A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots.

Dynamic transcriptome profiling of Bean Common Mosaic Virus (BCMV) infection in Common Bean (Phaseolus vulgaris L.).

BACKGROUND: Bean common mosaic virus (BCMV) is widespread, with Phaseolus species as the primary host plants. Numerous BCMV strains have been identified on the basis of a panel of bean varieties that distinguish the pathogenicity types with respect to the viral strains. The molecular responses in Phaseolus to BCMV infection have not yet been well characterized. RESULTS: We report the transcriptional responses of a widely susceptible variety of common bean (Phaseolus vulgaris L., cultivar 'Stringless green refugee') to two BCMV strains, in a time-course experiment. We also report the genome sequence of a previously unreported BCMV strain. The interaction with the known strain NL1-Iowa causes moderate symptoms and large transcriptional responses, and the newly identified strain (Strain 2 or S2) causes severe symptoms and moderate transcriptional responses. The transcriptional profiles of host plants infected with the two isolates are distinct, and involve numerous differences in splice forms in particular genes, and pathway specific expression patterns. CONCLUSIONS: We identified differential host transcriptome response after infection of two different strains of Bean common mosaic virus (BCMV) in common bean (Phaseolus vulgaris L.). Virus infection initiated a suite of changes in gene expression level and patterns in the host plants. Pathways related to defense, gene regulation, metabolic processes, photosynthesis were specifically altered after virus infection. Results presented in this study can increase the understanding of host-pathogen interactions and provide resources for further investigations of the biological mechanisms in BCMV infection and defense.

Positive and negative roles for soybean MPK6 in regulating defense responses.

It has been well established that MPK6 is a positive regulator of defense responses in model plants such as Arabidopsis and tobacco. However, the functional importance of soybean MPK6 in disease resistance has not been investigated. Here, we showed that silencing of GmMPK6 in soybean using virus-induced gene silencing mediated by Bean pod mottle virus (BPMV) caused stunted growth and spontaneous cell death on the leaves, a typical phenotype of activated defense responses. Consistent with this phenotype, expression of pathogenesis-related (PR) genes and the conjugated form of salicylic acid were significantly increased in GmMPK6-silenced plants. As expected, GmMPK6-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants, indicating a negative role of GmMPK6 in disease resistance. Interestingly, overexpression of GmMPK6, either transiently in Nicotiana benthamiana or stably in Arabidopsis, resulted in hypersensitive response (HR)-like cell death. The HR-like cell death was accompanied by increased PR gene expression, suggesting that GmMPK6, like its counterpart in other plant species, also plays a positive role in cell death induction and defense response. Using bimolecular fluorescence complementation analysis, we determined that GmMKK4 might function upstream of GmMPK6 and GmMKK4 could interact with GmMPK6 independent of its phosphorylation status. Taken together, our results indicate that GmMPK6 functions as both repressor and activator in defense responses of soybean.

Replication protein A subunit 3 and the iron efficiency response in soybean.

In soybean [Glycine max (L.) Merr.], iron deficiency results in interveinal chlorosis and decreased photosynthetic capacity, leading to stunting and yield loss. In this study, gene expression analyses investigated the role of soybean replication protein A (RPA) subunits during iron stress. Nine RPA homologs were significantly differentially expressed in response to iron stress in the near isogenic lines (NILs) Clark (iron efficient) and Isoclark (iron inefficient). RPA homologs exhibited opposing expression patterns in the two NILs, with RPA expression significantly repressed during iron deficiency in Clark but induced in Isoclark. We used virus induced gene silencing (VIGS) to repress GmRPA3 expression in the iron inefficient line Isoclark and mirror expression in Clark. GmRPA3-silenced plants had improved IDC symptoms and chlorophyll content under iron deficient conditions and also displayed stunted growth regardless of iron availability. RNA-Seq comparing gene expression between GmRPA3-silenced and empty vector plants revealed massive transcriptional reprogramming with differential expression of genes associated with defense, immunity, aging, death, protein modification, protein synthesis, photosynthesis and iron uptake and transport genes. Our findings suggest the iron efficient genotype Clark is able to induce energy controlling pathways, possibly regulated by SnRK1/TOR, to promote nutrient recycling and stress responses in iron deficient conditions.

The requirement of multiple defense genes in soybean Rsv1-mediated extreme resistance to soybean mosaic virus.

