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.

Maize Yellow Mosaic Virus Interacts with Maize Chlorotic Mottle Virus and Sugarcane Mosaic Virus in Mixed Infections, But Does Not Cause Maize Lethal Necrosis.

A maize-infecting polerovirus, variously named maize yellow dwarf virus RMV2 (MYDV RMV2), MYDV-like, and maize yellow mosaic virus (MaYMV), is frequently found in mixed infections in plants also infected with maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV), known to synergistically cause maize lethal necrosis (MLN). MaYMV was discovered in deep sequencing studies precipitated by recent MLN emergence and is prevalent at global locations with MLN, but its role in or contribution to disease was not known. We examined how MaYMV impacted disease development in mixed infections with MCMV, SCMV, and both MCMV and SCMV compared with mock-inoculated plants. Results demonstrated that MaYMV symptoms included stunting as well as leaf reddening in single and mixed infections. MaYMV did not recapitulate MLN synergistic disease in double infections in which either MCMV or SCMV was missing (MaYMV + MCMV or MaYMV + SCMV), but did significantly enhance stunting in mixed infections and suppressed titers of both MCMV and SCMV in double infections. Interestingly, MaYMV strongly suppressed the SCMV-induced titer increase of MCMV in triple infections, but MLN symptoms still occurred with the reduced MCMV titer. These data indicate the potential disease impact of this newly discovered ubiquitous maize virus, alone and in the context of MLN.

Fundamental Aspects of Plant Viruses-An Overview on Focus Issue Articles.

Given the importance of and rapid research progress in plant virology in recent years, this Focus Issue broadly emphasizes advances in fundamental aspects of virus infection cycles and epidemiology. This Focus Issue comprises three review articles and 18 research articles. The research articles cover broad research areas on the identification of novel viruses, the development of detection methods, reverse genetics systems and functional genomics for plant viruses, vector and seed transmission studies, viral population studies, virus-virus interactions and their effect on vector transmission, and management strategies of viral diseases. The three review articles discuss recent developments in application of prokaryotic clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) technology for plant virus resistance, mixed viral infections and their role in disease synergism and cross-protection, and viral transmission by whiteflies. The following briefly summarizes the articles appearing in this Focus Issue.

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.

Barley stripe mosaic virus (BSMV) as a virus-induced gene silencing vector in maize seedlings.

Barley stripe mosaic virus (BSMV) was the first reported and still widely used virus-induced gene silencing (VIGS) vector for monocotyledons including wheat and barley. Despite BSMV's reported infectivity on maize (Zea mays), the use of the virus as a vector in maize has not been optimized. Here, we assayed infectivity of BSMV in different maize cultivars by vascular puncture inoculation. Through knockdown of the endogenous host phytoene desaturase gene, we demonstrate for the first time that BSMV can be used as a VIGS vector in maize. This adds BSMV to the repertoire of tools available for functional studies in maize.

Complete sequence and diversity of a maize-associated Polerovirus in East Africa.

Since 2011-2012, Maize lethal necrosis (MLN) has emerged in East Africa, causing massive yield loss and propelling research to identify viruses and virus populations present in maize. As expected, next generation sequencing (NGS) has revealed diverse and abundant viruses from the family Potyviridae, primarily sugarcane mosaic virus (SCMV), and maize chlorotic mottle virus (MCMV) (Tombusviridae), which are known to cause MLN by synergistic co-infection. In addition to these expected viruses, we identified a virus in the genus Polerovirus (family Luteoviridae) in 104/172 samples selected for MLN or other potential virus symptoms from Kenya, Uganda, Rwanda, and Tanzania. This polerovirus (MF974579) nucleotide sequence is 97% identical to maize-associated viruses recently reported in China, termed 'maize yellow mosaic virus' (MaYMV) and maize yellow dwarf virus (MaYMV; KU291101, KU291107, MYDV-RMV2; KT992824); and 99% identical to MaYMV (KY684356) infecting sugarcane and itch grass in Nigeria; 83% identical to a barley-associated polerovirus recently identified in Korea (BVG; KT962089); and 79% identical to the U.S. maize-infecting polerovirus maize yellow dwarf virus (MYDV-RMV; KT992824). Nucleotide sequences from ORF0 of 20 individual East African isolates collected from Kenya, Uganda, Rwanda, and Tanzania shared 98% or higher identity, and were detected in 104/172 (60.5%) of samples collected for virus-like symptoms, indicating extensive prevalence but limited diversity of this virus in East Africa. We refer to this virus as "MYDV-like polerovirus" until symptoms of the virus in maize are known.

Johnsongrass mosaic virus Contributes to Maize Lethal Necrosis in East Africa.

