virus. RT-qPCR assays were used to validate differential transcript accumulation. Results/Significance: Feeding on MFSV-infected maize elicited a considerable transcriptional response in G. nigrifrons, with increased expression of cytoskeleton organization and immunity transcripts in infected leafhoppers. Differences between leafhoppers capable of transmitting MFSV, relative to non-transmitting but infected leafhoppers were more limited, which may reflect difficulties discerning between the two groups and/or the likelihood that the transmitter phenotype results from one or a few genetic differences. The ability of infected leafhoppers to transmit MFSV did not appear associated with virus transcript accumulation in the infected leafhoppers or sequence polymorphisms in the viral genome. However, the non-structural MFSV 3 gene was expressed at unexpectedly high levels in infected leafhoppers, suggesting it plays an active role in the infection of the insect host. The results of this study begin to define the functional roles of specific G. nigrifrons and MFSV genes in the viral transmission process. [ABSTRACT FROM AUTHOR]

Background Insects are the most important epidemiological factors for plant virus disease spread, with >75% of viruses being dependent on insects for transmission to new hosts. The black-faced leafhopper (Graminella nigrifrons Forbes) transmits two viruses that use different strategies for transmission: Maize chlorotic dwarf virus (MCDV) which is semi-persistently transmitted and Maize fine streak virus (MFSV) which is persistently and propagatively transmitted. To date, little is known regarding the molecular and cellular mechanisms in insects that regulate the process and efficiency of transmission, or how these mechanisms differ based on virus transmission strategy. Results RNA-Seq was used to examine transcript changes in leafhoppers after feeding on MCDV-infected, MFSV-infected and healthy maize for 4 h and 7 d. After sequencing cDNA libraries constructed from whole individuals using Illumina next generation sequencing, the Rnnotator pipeline in Galaxy was used to reassemble the G. nigrifrons transcriptome. Using differential expression analyses, we identified significant changes in transcript abundance in G. nigrifrons. In particular, transcripts implicated in the innate immune response and energy production were more highly expressed in insects fed on virus-infected maize. Leafhoppers fed on MFSV-infected maize also showed an induction of transcripts involved in hemocoel and cell-membrane linked immune responses within four hours of feeding. Patterns of transcript expression were validated for a subset of transcripts by quantitative real-time reverse transcription polymerase chain reaction using RNA samples collected from insects fed on healthy or virus-infected maize for between a 4 h and seven week period. Conclusions We expected, and found, changes in transcript expression in G. nigrifrons feeding of maize infected with a virus (MFSV) that also infects the leafhopper, including induction of immune responses in the hemocoel and at the cell membrane. The significant induction of the innate immune system in G. nigrifrons fed on a foregut-borne virus (MCDV) that does not infect leafhoppers was less expected. The changes in transcript accumulation that occur independent of the mode of pathogen transmission could be key for identifying insect factors that disrupt vector-mediated plant virus transmission. [ABSTRACT FROM AUTHOR]

Background: Leafhoppers (Hemiptera: Cicadellidae) are plant-phloem feeders that are known for their ability to vector plant pathogens. The black-faced leafhopper (Graminella nigrifrons) has been identified as the only known vector for the Maize fine streak virus (MFSV), an emerging plant pathogen in the Rhabdoviridae. Within G. nigrifrons populations, individuals can be experimentally separated into three classes based on their capacity for viral transmission: transmitters, acquirers and non-acquirers. Understanding the molecular interactions between vector and virus can reveal important insights in virus immune defense and vector transmission. Results: RNA sequencing (RNA-Seq) was performed to characterize the transcriptome of G. nigrifrons. A total of 38,240 ESTs of a minimum 100 bp were generated from two separate cDNA libraries consisting of virus transmitters and acquirers. More than 60% of known D. melanogaster, A. gambiae, T. castaneum immune response genes mapped to our G. nigrifrons EST database. Real time quantitative PCR (RT-qPCR) showed significant down-regulation of three genes for peptidoglycan recognition proteins (PGRP - SB1, SD, and LC) in G. nigrifrons transmitters versus control leafhoppers. Conclusions: Our study is the first to characterize the transcriptome of a leafhopper vector species. Significant sequence similarity in immune defense genes existed between G. nigrifrons and other well characterized insects. The down-regulation of PGRPs in MFSV transmitters suggested a possible role in rhabdovirus transmission. The results provide a framework for future studies aimed at elucidating the molecular mechanisms of plant virus vector competence. [ABSTRACT FROM AUTHOR]