Soybean mosaic virus (SMV) is a major viral pathogen of soybean. Among the three SMV resistance genes, Rsv1 mediates extreme resistance (ER) against most SMV strains, including the ß-glucuronidase-tagged G2 isolate that was previously used in studies of Rsv1. Using virus-induced gene silencing (VIGS), we screened 82 VIGS constructs to identify genes that play a role in Rsv1-mediated ER to SMV infection. The target genes included putative Rsv1 candidate genes, soybean orthologs to known defense-signaling genes, and 62 WRKY transcription factors. We identified eight VIGS constructs that compromised Rsv1-mediated resistance when the target genes were silenced, including GmEDR1, GmEDS1, GmHSP90, GmJAR1, GmPAD4, and two WRKY transcription factors. Together, our results provide new insight into the soybean signaling network required for ER against SMV.

Soybean homologs of MPK4 negatively regulate defense responses and positively regulate growth and development.

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). However, the importance of MAPK signaling pathways in the disease resistance of crops is still largely uninvestigated. To better understand the role of MAPK signaling pathways in disease resistance in soybean (Glycine max), 13, nine, and 10 genes encoding distinct MAPKs, MAPKKs, and MAPKKKs, respectively, were silenced using virus-induced gene silencing mediated by Bean pod mottle virus. Among the plants silenced for various MAPKs, MAPKKs, and MAPKKKs, those in which GmMAPK4 homologs (GmMPK4s) were silenced displayed strong phenotypes including stunted stature and spontaneous cell death on the leaves and stems, the characteristic hallmarks of activated defense responses. Microarray analysis showed that genes involved in defense responses, such as those in salicylic acid (SA) signaling pathways, were significantly up-regulated in GmMPK4-silenced plants, whereas genes involved in growth and development, such as those in auxin signaling pathways and in cell cycle and proliferation, were significantly down-regulated. As expected, SA and hydrogen peroxide accumulation was significantly increased in GmMPK4-silenced plants. Accordingly, GmMPK4-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants. Using bimolecular fluorescence complementation analysis and in vitro kinase assays, we determined that GmMKK1 and GmMKK2 might function upstream of GmMPK4. Taken together, our results indicate that GmMPK4s negatively regulate SA accumulation and defense response but positively regulate plant growth and development, and their functions are conserved across plant species.

Functional analysis of the Asian soybean rust resistance pathway mediated by Rpp2.

Asian soybean rust is an aggressive foliar disease caused by the obligate biotrophic fungus Phakopsora pachyrhizi. On susceptible plants, the pathogen penetrates and colonizes leaf tissue, resulting in the formation of necrotic lesions and the development of numerous uredinia. The soybean Rpp2 gene confers resistance to specific isolates of P. pachyrhizi. Rpp2-mediated resistance limits the growth of the pathogen and is characterized by the formation of reddish-brown lesions and few uredinia. Using virus-induced gene silencing, we screened 140 candidate genes to identify those that play a role in Rpp2 resistance toward P. pachyrhizi. Candidate genes included putative orthologs to known defense-signaling genes, transcription factors, and genes previously found to be upregulated during the Rpp2 resistance response. We identified 11 genes that compromised Rpp2-mediated resistance when silenced, including GmEDS1, GmNPR1, GmPAD4, GmPAL1, five predicted transcription factors, an O-methyl transferase, and a cytochrome P450 monooxygenase. Together, our results provide new insight into the signaling and biochemical pathways required for resistance against P. pachyrhizi.

The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

Landscape epidemiology of bean pod mottle comovirus: molecular evidence of heterogeneous sources.

Bean pod mottle virus (BPMV) RNAs are grouped into subgroups (sgI and sgII). A BPMV partial diploid reassortant (IA-Di1) from the perennial Desmodium illinoense contained both RNA1 subgroups and an RNA1 recombinant. The RNA2 of IA-Di1 was characteristic of sgII. Additionally, ten BPMV isolates from a soybean field adjacent to the locality of IA-Di1 shared >98.5% nucleotide identity with RNA1 sgII of IA-Di1. The data demonstrate the co-existence of two differing consensus BPMV RNA1 subgroups in adjacent habitats and illustrate variation in virus genetic structure that can occur in a contiguous plant community.

Development and use of an efficient DNA-based viral gene silencing vector for soybean.

