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.

ORF43 of maize rayado fino virus is dispensable for systemic infection of maize and transmission by leafhoppers.

Maize rayado fino virus (MRFV) possesses an open reading frame (ORF43) predicted to encode a 43 kDa protein (p43) that has been postulated to be a viral movement protein. Using a clone of MRFV (pMRFV-US) from which infectious RNA can be produced, point mutations were introduced to either prevent initiation from three potential AUG initiation codons near the 5'-end of ORF43 or prematurely terminate translation of ORF43. Inoculation of maize seed via vascular puncture inoculation (VPI) resulted in plants exhibiting symptoms typical of MRFV infection for all mutants tested. Furthermore, corn leafhoppers (Dalbulus maidis) transmitted the virus mutants to healthy plants at a frequency similar to that for wild-type MRFV-US. Viral RNA recovered from plants infected with mutants both prior to and after leafhopper transmission retained mutations blocking ORF43 expression. The results indicate that ORF43 of MRFV is dispensable for both systemic infection of maize and transmission by leafhoppers.

Feeding Behavior of Soybean Aphid (Hemiptera: Aphididae) Biotype 2 on Resistant and Susceptible Soybean.

Host plant resistance to the soybean aphid, Aphis glycines Matsumura, is an effective means of controlling populations of this introduced pest species in the United States. Rag (Resistance to Aphis glycines) genes identified in soybean germplasm have been incorporated into commercial cultivars, but differential responses by soybean aphid biotypes to the Rag genes have made understanding mechanisms underlying resistance associated with Rag genes increasingly important. We compared the behavior of biotype 2 aphids on the resistant soybean line PI243540, which is a source of Rag2, and the susceptible cultivar Wyandot. Scanning electron microscopy revealed that the abaxial surface of leaves from resistant plants had a higher density of both long and glandulartrichomes, which might repel aphids, on veins. Time-lapse animation also suggested a repellent effect of resistant plants on aphids. However, electropenatography (EPG) indicated that the time to first probe did not differ between aphids feeding on the resistant and susceptible lines. EPG also indicated that fewer aphids feeding on resistant plants reached the phloem, and the time before reaching the phloem was much longer relative to susceptible soybean. For aphids that reached the phloem, there was no difference in either number of feedings or their duration in phloem. However, aphids feeding on resistant soybean had fewer prolonged phases of active salivation (E1) and many more pathway activities and non-probing intervals. Together, the feeding behavior of aphids suggested that Rag2 resistance has strong antixenosis effects, in addition to previously reported antibiosis, and was associated with epidermal and mesophyll tissues.

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.

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.

Soybean Aphid (Hemiptera: Aphididae) Feeding Behavior is Largely Unchanged by Soybean Mosaic Virus but Significantly Altered by the Beetle-Transmitted Bean Pod Mottle Virus.

The soybean aphid (Aphis glycines Matsumura) is an economically important invasive pest of soybean. In addition to damage caused by soybean aphid feeding on the phloem sap, this insect also transmits many plant viruses, including soybean mosaic virus (SMV). Previous work has shown that plant viruses can change plant host phenotypes to alter the behavior of their insect vectors to promote virus spread, known as the vector manipulation hypothesis. In this study, we used electropenetography (EPG) to examine the effects of two plant viruses on soybean aphid feeding behavior: SMV, which is transmitted by many aphid species including the soybean aphid, and bean pod mottle virus (BPMV), which is transmitted by chrysomelid and some coccinellid beetles but not aphids. These two viruses often co-occur in soybean production and can act synergistically. Surprisingly, our results showed little to no effect of SMV on soybean aphid feeding behaviors measured by EPG, but profound differences were observed in aphids feeding on BPMV-infected plants. Aphids took longer to find the vascular bundle of BPMV-infected plants, and once found, spent more time entering and conditioning the phloem than ingesting phloem sap. Interestingly, these observed alterations are similar to those of aphids feeding on insect-resistant soybean plants. The cause of these changes in feeding behavior is not known, and how they impact virus transmission and soybean aphid populations in the field will require further study.

Transcriptome of the Maize Leafhopper (Dalbulus maidis) and Its Transcriptional Response to Maize Rayado Fino Virus (MRFV), Which It Transmits in a Persistent, Propagative Manner.

The corn leafhopper (Dalbulus maidis) is an important vector of maize rayado fino virus (MRFV), a positive-strand RNA (+ssRNA) marafivirus which it transmits in a persistent propagative manner. The interaction of D. maidis with MRFV, including infection of the insect and subsequent transmission to new plants, is not well understood at the molecular level. To examine the leafhopper-virus interaction, a D. maidis transcriptome was assembled and differences in transcript abundance between virus-exposed and naive D. maidis were examined at two time points (4 h and 7 days) post exposure to MRFV. The D. maidis transcriptome contained 56,116 transcripts generated from 1,727,369,026 100-nt paired-end reads from whole adult insects. The transcriptome of D. maidis shared highest identity and most orthologs with the leafhopper Graminella nigrifrons (65% of transcripts had matches with E values of <10-5) versus planthoppers Sogatella furcifera (with 23% of transcript matches below the E value cutoff) and Peregrinus maidis (with 21% transcript matches below the E value cutoff), as expected based on taxonomy. D. maidis expressed genes in the Toll, Imd, and Jak/Stat insect immune signaling pathways, RNA interference (RNAi) pathway genes, prophenoloxidase-activating system pathways, and immune recognition protein-encoding genes such as peptidoglycan recognition proteins (PGRPs), antimicrobial peptides, and other effectors. Statistical analysis (performed by R package DESeq2) identified 72 transcripts at 4 h and 67 at 7 days that were significantly responsive to MRFV exposure. Genes expected to be favorable for virus propagation, such as protein synthesis-related genes and genes encoding superoxide dismutase, were significantly upregulated after MRFV exposure. IMPORTANCE The transcriptome of the corn leafhopper, D. maidis, revealed conserved biochemical pathways for immunity and discovered transcripts responsive to MRFV-infected plants at two time points, providing a basis for functional identification of genes that either limit or promote the virus-vector interaction. Compared to other hopper species and the propagative plant viruses they transmit, D. maidis shared 15 responsive transcripts with S. furcifera (to southern rice black-streaked dwarf virus [SRBSDV]), one with G. nigrifrons (to maize fine streak virus [MFSV]), and one with P. maidis (to maize mosaic virus [MMV]), but no virus-responsive transcripts identified were shared among all four hopper vector species.

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.

Plant host range and leafhopper transmission of maize 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.

Propranolol treatment for hemangioma of infancy: risks and recommendations.

Hemangioma of infancy is a condition that may be associated with significant morbidity. While evidence most supports the use of corticosteroids, there is no well-defined or Federal Drug Administration (FDA)-approved systemic therapy for hemangioma of infancy. All currently used treatments have significant risks. Dramatic improvement of complicated hemangioma of infancy to propranolol was recently reported, but details for initiating therapy, monitoring, and potential risks were not included. We present two infants treated with propranolol, who suffered complications and propose a treatment protocol to minimize potential adverse events.