The rhabdoviruses form a large family (Rhabdoviridae) whose host ranges include humans, other vertebrates, invertebrates, and plants. There are at least 90 plant-infecting rhabdoviruses, several of which are economically important pathogens of various crops. All definitive plant-infecting and many vertebrate-infecting rhabdoviruses are persistently transmitted by insect vectors, and a few putative plant rhabdoviruses are transmitted by mites. Plant rhabdoviruses replicate in their plant and arthropod hosts, and transmission by vectors is highly specific, with each virus species transmitted by one or a few related insect species, mainly aphids, leafhoppers, or planthoppers. Here, we provide an overview of plant rhabdovirus interactions with their insect hosts and of how these interactions compare with those of vertebrate-infecting viruses and with the Sigma rhabdovirus that infects Drosophila flies. We focus on cellular and molecular aspects of vector/host specificity, transmission barriers, and virus receptors in the vectors. In addition, we briefly discuss recent advances in understanding rhabdovirus-plant interactions. [ABSTRACT FROM AUTHOR]

To determine the most economical and efficient means to maintain cultures of Maize chlorotic dwarf virus (MCDV) and to screen for host plant resistance to MCDV, we evaluated the effects of temperature, light intensity, daylength, atmospheric pressure, and leafhopper gender on the frequency of transmission of MCDV by Graminella nigrifrons Forbes (Homoptera: Cicadellidae). Female leafhoppers transmitted at higher frequencies than males under most conditions. In temperature studies, transmission rates for both male and female leafhoppers progressively increased as temperatures rose from 20 to 30 ° C. At high light intensities, both males and females transmitted at greater frequencies than they did at low. Similarly, longer day lengths were correlated with higher transmission rates for both sexes. No significant differences in transmission rates were observed in response to differences in atmospheric pressure. The results also showed that transmission rates under most conditions are high enough to overcome potential ambiguities caused by inoculated susceptible plants that do not become infected (disease escapes) when screening for resistance. [ABSTRACT FROM AUTHOR]

Maize fine streak virus (MFSV), an emerging Rhabdovirtls sp. in the genus Nueleorhabdovirus, is persistently transmitted by the black-faced leafhopper. Graminella nigrifrons (Forbes). MFSV was transmitted to maize, wheat, oat, rye, barley, foxtail, annual ryegrass, and quackgrass by G. nigrtfrons. Parameters affecting efficiency of MFSV acquisition (infection) and transmission (inoculation) to maize were evaluated using single-leathopper inoculations and enzyme-linked immunosorbent assay. MFSV was detected in ∼20% of leafhoppers that fed on infected plants but <10% of insects transmitted the virus. Nymphs became infected earlier and supported higher viral titers than adults but developmental stage at aquisition did not affect the rate of MFSV transmission. Viral titer and transmission also increased with longer post-first access to diseased periods (PADPs) (the sum of the intervals from the beginning of the acquisition access period to the end of the inoculation access period). Length of the acquisition access period was more important for virus accumulation in adults, whereas length of the interval between acquisition access and inoculation access was more important in nymphs. A threshold viral titer was needed for transmission but no transmission occurred, irrespective of titer, with a PADP of <4 weeks. MFSV was first detected by immunofluorescence confocal laser scanning microscopy at 2-week PADPs in midgut cells, hemocytes, and neural tissues; 3-week PADPs in tracheal cells; and 4-week PADPs in salivary glands, coinciding with the time of transmission to plants. [ABSTRACT FROM AUTHOR]