Maize lethal necrosis (MLN), a severe virus disease of maize, has emerged in East Africa in recent years with devastating effects on production and food security where maize is a staple subsistence crop. In extensive surveys of MLN-symptomatic plants in East Africa, sequences of Johnsongrass mosaic virus (JGMV) were identified in Uganda, Kenya, Rwanda, and Tanzania. The East African JGMV is distinct from previously reported isolates and infects maize, sorghum, and Johnsongrass but not wheat or oat. This isolate causes MLN in coinfection with Maize chlorotic mottle virus (MCMV), as reported for other potyviruses, and was present in MLN-symptomatic plants in which the major East African potyvirus, Sugarcane mosaic virus (SCMV), was not detected. Virus titers were compared in single and coinfections by quantitative reverse transcription-polymerase chain reaction. MCMV titer increased in coinfected plants whereas SCMV, Maize dwarf mosaic virus, and JGMV titers were unchanged compared with single infections at 11 days postinoculation. Together, these results demonstrate the presence of an East African JGMV that contributes to MLN in the region.

Soil-borne wheat mosaic virus infectious clone and manipulation for gene-carrying capacity.

A full-length infectious cDNA clone of soil-borne wheat mosaic virus (SBWMV; genus Furovirus; family Virgaviridae) was developed for agrobacterium delivery. The cloned virus can be agroinfiltrated to Nicotiana benthamiana for subsequent infection of wheat (Triticum aestivum, L.). The utility of the virus as a vector for gene silencing and expression was assessed through sequence insertions in multiple sites of RNA2. Virus-induced photobleaching was observed in N. benthamiana but not in wheat, despite the stability of the inserts. The SBWMV infectious clone can be used for further studies to investigate the biology of SBWMV through mutagenesis.

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.

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 Lethal Necrosis (MLN), an Emerging Threat to Maize-Based Food Security in Sub-Saharan Africa.

In sub-Saharan Africa, maize is a staple food and key determinant of food security for smallholder farming communities. Pest and disease outbreaks are key constraints to maize productivity. In September 2011, a serious disease outbreak, later diagnosed as maize lethal necrosis (MLN), was reported on maize in Kenya. The disease has since been confirmed in Rwanda and the Democratic Republic of Congo, and similar symptoms have been reported in Tanzania, Uganda, South Sudan, and Ethiopia. In 2012, yield losses of up to 90% resulted in an estimated grain loss of 126,000 metric tons valued at $52 million in Kenya alone. In eastern Africa, MLN was found to result from coinfection of maize with Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), although MCMV alone appears to cause significant crop losses. We summarize here the results of collaborative research undertaken to understand the biology and epidemiology of MLN in East Africa and to develop disease management strategies, including identification of MLN-tolerant maize germplasm. We discuss recent progress, identify major issues requiring further research, and discuss the possible next steps for effective management of MLN.

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

BACKGROUND: Insects 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. RESULTS: RNA-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. CONCLUSIONS: We 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.

Co-infection and Disease Severity of Ohio Maize dwarf mosaic virus and Maize chlorotic dwarf virus Strains.

Two major maize viruses have been reported in the United States: Maize dwarf mosaic virus (MDMV) and Maize chlorotic dwarf virus (MCDV). These viruses co-occur in regions where maize is grown, such that co-infections are likely. Co-infection of different strains of MCDV is also observed, and a synergistic enhancement of symptoms in co-infected plants was previously reported. Here, we examined the impact of co-infections of two strains of MCDV (MCDV-S and MCDV-M1, severe and mild, respectively), and co-infections of MCDV and MDMV in the sweet corn hybrid 'Spirit' in greenhouse experiments. Quantitative plant growth and development parameters were measured and virus accumulation was measured by reverse-transcriptase quantitative polymerase chain reaction. Virus symptoms were enhanced and plants showed no recovery over time in co-infections of MDMV-OH and MCDV-S but virus titers and quantitative growth parameters did not indicate synergy in co-infected plants. MCDV-M1 co-infections with either MDMV-OH or MCDV-S did not show symptom enhancement or evidence of synergism.

Global phylogenetic analysis of soybean dwarf virus isolates and their associations with aphid vectors and severe disease in soybeans.

Soybean dwarf virus (SbDV) was first described in Japan as an agent of severe soybean disease transmitted by the foxglove aphid, Aulacorthum solani, with separable yellowing (Y) and dwarfing (D) strains. SbDV of both Y and D genotypes were later documented in other countries. For three decades, SbDV isolates were assessed to evaluate risk to U.S. soybean production. U.S. SbDV isolates were transmitted by the pea aphid Acyrthosiphum pisum and showed limited disease in soybeans, suggesting it was not a major threat to U.S. soybean production. Here we report 21 new full-length SbDV genome sequences including those of the originally described Japanese Y and D isolates, isolates from Syria and New Zealand associated with severe disease, and 17 isolates from U.S. field collections. Using these new full-length genomes, a global phylogeny was assembled and used to revisit risk assessment based on sequence similarities, isolate pathogenicity, and vector specificity.

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.

Critical residues for proteolysis activity of maize chlorotic dwarf virus (MCDV) 3C-like protease and comparison of activity of orthologous waikavirus proteases.

Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined.

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.

A mutation in the Lettuce infectious yellows virus minor coat protein disrupts whitefly transmission but not in planta systemic movement.

The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.

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.

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.