Transcriptome Analysis Reveals Functional Diversity in Salivary Glands of Plant Virus Vector, Graminella nigrifrons

Insect salivary glands play an important role for host feeding, specifically by secreting salivary proteins for digestion and potentially modulating host defenses. Compared to other hemipterans, the significance of salivary glands is less studied in the black-faced leafhopper, Graminella nigrifrons, a crop pest that vectors several agronomically important plant viruses. To identify functionally important genes in the salivary glands of the black-faced leafhopper, we compared transcriptomes between adult salivary glands (SG) and the remaining carcasses. We identified 14,297 salivary gland-enriched transcripts and 195 predicted secretory peptides (i.e., with a signal peptide and extracellular localization characteristics). Overall, the SG transcriptome included functions such as ‘oxidoreduction’, ‘membrane transport’, and ‘ATP-binding’, which might be important for the fundamental physiology of this tissue. We further evaluated transcripts with potential contributions in host feeding using RT-qPCR. Two SG-enriched transcripts (log2 fold change > 5), GnP19 and GnE63 (a putative calcium binding protein), were significantly upregulated in maize-fed adults relative to starved adults, validating their importance in feeding. The SG-enriched transcripts of the black-faced leafhopper could play a potential role for interacting with maize and could be targets of interest for further functional studies and improve pest control and disease transmission.

Infectious Maize rayado fino virus from Cloned cDNA

A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

Maize necrotic streak virus, a New Maize Virus with Similarity to Species of the Family Tombusviridae

A new virus was isolated from maize (Zea mays L.) leaves showing mild mosaic symptoms and coinfected with Maize dwarf mosaic virus. The virus was readily transmitted by vascular puncture inoculation (VPI) but not leaf-rub inoculation. Virus symptoms on susceptible maize included pale green, yellow, or cream-colored spots and streaks measuring 1 to 2 mm on emerging leaves 5 to 7 days post-VPI. As leaves developed, the spots and streaks became spindle-shaped, then coalesced into long, chlorotic bands. These bands became translucent and necrotic around the edges. There was a distinctive chlorosis on the stalks that became necrotic. Based on these distinctive symptoms, the new virus was named Maize necrotic streak virus (MNeSV). The virus was not transmitted by Aphis maidis-radicus, Myzus persicae, Macrosiphum euphorbiae, Rhopalosiphum padi, Dalbulus maidis, Graminella nigrifrons, Perigrinus maidis, or Diabrotica virgifera virgifera under persistent or nonpersistent conditions. Both susceptible and resistant maize genotypes were identified following VPI with MNeSV. The isolated virus had isometric (32 nm) virions and a single 29.5-kDa coat protein. MNeSV was serologically distinct from morphologically similar maize viruses. The 4.3-kb single-stranded RNA genome had 25 to 53% sequence identity with species in the family Tombusviridae.

Transmission of Sorghum Stunt Mosaic Rhabdovirus by the Leafhopper Vector, Graminella sonora (Homoptera: Cicadellidae)

The transmission parameters of sorghum stunt mosaic rhabdovirus (SSMV) by the leafhopper Graminella sonora were determined. The minimum acquisition and inoculation times were 6 and 1 h, respectively, while 100% transmission was obtained from a 48-h acquisition access or a 24-h inoculation access period. Transmission efficiency was highest at temperatures of 24 to 36°C. The minimum latent period prior to transmission was 9 days at 30°C, with an 11-day incubation period necessary for 100% transmission. After a 14-day incubation period at 16°C, no transmission was obtained; however, doubling the incubation period to 28 days gave high levels of transmission. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of individual leafhoppers held at 16°C for a 14-day incubation period detected no virus in leafhoppers, whereas over 50% of the leafhoppers had detectable virus after a 28-day incubation period. Graminella nigrifrons and Peregrinus maidis were not able to transmit SSMV.