Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation.

Despite their economic significance in agricultural cropping systems, a lack of suitable molecular tools for manipulating gene expression has hindered progress in the functional genomics of plant parasitic nematodes (PPN). Obligate sexual reproduction and the obligate nature of PPN-host interactions further complicate the development of in vivo gene delivery and expression systems in these pests. Methods such as microinjection and microprojectile bombardment have been developed for introducing gene constructs into the free-living nematode, Caenorhabditis elegans. However, these procedures can be laborious and inefficient. Electroporation has been used extensively to introduce macromolecules, including single-stranded RNAs, into eukaryotic and prokaryotic cells. The technique has also been used for the delivery of DNA and double-stranded RNA constructs into nematodes by whole-animal electroporation. Here, we describe methods for the expression of a nematode-optimized NanoLuc luciferase mRNA in the form of in vitro transcripts following whole-animal electroporation of Heterodera glycines, Meloidogyne incognita, and C. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management.

Discovery of a Novel Member of the Carlavirus Genus from Soybean (Glycine max L. Merr.).

A novel member of the Carlavirus genus, provisionally named soybean carlavirus 1 (SCV1), was discovered by RNA-seq analysis of randomly collected soybean leaves in Illinois, USA. The SCV1 genome contains six open reading frames that encode a viral replicase, triple gene block proteins, a coat protein (CP) and a nucleic acid binding protein. The proteins showed highest amino acid sequence identities with the corresponding proteins of red clover carlavirus A (RCCVA). The predicted amino acid sequence of the SCV1 replicase was only 60.6% identical with the replicase of RCCVA, which is below the demarcation criteria for a new species in the family Betaflexiviridae. The predicted replicase and CP amino acid sequences of four SCV1 isolates grouped phylogenetically with those of members of the Carlavirus genus in the family Betaflexiviridae. The features of the encoded proteins, low nucleotide and amino acid sequence identities of the replicase with the closest member, and the phylogenetic grouping suggest SCV1 is a new member of the Carlavirus genus.

Soybean Thrips (Thysanoptera: Thripidae) Harbor Highly Diverse Populations of Arthropod, Fungal and Plant Viruses.

Soybean thrips (Neohydatothrips variabilis) are one of the most efficient vectors of soybean vein necrosis virus, which can cause severe necrotic symptoms in sensitive soybean plants. To determine which other viruses are associated with soybean thrips, the metatranscriptome of soybean thrips, collected by the Midwest Suction Trap Network during 2018, was analyzed. Contigs assembled from the data revealed a remarkable diversity of virus-like sequences. Of the 181 virus-like sequences identified, 155 were novel and associated primarily with taxa of arthropod-infecting viruses, but sequences similar to plant and fungus-infecting viruses were also identified. The novel viruses were predicted to have positive-sense RNA, negative-stranded RNA, double-stranded RNA, and single-stranded DNA genomes. The assembled sequences included 100 contigs that represented at least 95% coverage of a virus genome or genome segment. Sequences represented 12 previously described arthropod viruses including eight viruses reported from Hubei Province in China, and 12 plant virus sequences of which six have been previously described. The presence of diverse populations of plant viruses within soybean thrips suggests they feed on and acquire viruses from multiple host plant species that could be transmitted to soybean. Assessment of the virome of soybean thrips provides, for the first time, information on the diversity of viruses present in thrips.

Aphis glycines virus 1, a new bicistronic virus with two functional internal ribosome entry sites, is related to a group of unclassified viruses in the Picornavirales.

A novel picorna-like virus, provisionally named Aphis glycines virus 1 (ApGlV1) was discovered by high-throughput sequencing of soybean total RNAs and detected in suction trap-collected Aphis glycines. The ApGlV1 genome contains two large ORFs organized similar to those of dicipiviruses in the Picornaviridae where ORFs 1 and 2 encode structural and nonstructural proteins, respectively. Both ORFs are preceded by internal ribosome entry site (IRES) elements. The 5' IRES was more active in dual luciferase activity assays than the IRES in the intergenic region. The ApGlV1 genome was predicted to encode a serine protease instead of a cysteine protease and showed very low aa sequence identities to recognized members of the Picornavirales. In phylogenetic analyses based on capsid protein and RNA-dependent RNA polymerase sequences, ApGlV1 consistently clustered with a group of unclassified bicistronic picorna-like viruses discovered from arthropods and plants that may represent a novel family in the order Picornavirales.

Strain-specific association of soybean dwarf virus small subgenomic RNA with virus particles.

Soybean dwarf virus (SbDV) produces a large subgenomic RNA (LsgRNA) for expression of structural and movement proteins and a small subgenomic RNA (SsgRNA) that does not contain an open reading frame. Sucrose gradient-purified SbDV virions from soybean plants systemically infected with SbDV by aphids and Nicotiana benthamiana leaves agroinfiltrated with infectious clones of two red clover SbDV isolates encapsidated genomic RNA and were associated with SsgRNA in a strain-specific manner. The LsgRNA was protected from RNase degradation, but not packaged into virions as indicated by its presence primarily in ELISA-negative fractions near the tops of sucrose gradients even in mutants that did not express coat protein. Nucleotide differences in the SsgRNA region between isolates conferred differential association of SsgRNA with virions.

