Experimental host and vector ranges of the emerging maize yellow mosaic polerovirus.

Maize yellow mosaic virus (MaYMV) is an emerging polerovirus that has been detected in maize, other cereal crops and weedy grass species in Asia, Africa, and the Americas. Disease symptoms in maize include prominent leaf tip reddening and stunting. Infection by MaYMV has been reported to reduce plant growth and yields by 10-30% in some instances. In this study, an experimental host range for MaYMV among agronomically important cereal crops and common grasses was established. Additional aphid species were assessed as potential vectors for MaYMV and their transmission efficiencies were determined. Here we report oats, foxtail millet, barley, and rye as new experimental cereal crop hosts of MaYMV in addition to confirming the previously reported hosts of corn, sorghum, wheat, and broom millet. Four of the nine other grass species evaluated were also identified as suitable experimental hosts for MaYMV: ryegrass, switchgrass, green foxtail, and sand love grass. Interestingly, no visible symptoms were present in any of the infected hosts besides the susceptible maize control. Vector range studies identified the greenbug aphid, Schizaphis graminum, as a new vector of MaYMV, though transmission efficiency was lower than the previously reported Rhopalosiphum maidis vector and similar to the other known aphid vector, R. padi. Given MaYMV's global ubiquity, ability to evade detection, and broad host range, further characterization of yield impacts and identification of viable control strategies are desirable.

Coat protein expression strategy of maize rayado fino virus and evidence for requirement of CP1 for leafhopper transmission.

Marafiviruses, including maize rayado fino virus (MRFV) and oat blue dwarf virus (OBDV), encode two carboxy co-terminal coat proteins, CP1 and CP2, which encapsidate the genome to form icosahedral virions. While CP2 expression is expected to be solely driven from a second start codon of a subgenomic RNA under a marafibox promoter sequence, the larger CP1 with an in-frame N-terminal extension relative to CP2 could potentially be expressed either by proteolytic release from the MRFV polyprotein or from subgenomic RNA translation. We examined MRFV CP expression strategy with a series of mutations in the CP coding region and identified mutants viable and nonviable for systemic plant infection. Polyprotein expression of MRFV CP1 was minimal. Mutants blocking CP2 expression failed to establish systemic infection, while mutants depleted in CP1 exhibited systemic infection and formation of virus-like particles but lost leafhopper transmissibility, indicating that CP1 is required for leafhopper transmission.

Simultaneous gene expression and multi-gene silencing in Zea mays using maize dwarf mosaic virus.

BACKGROUND: Maize dwarf mosaic virus (MDMV), a member of the genus Potyvirus, infects maize and is non-persistently transmitted by aphids. Several plant viruses have been developed as tools for gene expression and gene silencing in plants. The capacity of MDMV for both gene expression and gene silencing were examined. RESULTS: Infectious clones of an Ohio isolate of MDMV, MDMV OH5, were obtained, and engineered for gene expression only, and for simultaneous marker gene expression and virus-induced gene silencing (VIGS) of three endogenous maize target genes. Single gene expression in single insertion constructs and simultaneous expression of green fluorescent protein (GFP) and silencing of three maize genes in a double insertion construct was demonstrated. Constructs with GFP inserted in the N-terminus of HCPro were more stable than those with insertion at the N-terminus of CP in our study. Unexpectedly, the construct with two insertion sites also retained insertions at a higher rate than single-insertion constructs. Engineered MDMV expression and VIGS constructs were transmissible by aphids (Rhopalosiphum padi). CONCLUSIONS: These results demonstrate that MDMV-based vector can be used as a tool for simultaneous gene expression and multi-gene silencing in maize.

First report of cDNA clone-launched infection of maize plants with the polerovirus maize yellow mosaic virus (MaYMV).

An East African isolate of the maize-associated polerovirus, maize yellow mosaic virus (MaYMV) was previously shown to cause leaf reddening on singly infected maize plants (Zea mays). Here we describe the construction of a full-length infectious clone of an East African isolate and, for the first time, show infectivity of clone-derived transcripts in the primary host, maize, through vascular puncture inoculation (VPI), as well as in the dicotyledonous research model plant species, Nicotiana benthamiana, through agrobacterium inoculation. Characteristic leaf reddening symptoms were observed in a subset of maize plants inoculated with clone-derived transcripts, and infection was confirmed by RT-PCR and Northern blot analyses. In N. benthamiana plants, infections were entirely asymptomatic even at high virus titers, as was also reported for the cloned Chinese isolate. In this study, however, we demonstrated that N. benthamiana can serve as a clone launching platform for maize infection, as VPI of sap of infected N. benthamiana plants into maize kernels resulted in infection and the typical red leaf symptoms. We further demonstrated that the cloned East African isolate virus was aphid transmissible to maize, with experimental transmission rates up to 97 %, comparable to that shown previously for the native virus. Interestingly, our data additionally showed a definitive correlation of leaf reddening symptoms with increased expression of chalcone synthase, thus suggesting upregulation of the flavonoid biosynthesis pathway as the molecular basis for symptom induction in maize. As the first report of experimental infection of maize with transcripts from a cloned polerovirus, this work constitutes a breakthrough for studies on molecular maize-polerovirus-aphid interactions.

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.

A Recently Discovered Maize Polerovirus Causes Leaf Reddening Symptoms in Several Maize Genotypes and is Transmitted by Both the Corn Leaf Aphid (Rhopalosiphum maidis) and the Bird Cherry-Oat Aphid (Rhopalosiphum padi).

A maize-infecting polerovirus variously named maize yellow dwarf virus RMV2 (MYDV-RMV2) and maize yellow mosaic virus (MaYMV) has been discovered and previously described in East Africa, Asia, and South America. It was identified in virus surveys in these locations instigated by outbreaks of maize lethal necrosis (MLN), known to be caused by coinfections of unrelated maize chlorotic mottle virus (MCMV) and any of several maize-infecting potyviruses, and was often found in coinfections with MLN viruses. Although sequenced in many locations globally and named for symptoms of related or coinfecting viruses, and with an infectious clone reported that experimentally infects Nicotiana benthamiana, rudimentary biological characterization of MaYMV in maize, including insect vector(s) and symptoms in single infections, has not been reported until now. We report isolation from other viruses and leaf tip reddening symptoms in several maize genotypes, along with transmission by two aphids, Rhopalosiphum padi and Rhopalosiphum maidis. This is important information distinguishing this virus and demonstrating that in single infections it causes symptoms distinct from those of potyviruses or MCMV in maize, and identification of vectors provides an important framework for determination of potential disease impact and management.