Graminella Nigrifrons Research Articles

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.

Engineering Maize rayado fino virus for virus-induced gene silencing.

Maize rayado fino virus (MRFV) is the type species of the genus Marafivirus in the family Tymoviridae. It infects maize (Zea mays), its natural host, to which it is transmitted by leafhoppers including Dalbulus maidis and Graminella nigrifrons in a persistent‐propagative manner. The MRFV monopartite RNA genome encodes a precursor polyprotein that is processed into replication‐associated proteins. The genome is encapsidated by two carboxy co‐terminal coat proteins, CP1 and CP2. Cloned MRFV can be readily transmitted to maize by vascular puncture inoculation (VPI), and such virus systems that can be used in maize are valuable to examine plant gene function by gene silencing. However, the efficacy of marafiviruses for virus‐induced gene silencing (VIGS) has not been investigated to date. To this end, MRFV genomic loci were tested for their potential to host foreign insertions without attenuating virus viability. This was done using infectious MRFV clones engineered to carry maize phytoene desaturase (PDS) gene fragments (ZmPDS) at various genomic regions. Several MRFV‐PDS constructs were generated and tested for infectivity and VIGS in maize. This culminated in identification of the helicase/polymerase (HEL/POL) junction as a viable insertion site that preserved virus infectivity, as well as several sites at which sequence insertion caused loss of virus infectivity. Transcripts of viable constructs, carrying PDS inserts in the HEL/POL junction, induced stable local and systemic MRFV symptoms similar to wild‐type infections, and triggered PDS VIGS initiating in veins and spreading into both inoculated and noninoculated leaves. These constructs were remarkably stable, retaining inserted sequences for at least four VPI passages while maintaining transmissibility by D. maidis. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize.

Isolation and characterization of a new iflavirus from Armigeres spp. mosquitoes in the Philippines.

During an entomological surveillance for arthropod-borne viruses in the Philippines, we isolated a previously unrecognized virus from female Armigeres spp. mosquitoes. Whole-genome sequencing, genetic characterization and phylogenetic analysis revealed that the isolated virus, designated Armigeres iflavirus (ArIFV), is a novel member of the iflaviruses (genus Iflavirus, family Iflaviridae) and phylogenetically related to Moku virus, Hubei odonate virus 4, slow bee paralysis virus and Graminella nigrifrons virus 1. To our knowledge, this is the first successful isolation of iflavirus from a dipteran insect. Spherical ArIFV particles of approximately 30 nm in diameter contained at least three major structural proteins. ArIFV multiplied to high titres (~109 p.f.u. ml-1) and formed clear plaques in a mosquito cell line, C6/36. Our findings provide new insights into the infection mechanism, genetic diversity and evolution of the Iflaviridae family.

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.

Genome Sequence of a Novel Iflavirus from the Leafhopper Graminella nigrifrons.

A novel iflavirus was discovered from the transcriptome of the leafhopper Graminella nigrifrons. The assembled virus genome has 9,700 nucleotides (nt) and encodes a 3,035-amino-acid polyprotein. Sanger sequencing was used to confirm the sequence encoding the polyprotein and indicated a genome length of 9,617 nt with a polyadenylated tail.

Molecular interactions and immune responses between Maize fine streak virus and the leafhopper vector Graminella nigrifrons through differential expression and RNA interference.

Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: 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 G.nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G.nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults 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, and may provide approaches for developing novel targets of RNAi-based pest control.

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

BackgroundMost 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.MethodologyRNA-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/SignificanceFeeding 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.

Virus-independent and common transcriptome responses of leafhopper vectors feeding on maize infected with semi-persistently and persistent propagatively transmitted viruses

BackgroundInsects 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.ResultsRNA-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.ConclusionsWe 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.

Transcriptome of the Plant Virus Vector Graminella nigrifrons, and the Molecular Interactions of Maize fine streak rhabdovirus Transmission

BackgroundLeafhoppers (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.ResultsRNA 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.ConclusionsOur 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.

Identification of Graminella nigrifrons as a potential vector for phytoplasmas affecting Prunus and Pyrus species in Canada

Prunus and Pyrus species affected with phytoplasma diseases, as well as leafhopper species collected from Prunus and Pyrus fields in Ontario, Canada were tested for presence of phytoplasmas. Preliminary results showed that Graminella nigrifrons is a potential vector for phytoplasma groups 16SrI-W (‘Candidatus Phytoplasma asteris’), and 16SrVII-A (‘Candidatus Phytoplasma fraxini’) to a variety of plant hosts, including peach, apricot, plum and pear. Results showed that G. nigrifrons may be able to transmit both phytoplasma groups simultaneously within the same location and suggest that G. nigrifrons populations appear to have a complex ecology.

