Genetic insights into Graminella nigrifrons Competence for maize fine streak virus infection and transmission.

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.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.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

DAVID functional annotation clusters of <i>G. nigrifrons</i> transcripts upregulated in transmitters and acquirers relative to non-acquirers and the healthy control.

<p>DAVID functional annotation clusters of <i>G. nigrifrons</i> transcripts upregulated in transmitters and acquirers relative to non-acquirers and the healthy control.</p

MFSV transcript accumulation in <i>G. nigrifrons</i> transmitters and acquirers.

<p>Accumulation of MFSV transcripts relative to the N gene was determined using RT-qPCR. Transcript accumulation was calculated according to the 2^−ΔΔCt algorithm using the <i>ribosomal protein S13</i> (RPS13) gene expression as the calibrator. Means for relative accumulation of each gene are shown. Bars with different letters were significantly different using LSD (<i>P</i><0.05).</p

Heat map for expression of 891 differentially expressed <i>G. nigrifrons</i> transcripts.

<p>Differentially expressed transcripts were identified using one-way ANOVA (<i>P</i><0.05; RPKM change >2). Each row represents an individual transcript; each column labeled 1–3 represent replicate samples of non-acquirers, acquirers, transmitters, or the healthy control, as outlined in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113529#s2" target="_blank">Methods</a>. For each transcript (row), the relative expression level for each sample is represented by a color that reflects its z-score (shown in the redgreen key), calculated by subtracting the mean expression value for the row from the sample value and dividing by the standard deviation for the row.</p

Comparison of abundance for six differentially expressed transcripts in <i>G. nigrifrons</i> transmitters or acquirers relative to the healthy control using RNA-Seq and RT-qPCR.

<p>Comparison of abundance for six differentially expressed transcripts in <i>G. nigrifrons</i> transmitters or acquirers relative to the healthy control using RNA-Seq and RT-qPCR.</p