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.

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.

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.

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.

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.

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.

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.

Maize lethal necrosis viruses and other maize viruses in Rwanda.

Maize lethal necrosis (MLN) is emergent in East Africa, first reported in 2011 in Kenya, and is devastating to maize production in the region. MLN is caused by coinfection of maize with the emergent maize chlorotic mottle virus (MCMV) and any of several maize-infecting potyviruses endemic in East Africa and worldwide. Here, we examined the distribution of MCMV and sugarcane mosaic virus (SCMV), the major viruses contributing to MLN in Rwanda. These and other viruses in maize across Rwanda were further characterized by deep sequencing. When identified, MCMV had high titres and minimal sequence variability, whereas SCMV showed moderate titres and high sequence variability. Deep sequencing also identified maize streak virus and other maize-associated viruses, including a previously described polerovirus, maize yellow mosaic virus, and barley yellow dwarf virus, diverse maize-associated totiviruses, maize-associated pteridovirus, Zea mays chrysovirus 1, and a maize-associated betaflexivirus. Detection of each virus was confirmed in maize samples by reverse transcription polymerase chain reaction.

Maize Lethal Necrosis: An Emerging, Synergistic Viral Disease.

Maize lethal necrosis (MLN) is a disease of maize caused by coinfection of maize with maize chlorotic mottle virus (MCMV) and one of several viruses from the Potyviridae, such as sugarcane mosaic virus, maize dwarf mosaic virus, Johnsongrass mosaic virus or wheat streak mosaic virus. The coinfecting viruses act synergistically to result in frequent plant death or severely reduce or negligible yield. Over the past eight years, MLN has emerged in sub-Saharan East Africa, Southeast Asia, and South America, with large impacts on smallholder farmers. Factors associated with MLN emergence include multiple maize crops per year, the presence of maize thrips ( Frankliniella williamsi), and highly susceptible maize crops. Soil and seed transmission of MCMV may also play significant roles in development and perpetuation of MLN epidemics. Containment and control of MLN will likely require a multipronged approach, and more research is needed to identify and develop the best measures.

The Role of Viruses in the Phytobiome.

Viruses are an important but sequence-diverse and often understudied component of the phytobiome. We succinctly review current information on how plant viruses directly affect plant health and physiology and consequently have the capacity to modulate plant interactions with their biotic and abiotic environments. Virus interactions with other biota in the phytobiome, including arthropods, fungi, and nematodes, may also impact plant health. For example, viruses interact with and modulate the interface between plants and insects. This has been extensively studied for insect-vectored plant viruses, some of which also infect their vectors. Other viruses have been shown to alter the impacts of plant-interacting phytopathogenic and nonpathogenic fungi and bacteria. Viruses that infect nematodes have also recently been discovered, but the impact of these and phage infecting soil bacteria on plant health remain largely unexplored.

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.

Complete Genome Sequence of a New Maize-Associated Cytorhabdovirus.

A new 11,877-nucleotide cytorhabdovirus sequence with 6 open reading frames has been identified in a maize sample. It shares 50 and 51% genome-wide nucleotide sequence identity with northern cereal mosaic cytorhabdovirus and barley yellow striate mosaic cytorhabdovirus, respectively.

Identification of a maize chlorotic dwarf virus silencing suppressor protein.

Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389kDa proteolytically processed polyprotein. We show that the N-terminal 78kDa polyprotein (R78) of MCDV acts as a suppressor of RNA silencing in a well-established assay system. We further demonstrate that R78 is cleaved by the viral 3C-like protease into 51 and 27kDa proteins (p51 and p27), and that p51 is responsible for silencing suppressor activity. Silencing suppressor activity of R78 is conserved in three divergent MCDV strains (MCDV-Severe, MCDV-M1, and MCDV-Tennessee), as well as the waikavirus Bellflower vein chlorosis virus, but was not detected for orthologous protein of Rice tungro spherical virus (RTSV-A) or the similarly-positioned protein from the sequivirus Parsnip yellow fleck virus (PYFV). This is the first identification of a virus suppressor of RNA silencing encoded by a waikavirus.

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.