Virus-induced gene silencing (VIGS) is increasingly being used as a reverse genetics tool to study functions of specific plant genes. It is especially useful for plants, such as soybean, that are recalcitrant to transformation. Previously, Bean pod mottle virus (BPMV) was shown to be an effective VIGS vector for soybean. However, the reported BPMV vector requires in vitro RNA transcription and inoculation, which is not reliable or amenable to high-throughput applications. To increase the efficiency of the BPMV vector for soybean functional genomics, a DNA-based version was developed. Reported here is the construction of a Cauliflower mosaic virus 35S promoter-driven BPMV vector that is efficient for the study of soybean gene function. The selection of a mild rather than a severe BPMV strain greatly reduced the symptom interference caused by virus infection. The DNA-based BPMV vector was used to silence soybean homologues of genes involved in plant defense, translation, and the cytoskeleton in shoots and in roots. VIGS of the Actin gene resulted in reduced numbers of Soybean mosaic virus infection foci. The results demonstrate the utility of this new vector as an efficient tool for a wide range of applications for soybean functional genomics.

A sugarcane mosaic virus vector for gene expression in maize.

Zea mays L. ssp. mays (maize) is an important crop plant as well as model system for genetics and plant biology. The ability to select among different virus-based platforms for transient gene silencing or protein expression experiments is expected to facilitate studies of gene function in maize and complement experiments with stable transgenes. Here, we describe the development of a sugarcane mosaic virus (SCMV) vector for the purpose of protein expression in maize. An infectious SCMV cDNA clone was constructed, and heterologous genetic elements were placed between the protein 1 (P1) and helper component-proteinase (HC-Pro) cistrons in the SCMV genome. Recombinant SCMV clones engineered to express green fluorescent protein (GFP), ß-glucuronidase (GUS), or bialaphos resistance (BAR) protein were introduced into sweet corn (Golden × Bantam) plants. Documentation of developmental time courses spanning maize growth from seedling to tasseling showed that the SCMV genome tolerates insertion of foreign sequences of at least 1,809 nucleotides at the P1/HC-Pro junction. Analysis of insert stability showed that the integrity of GFP and BAR coding sequences was maintained longer than that of the much larger GUS coding sequence. The SCMV isolate from which the expression vector is derived is able to infect several important maize inbred lines, suggesting that this SCMV vector has potential to be a valuable tool for gene functional analysis in a broad range of experimentally important maize genotypes.

Identification and molecular characterization of two naturally occurring Soybean mosaic virus isolates that are closely related but differ in their ability to overcome Rsv4 resistance.

A naturally occurring Rsv4 resistance-breaking isolate (L-RB) and a closely related non-resistance-breaking isolate (L) of Soybean mosaic virus (SMV) were identified in soybean fields in London, Ontario, Canada. The viral genomes of L and L-RB were completely sequenced. Each isolate has a 9585-nucleotide genome with a single open reading frame encoding a polyprotein of approximately 350 kDa. L-RB and L have a very high sequence similarity (99.6%) at both the nucleotide and amino acid levels. Phylogenetic analysis showed that the two isolates belong to the G2 pathotype. Pathogenicity predictions of all virus/soybean combinations, based on the phylogenetic profile, were confirmed by pathogenicity tests using L and L-RB isolates and soybeans carrying different resistance genes, with an exception that L-RB infected a soybean cultivar carrying Rsv4 resistance. The temporal and spatial proximity of L and L-RB and their high sequence similarity suggest L-RB was likely derived from the SMV-L quasispecies. Recombination analysis did not reveal the evidence of genetic recombination for the emergence of L-RB. Mutations introduced by virus-encoded RNA-dependent RNA polymerase during viral genome replication and selection pressure probably contributed to the occurrence of L-RB.

Evaluation of management strategies for bean leaf beetles (Coleoptera: Chrysomelidae) and Bean pod mottle virus (Comoviridae) in soybean.

Cerotoma trifurcata Förster (Coleoptera: Chrysomelidae) and Bean pod mottle virus (Comoviridae) (BPMV) both can reduce yield and seed quality of soybean, Glycine max (L.) Merr. Field experiments were conducted to evaluate the effects of systemic, seed-applied, and foliar-applied insecticides for the management of this pest complex at three locations in central, northeastern, and northwestern Iowa during 2002-2004. Seed-applied insecticide was evaluated according to a currently recommended management program for Iowa (i.e., insecticide applications that target emerging overwintered beetles, F0, and the first seasonal generation, F1 ). The experimental treatments included seed-applied (thiamethoxam, 0.3-0.5 g [AI] kg(-1)] or clothianidin, 47.32 ml [AI] kg(-1)) and foliar-applied (A-cyhalothrin, 16.83-28.05 g [AI] ha(-1)) or esfenvalerate (43.74-54.69 g [AI] ha(-1)) insecticides. Applications of the foliar insecticides were timed to target F0, F1 or both F0 and F1 populations of C. trifurcata. Our results confirm that insecticides timed at F0 and F1 populations of C. trifurcata can reduce vector populations throughout the growing season, provide limited reduction in virus incidence, and improve both yield and seed coat color. Furthermore, seed-applied insecticides may be the more reliable option for an F0-targeted insecticide if used within this management strategy. An F0-targeted insecticide by itself only gave a yield improvement in one out of eight location-years. However, by adding an F1-targeted insecticide, there was a yield gain of 1.42-1.67 quintal ha(-1), based on contrast comparisons at three location-years.