Plant Host Range and Leafhopper Transmission ofMaize fine streak virus

Maize fine streak virus (MFSV), an emerging Rhabdovirus sp. in the genus Nucleorhabdovirus, 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. nigrifrons. Parameters affecting efficiency of MFSV acquisition (infection) and transmission (inoculation) to maize were evaluated using single-leafhopper 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.

Spiroplasma-like organisms closely associated with the gut in five leafhopper species (Hemiptera: Cicadellidae)

Spiroplasmas are bacteria in the Class Mollicutes that are frequently associated with insects and/or plants. Here, we describe the ultrastructure, localization, and occurrence of apparent commensal/symbiotic spiroplasma-like organisms (SLOs) in the midgut and hindgut of five leafhopper species from laboratory-reared colonies. Those found in Dalbulus elimatus, Endria inimica, and Macrosteles quadrilineatus were long and tubular shaped, whereas those in Dalbulus maidis and Graminella nigrifrons were shorter and mostly rod-shaped in their host organisms. These SLOs were found in great numbers in the gut lumen frequently associated with the gut microvilli, but unlike the plant-pathogenic mollicutes, they did not seem to invade the gut epithelium or other tissues in any of these five leafhopper species. Large accumulations of these gut-associated organisms were more commonly found by confocal laser scanning microscopy in males than in females and in crowded than in singly reared leafhoppers. Ultrastructural evidence suggests that these SLOs may be horizontally transmitted between leafhoppers by contamination of the mouth parts with leafhopper excretions.

Identification of quantitative trait loci controlling resistance to maize chlorotic dwarf virus

Ineffective screening methods and low levels of disease resistance have hampered genetic analysis of maize (Zea mays L.) resistance to disease caused by maize chlorotic dwarf virus (MCDV). Progeny from a cross between the highly resistant maize inbred line Oh1VI and the susceptible inbred line Va35 were evaluated for MCDV symptoms after multiple virus inoculations, using the viral vector Graminella nigrifrons. Symptom severity scores from three rating dates were used to calculate area under the disease progress curve (AUDPC) scores for vein banding, leaf twist and tear, and whorl chlorosis. AUDPC scores for the F(2) population indicated that MCDV resistance was quantitatively inherited. Genotypic and phenotypic analyses of 314 F(2) individuals were compared using composite interval mapping (CIM) and analysis of variance. CIM identified two major quantitative trait loci (QTL) on chromosomes 3 and 10 and two minor QTL on chromosomes 4 and 6. Resistance was additive, with alleles from Oh1VI at the loci on chromosomes 3 and 10 contributing equally to resistance.

Accumulation of Maize chlorotic dwarf virus proteins in its plant host and leafhopper vector

The genome of Maize chlorotic dwarf virus (MCDV; genus Waikavirus; family Sequiviridae) consists of a monopartite positive-sense RNA genome encoding a single large polyprotein. Antibodies were produced to His-fusions of three undefined regions of the MCDV polyprotein: the N-terminus of the polyprotein (R78), a region between coat proteins (CPs) and the nucleotide-binding site (NBS) (R37), and a region between the NBS and a 3C-like protease (R69). The R78 antibodies react with proteins of 50 kDa (P50), 35 kDa (P35), and 25 kDa (P25) in virus preparations, and with P35 in plant extracts. In extracts of the leafhopper vector Graminella nigrifrons fed on MCDV-infected plants, the R78 antibodies reacted with P25 but not with P50 and P35. The R69 antibodies bound proteins of approximately 36 kDa (P36), 30 kDa (P30), and 26 kDa (P26) in virus preparations, and P36 and P26 in plant extracts. Antibodies to R37 reacted with a 26-kDa protein in purified virus preparations, but not in plant extracts. Neither the R69 nor the R37 antibodies bound any proteins in G. nigrifrons. Thus, in addition to the three CPs, cysteine protease and RNA-dependent RNA polymerase, the MCDV polyprotein is apparently post-transitionally cleaved into P50, P35, P25, P36, P30, and P26.

Effect of environmental conditions and leafhopper gender on Maize chlorotic dwarf virus transmission by Graminella nigrifrons (Homoptera: Cicadellidae).

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 Grarminella 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 degrees 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.

Maize fine streak virus, a New Leafhopper-Transmitted Rhabdovirus

ABSTRACT A previously uncharacterized virus was isolated from fall-planted sweet corn (Zea mays L., Syngenta GSS 0966) leaves showing fine chlorotic streaks. Symptomatic plants were negative in enzyme-linked immunosorbent assay against many maize viruses, but reacted weakly with antisera to Sorghum stunt mosaic virus suggesting a distant relationship between the viruses. The virus was readily transmitted by vascular puncture inoculation (VPI), but not by leaf-rub inoculation. Symptoms on maize included dwarfing and fine chlorotic streaks along intermediate and small veins that developed 12 to 17 days post-VPI. The isolated virus was bacilliform (231 +/- 5 nm long and 71 +/- 2 nm wide), with a knobby surface, and obvious helical structure typical of rhabdovirus morphology. Nucleorhabdovirus virions were observed by transmission electron microscopy of infected maize leaf tissue sections. Proteins unique to infected plants were observed in extracts of infected leaves, and the isolated virion contained three proteins with molecular masses 82 +/- 2, 50 +/- 3, and 32 +/- 2 kDa. Preliminary sequence analysis indicated the virus had similarity to members of the family Rhabdoviridae. The virus was transmitted by Graminella nigrifrons under persistent conditions. The data indicate the virus, provisionally designated Maize fine streak virus, is a new species in the genus Nucleorhabdovirus.

Regulation of Chorusing in the Vibrational Communication System of the LeafhopperGraminella nigrifrons

Male Graminella nigrifrons participate in alternating choruses. Vibrational calls emitted by males consist of three sections (S1, S2, and S3) that differ in pattern of amplitude modulation. In this study we examined the response of single males to synthetic choruses and to isolated call components to gain insight into the regulation of chorus structure. Males initiated calls primarily during the silent periods within synthetic choruses. In all 15 trials the number of overlapping calls and the duration of overlap was significantly less than expected if males call at random. Playback of S2, S3, or random noise while males emitted S1 caused males to interrupt calling, whereas males continued to call when S1 or no signal was played. In a related experiment, we played S2 or no signal while males were beginning to emit the S1, S2, or S3 phase of their calls. In response to this playback the duration of S1 and S3 was reduced, but the duration of S2 was not affected. These results suggest that an inhibitory-resetting mechanism may result in alternation of calls in this leafhopper.

Regulation of Chorusing in the Vibrational Communication System of the Leafhopper Graminella nigrifrons1

Male Graminella nigrifrons participate in alternating choruses. Vibrational calls emitted by males consist of three sections (S1, S2, and S3) that differ in pattern of amplitude modulation. In this study we examined the response of single males to synthetic choruses and to isolated call components to gain insight into the regulation of chorus structure. Males initiated calls primarily during the silent periods within synthetic choruses. In all 15 trials the number of overlapping calls and the duration of overlap was significantly less than expected if males call at random. Playback of S2, S3, or random noise while males emitted S1 caused males to interrupt calling, whereas males continued to call when S1 or no signal was played. In a related experiment, we played S2 or no signal while males were beginning to emit the S1, S2, or S3 phase of their calls. In response to this playback the duration of S1 and S3 was reduced, but the duration of S2 was not affected. These results suggest that an inhibitory-resetting mechanism may result in alternation of calls in this leafhopper.

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.

Quantification of Viral Disease Incidence in Corn (Zea mays) as Affected by Johnsongrass (Sorghum halepense) Control

Field and laboratory experiments were conducted to evaluate the effect of postemergence johnsongrass control on incidence of maize chlorotic dwarf virus (MCDV) and maize dwarf mosaic virus (MDMV) in corn and to confirm the presence and movement of the blackfaced leafhopper, the insect vector of MCDV. Corn plants surrounded by MCDV- and MDMV-infected rhizomatous johnsongrass were either treated or not treated with nicosulfuron at 35 g ai/ha. Corn tissue samples were taken at the time of treatment and 4, 9, 14, and 21 d after treatment and the presence of MCDV and MDMV was determined by enzyme-linked immunosorbent assay (ELISA). Virus incidence in treated experimental units was higher at the later sampling dates relative to the nontreated. Earlier differences in incidence of MCDV and MDMV double infection in corn were detected where johnsongrass was controlled. Movement of the insect vector of MCDV was observed within the experimental area after johnsongrass was controlled, but was not significantly different from that in nontreated areas.