Molecular characterization of a new soybean-infecting member of the genus Nepovirus identified by high-throughput sequencing.

The complete nucleotide sequence of a new soybean-infecting member of the genus Nepovirus (provisionally named "soybean latent spherical virus" [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788 nt) were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3' nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus.

Transfection of Sclerotinia sclerotiorum with in vitro transcripts of a naturally occurring interspecific recombinant of Sclerotinia sclerotiorum hypovirus 2 significantly reduces virulence of the fungus.

UNLABELLED: A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5'- and 3'-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5' terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related to Valsa ceratosperma hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strain Sclerotinia sclerotiorum hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession number KF898354. Sclerotinia sclerotiorum isolate 328 was coinfected with a strain of Sclerotinia sclerotiorum endornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in S. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized in vitro and transfected into a virus-free isolate of S. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L. IMPORTANCE: A cosmopolitan fungus, Sclerotinia sclerotiorum infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in S. sclerotiorum were lacking. By establishing a cause-and-effect relationship between Sclerotinia sclerotiorum hypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and S. sclerotiorum, a widespread fungal pathogen of plants.

A novel flavivirus in the soybean cyst nematode.

Heterodera glycines, the soybean cyst nematode (SCN), is a subterranean root pathogen that causes the most damaging disease of soybean in the USA. A novel nematode virus genome, soybean cyst nematode virus 5 (SbCNV-5), was identified in RNA sequencing data from SCN eggs and second-stage juveniles. The SbCNV-5 RNA-dependent RNA polymerase and RNA helicase domains had homology to pestiviruses in the family Flaviviridae, suggesting that SbCNV-5 is a positive-polarity ssRNA virus. SbCNV-5 RNA was present in all nematode developmental stages, indicating a transovarial mode of transmission, but is also potentially sexually transmitted via the male. SbCNV-5 was common in SCN laboratory cultures and in nematode populations isolated from the field. Transmission electron microscopy of sections from a female SCN showed virus particles budding from the endoplasmic reticulum and in endosomes. The size of the viral genome was 19 191 nt, which makes it much larger than other known pestiviruses. Additionally, the presence of a methyltransferase in the SbCNV-5 genome is atypical for a pestivirus. When cDNA sequences were mapped to the genome of SbCNV-5, a disproportionate number aligned to the 3' NTR, suggesting that SbCNV-5 produces a subgenomic RNA, which was confirmed by RNA blot analysis. As subgenomic RNAs and methyltransferases do not occur in pestiviruses, we conclude that SbCNV-5 is a new flavivirus infecting SCNs.

Identification of novel double-stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence.

Virulence and double-stranded RNA (dsRNA) profiles of 44 isolates of Fusarium virguliforme were compared. When grouped according to dsRNA profiles, isolates with large dsRNAs were significantly (P≤0.05) less virulent than isolates without dsRNAs. High-throughput sequence analysis of total RNA prepared from cultures with large dsRNAs identified two novel RNA viruses with genome sequences of approximately 9.3 kbp, which were named Fusarium virguliforme dsRNA mycovirus 1 and Fusarium virguliforme dsRNA mycovirus 2. The new viruses were most closely related to a group of unclassified viruses that included viruses of F. graminearum and Phlebiopsis gigantea and are related to members of the family Totiviridae.

Multiple loci condition seed transmission of soybean mosaic virus (SMV) and SMV-induced seed coat mottling in soybean.

Infection of soybean plants with Soybean mosaic virus (SMV), which is transmitted by aphids and through seed, can cause significant reductions in seed production and quality. Because seedborne infections are the primary sources of inoculum for SMV infections in North America, host-plant resistance to seed transmission can limit the pool of plants that can serve as sources of inoculum. To examine the inheritance of SMV seed transmission in soybean, crosses were made between plant introductions (PIs) with high (PI88799), moderate (PI60279), and low (PI548391) rates of transmission of SMV through seed. In four F(2) populations, SMV seed transmission segregated as if conditioned by two or more genes. Consequently, a recombinant inbred line population was derived from a cross between PIs 88799 and 548391 and evaluated for segregation of SMV seed transmission, seed coat mottling, and simple sequence repeat markers. Chromosomal regions on linkage groups C1 and C2 were significantly associated with both transmission of isolate SMV 413 through seed and SMV-induced seed coat mottling, and explained ≈42.8 and 46.4% of the variability in these two traits, respectively. Chromosomal regions associated with seed transmission and seed coat mottling contained homologues of Arabidopsis genes DCL3 and RDR6, which encode enzymes involved in RNA-mediated transcriptional and posttranscriptional gene silencing.