The pathogenic development of Sclerotinia sclerotiorum in soybean requires specific host NADPH oxidases.

The plant membrane-localized NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), play crucial roles in various cellular activities, including plant disease responses, and are a major source of reactive oxygen species (ROS). Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen that causes Sclerotinia stem rot (SSR) in soybean. Via a key virulence factor, oxalic acid, it induces programmed cell death (PCD) in the host plant, a process that is reliant on ROS generation. In this study, using protein sequence similarity searches, we identified 17 soybean RBOHs (GmRBOHs) and studied their contribution to SSR disease development, drought tolerance and nodulation. We clustered the soybean RBOH genes into six groups of orthologues based on phylogenetic analysis with their Arabidopsis counterparts. Transcript analysis of all 17 GmRBOHs revealed that, of the six identified groups, group VI (GmRBOH-VI) was specifically and drastically induced following S. sclerotiorum challenge. Virus-induced gene silencing (VIGS) of GmRBOH-VI using Bean pod mottle virus (BPMV) resulted in enhanced resistance to S. sclerotiorum and markedly reduced ROS levels during disease development. Coincidently, GmRBOH-VI-silenced plants were also found to be drought tolerant, but showed a reduced capacity to form nodules. Our results indicate that the pathogenic development of S. sclerotiorum in soybean requires the active participation of specific host RBOHs, to induce ROS and cell death, thus leading to the establishment of disease.

Potential for Integrated Management of Soybean Virus Disease.

The recent introduction of the colonizing soybean aphid (Aphis glycines) to soybean in the northern United States has raised concern for potential increased disease caused by the nonpersistently aphid-transmitted Soybean mosaic virus (SMV). This study was conducted to examine the potential integration of host plant resistance and insecticide tactics for control of virus disease. Research from four location-years demonstrated that foliar application of the pyrethroid insecticide lambda-cyhalothrin (Warrior) or the organophosphate chlorpyrifos (Lorsban 4E) timed to suppress soybean aphid populations does not reduce SMV. Therefore, the introduction of a colonizing aphid to the array of migratory noncolonizing aphids that transmit SMV does not result in potential for disease control through vector suppression by foliar insecticides. Treatment also did not result in management of Bean pod mottle virus (BPMV), transmitted by the bean leaf beetle (Cerotoma trifurcata), presumably because of issues related to different phenologies of the insect vectors. Soybean cultivars with the lowest virus titer in seed produced the highest grain yield and, thus, were rated as field tolerant compared with cultivars with the highest virus titer in seed. Host plant resistance, not vector control, is the most effective tactic to control SMV.

No-choice preference of cerotoma trifurcata (coleoptera: chrysomelidae) to potential host plants of bean pod mottle virus (Comoviridae) in Iowa.

To better understand the naturally occurring host range of Bean pod mottle virus (family Comoviridae, genus Comovirus, BPMV) and its principal vector Cerotoma trifurcata (Förster) (Coleoptera: Chrysomelidae), 18 field-collected perennial plant species were tested for the presence of BPMV. By using no-choice assays, we determined the preference of these plants by bean leaf beetle, by measuring their level of herbivory relative to soybean, Glycine max (L.). New food hosts for adult bean leaf beetles include Lespedeza capitata (Michaux), Lotus corniculatus L., Trifolium alexandrinum L., Trifolium ambiguum Bieberstein, and Trifolium incarnatum L. Desmodium illinoense Gray is discovered as a new naturally occurring host for BPMV.

Potential Primary Inoculum Sources of Bean pod mottle virus in Iowa.

A survey of Bean pod mottle virus (BPMV) in Iowa counties was conducted and the virus was found throughout the state. A long-term monitoring study (1989 to 2002) of the main BPMV vector, the bean leaf beetle, Cerotoma trifurcata, indicated that, in 2002, populations reached the highest abundance recorded in 14 years. Three potential sources for an early season primary inoculum source were found: (i) soybean (Glycine max) seed, (ii) overwintered bean leaf beetles, and (iii) alternate BPMV host plants. Examination of 5,804 and 8,064 soybean seedlings of two cultivars yielded 0 and 3 seedlings, respectively, infected with BPMV. In a separate test, BPMV was detected in mottled and nonmottled soybean seed. Some mottled seed did not contain BPMV, indicating that soybean seed coat mottling is an unreliable indicator for presence of the virus in seed. Of 194 naturally overwintered bean leaf beetles, only 1 transmitted BPMV to soybean. BPMV was detected serologically only in 1 alternate host, Desmodium canadense, out of 23 naturally occurring plant species collected from the field. The three inoculum sources discovered in Iowa in this study could be important primary sources when vector populations are high and indicate starting points for future epidemiological investigations.

Bean leaf beetle (Coleoptera: Chrysomelidae) management for reduction of bean pod mottle virus.

Bean pod mottle virus (BPMV) is a management concern for soybean, Glycine max (L.), producers in the North Central states because it can cause yield loss and reduce seed quality by induction of seed coat mottling. The main vector of BPMV is the bean leaf beetle, Cerotoma trifurcata (Forster). An experiment was conducted in 2000 and 2001 at two locations in northwestern and central Iowa to test three insecticide treatments for suppression of bean leaf beetles, and subsequently, BPMV. Treatments of insecticide applications with lambda-cyhalothrin were 1) a single early-season application (23 g [AI] /ha) (2.5 oz/acre) at the VE-VC soybean developmental stage; 2) two early-season applications, the first the same as treatment 1 and a second at the same rate 9-13 d later; 3) a single early-season application the same as treatment 1, followed by a mid-season application (28 g [AI] /ha (3.2 oz/acre) at approximately R2 (flowering, near 15 July); and 4) an unsprayed control. Application of lambda-cyhalothrin after soybean emergence and again as first-generation bean leaf beetles emerged in northwestern Iowa in 2000 (treatment 3) significantly reduced beetle densities through mid-season, BPMV field incidence by 31.5%, and seed coat mottling by 31.2%, compared with the unsprayed control. Similar effects were measured at the same location when insecticide was applied twice at early season (treatment 2). Yield was 453.7 kg/ha (6.74 bu/acre) greater in treatment 2 and 525.20 kg/ha (7.80 bu/acre) greater in treatment 3 than in the unsprayed control at the northwestern site in 2000. At both locations in 2001 fewer treatment effects were observed, which was likely related to lower beetle populations in that year. Early-season insecticide sprays targeted at overwintered beetles on VC-VE reduced the initial population of vector insects and may have contributed to a lower first-generation population because of reduced overwintered beetle oviposition. In 1 year at one location there was a benefit to an additional mid-season insecticide spray, although effectiveness of spraying at this time could vary based on the magnitude of the vector population.

Control of virus diseases in soybeans.

Soybean, one of the world's most important sources of animal feed and vegetable oil, can be infected by numerous viruses. However, only a small number of the viruses that can potentially infect soybean are considered as major economic problems to soybean production. Therefore, we consider management options available to control diseases caused by eight viruses that cause, or have the potential to cause, significant economic loss to producers. We summarize management tactics in use and suggest direction for the future. Clearly, the most important tactic is disease resistance. Several resistance genes are available for three of the eight viruses discussed. Other options include use of virus-free seed and avoidance of alternative virus hosts when planting. Attempts at arthropod vector control have generally not provided consistent disease management. In the future, disease management will be considerably enhanced by knowledge of the interaction between soybean and viral proteins. Identification of genes required for soybean defense may represent key regulatory hubs that will enhance or broaden the spectrum of basal resistance to viruses. It may be possible to create new recessive or dominant negative alleles of host proteins that do not support viral functions but perform normal cellular function. The future approach to virus control based on gene editing or exploiting allelic diversity points to necessary research into soybean-virus interactions. This will help to generate the knowledge needed for rational design of durable resistance that will maximize global production.

Virus-induced gene silencing in soybean and common bean.

Plant viral vectors are useful for transient gene expression as well as for downregulation of gene expression via virus-induced gene silencing (VIGS). When used in reverse genetics approaches, VIGS offers a convenient way of transforming genomic information into knowledge of gene function. Efforts to develop and improve plant viral vectors have expanded their applications and have led to substantial advances needed to facilitate gene function studies in major row crops. Here, we describe a DNA-based Bean pod mottle virus (BPMV) vector system for both gene expression and VIGS in soybean and common bean.