Soybean mosaic virus Helper Component-Protease Alters Leaf Morphology and Reduces Seed Production in Transgenic Soybean Plants.

ABSTRACT Transgenic soybean (Glycine max) plants expressing Soybean mosaic virus (SMV) helper component-protease (HC-Pro) showed altered vegetative and reproductive phenotypes and responses to SMV infection. When inoculated with SMV, transgenic plants expressing the lowest level of HC-Pro mRNA and those transformed with the vector alone initially showed mild SMV symptoms. Plants that accumulated the highest level of SMV HC-Pro mRNA showed very severe SMV symptoms initially, but after 2 weeks symptoms disappeared, and SMV titers were greatly reduced. Analysis of SMV RNA abundance over time with region-specific probes showed that the HC-Pro region of the SMV genome was degraded before the coat protein region. Transgenic soybean plants that expressed SMV HC-Pro showed dose-dependent alterations in unifoliate leaf morphologies and seed production where plants expressing the highest levels of HC-Pro had the most deformed leaves and the lowest seed production. Accumulation of microRNAs (miRNAs) and mRNAs putatively targeted by miRNAs was analyzed in leaves and flowers of healthy, HC-Pro-transgenic, and SMV-infected plants. Neither expression of SMV HC-Pro nor SMV infection produced greater than twofold changes in accumulation of six miRNAs. In contrast, SMV infection was associated with twofold or greater increases in the accumulation of four of seven miRNA-targeted mRNAs tested.

Similarities in Seed and Aphid Transmission Among Soybean mosaic virus Isolates.

Soybean mosaic virus (SMV) is an aphid- and seed-transmitted virus that infects soybean (Glycine max) plants and causes significant yield losses. Seed-borne infections are the primary sources of inoculum for SMV infections. The strain specificity of SMV transmission through seed and SMV-induced seed-coat mottling were investigated in field experiments. Six soybean plant introductions (PIs) were inoculated with eight SMV strains and isolates. Transmission of SMV through seed ranged from 0 to 43%, and isolate-by-soybean line interactions occurred in both transmission rates and percentages of mottled seeds. For example, SMV 746 was transmitted through 43% of seed in PI 229324, but was not transmitted through seed of PIs 68522, 68671, or 86449. In contrast, SMV 413 was transmitted through seed from all PIs. SMVs that were transmitted poorly by the Asian soybean aphid, Aphis glycines, also were transmitted poorly through seed. No predicted amino acid sequences within the helper-component protease or coat protein coding regions differentiated the two groups of SMV strains. The loss of aphid and seed transmissibility by repeated mechanical transmission suggests that constant selection pressure is needed to maintain the regions of the SMV genome controlling the two phenotypes from genetic drift and loss of function.

In vivo activity of Rhopalosiphum padi virus internal ribosome entry sites.

The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transcription and translation of plasmid templates containing the IRESs. In this system, the two RhPV IRESs directed initiation of translation from bicistronic plasmids with equal efficiency. Competition was observed between the two IRESs when they were in cis in a bicistronic plasmid. A mutation that disrupted the 3'-proximal pseudoknot of the intergenic (IG) IRES reduced translation initiation in vivo. Similarly, mutations in the RhPV IG IRES disrupted its ability to bind 80S particles in vitro. The two IRESs preferentially labelled proteins of different masses in UV cross-linking experiments, illustrating the different translation initiation mechanisms employed by the two elements.

Nucleotide sequence shows that Bean leafroll virus has a Luteovirus-like genome organization.

The complete nucleotide sequence of the Bean leafroll virus (BLRV) genomic RNA and the termini of its smallest subgenomic RNAs were determined to better understand its mechanisms of gene expression and replication and its phylogenetic position within the Luteoviridae: The number and placement of open reading frames (ORFs) within the BLRV genome was Luteovirus-like. The nucleotide and predicted amino acid sequences of BLRV were most similar to those of Soybean dwarf virus (SbDV). Phylogenetic analyses employing the neighbour-joining method and sister-scanning analysis indicated that the BLRV nonstructural proteins were closely related to those of Barley yellow dwarf virus-PAV (BYDV-PAV), a luteovirus: The region surrounding the frameshift at the junction between ORFs 1 and 2 also contained sequences very similar to those of BYDV-PAV and a Dianthovirus, Red clover necrotic mosaic virus. Similar analyses showed that the structural proteins were most similar to those of the Polerovirus genus. The 3'-noncoding regions downstream of ORF5 contained sequences similar to translational control elements identified in the BYDV-PAV genome. These data suggest that BLRV, like SbDV, is derived either through selection from a common ancestor with BYDV-PAV or that BLRV is the product of two recombination events between luteovirus-like and polerovirus-like ancestors where the 5' 2900 nt and 3' 700 nt of the BLRV genome are from a Luteovirus and the intervening sequences are derived from a Polerovirus: