Wendy M. Havens

An Oleic Acid–Mediated Pathway Induces Constitutive Defense Signaling and Enhanced Resistance to Multiple Pathogens in Soybean

Stearoyl-acyl carrier protein-desaturase (SACPD)-catalyzed synthesis of oleic acid (18:1) is an essential step in fatty acid biosynthesis. Arabidopsis mutants (ssi2) with reduced SACPD activity accumulate salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens. We show that reduced levels of 18:1 induce similar defense-related phenotypes in soybean. A Bean pod mottle virus (BPMV)-based vector was employed to effectively silence soybean SACPDs. The silenced plants contained reduced 18:1 and increased stearic acid, developed spontaneous cell death lesions, increased SA accumulation, and constitutively expressed pathogenesis-related genes. These plants also expressed elevated levels of resistance-like genes and showed resistance to bacterial and oomycete pathogens. Exogenous application of glycerol induced similar phenotypes, mimicking the effect of silencing SACPDs in healthy soybean plants. Overexpression of a soybean SACPD increased 18:1 levels in ssi2 but not in wild-type Arabidopsis plants, suggesting that the soybean enzyme was under feedback regulation similar to that of the Arabidopsis isozymes. These results suggest that soybean and Arabidopsis respond similarly to 18:1-derived cues by inducing a novel broad-spectrum resistance-conferring pathway, even though they differ significantly in their lipid biosynthetic pathways. We also demonstrate the efficacy of BPMV-induced gene silencing as a tool for functional studies in soybean.

Structure of Flexible Filamentous Plant Viruses

ABSTRACT Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.

Atomic structure reveals the unique capsid organization of a dsRNA virus.

For most dsRNA viruses, the genome-enclosing capsid comprises 120 copies of a single capsid protein (CP) organized into 60 icosahedrally equivalent dimers, generally identified as 2 nonsymmetricallyinteracting CP molecules with extensive lateral contacts. The crystal structure of a partitivirus, Penicillium stoloniferum virus F (PsV-F), reveals a different organization, in which the CP dimer is related by almost-perfect local 2-fold symmetry, forms prominent surface arches, and includes extensive structure swapping between the 2 subunits. An electron cryomicroscopy map of PsV-F shows that the disordered N terminus of each CP molecule interacts with the dsRNA genome and probably participates in its packaging or transcription. Intact PsV-F particles mediate semiconservative transcription, and transcripts are likely to exit through negatively charged channels at the icosahedral 5-fold axes. Other findings suggest that the PsV-F capsid is assembled from dimers of CP dimers, with an arrangement similar to flavivirus E glycoproteins.

RNA Sequence Determinants of a Coupled Termination-Reinitiation Strategy for Downstream Open Reading Frame Translation in Helminthosporium victoriae Virus 190S and Other Victoriviruses (Family Totiviridae)

ABSTRACT The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORFs stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.

The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication

ABSTRACT Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 Å resolution. The capsid protein has a high content of rod-like densities characteristic of α-helices, forming a repeated α-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the α-helical core resembles that found in the capsid protein of the L-A virus, a fungal totivirus with an undivided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells; whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability.

Overexpression of GmCaM4 in soybean enhances resistance to pathogens and tolerance to salt stress.

Plant diseases inflict heavy losses on soybean yield, necessitating an understanding of the molecular mechanisms underlying biotic/abiotic stress responses. Ca(2) (+) is an important universal messenger, and protein sensors, prominently calmodulins (CaMs), recognize cellular changes in Ca(2) (+) in response to diverse signals. Because the development of stable transgenic soybeans is laborious and time consuming, we used the Bean pod mottle virus (BPMV)-based vector for rapid and efficient protein expression and gene silencing. The present study focuses on the functional roles of the gene encoding the soybean CaM isoform GmCaM4. Overexpression of GmCaM4 in soybean resulted in enhanced resistance to three plant pathogens and increased tolerance to high salt conditions. To gain an understanding of the underlying mechanisms, we examined the potential defence pathways involved. Our studies revealed activation/increased expression levels of pathogenesis-related (PR) genes in GmCaM4-overexpressing plants and the accumulation of jasmonic acid (JA). Silencing of GmCaM4, however, markedly repressed the expression of PR genes. We confirmed the in vivo interaction between GmCaM4 and the CaM binding transcription factor Myb2, which regulates the expression of salt-responsive genes, using the yeast two-hybrid (Y2H) system and bimolecular fluorescence complementation assays. GmCaM4 and Glycine max CaM binding receptor-like kinase (GmCBRLK) did not interact in the Y2H assays, but the interaction between GmCaM2 and GmCBRLK was confirmed. Thus, a GmCaM2-GmCBRLK-mediated salt tolerance mechanism, similar to that reported in Glycine soja, may also be functional in soybean. Confocal microscopy showed subcellular localization of the green fluorescent protein (GFP)-GmCaM4 fusion protein in the nucleus and cytoplasm.

Structure of Fusarium poae virus 1 shows conserved and variable elements of partitivirus capsids and evolutionary relationships to picobirnavirus

Filamentous fungus Fusarium poae is a worldwide cause of the economically important disease Fusarium head blight of cereal grains. The fungus is itself commonly infected with a bisegmented dsRNA virus from the family Partitiviridae. For this study, we determined the structure of partitivirus Fusarium poae virus 1 (FpV1) to a resolution of 5.6Å or better by electron cryomicroscopy and three-dimensional image reconstruction. The main structural features of FpV1 are consistent with those of two other fungal partitiviruses for which high-resolution structures have been recently reported. These shared features include a 120-subunit T=1 capsid comprising 60 quasisymmetrical capsid protein dimers with both shell and protruding domains. Distinguishing features are evident throughout the FpV1 capsid, however, consistent with its more massive subunits and its greater phylogenetic divergence relative to the other two structurally characterized partitiviruses. These results broaden our understanding of conserved and variable elements of fungal partitivirus structure, as well as that of vertebrate picobirnavirus, and support the suggestion that a phylogenetic subcluster of partitiviruses closely related to FpV1 should constitute a separate taxonomic genus.

The Helminthosporium victoriae 190S mycovirus has two forms distinguishable by capsid protein composition and phosphorylation state.

Purified preparations of the Helminthosporium victoriae 190S (Hv190S) virus contain two sedimenting components that differ in capsid structure. The slower sedimenting component (190S-1) contained p88 and p83 as the major capsid proteins; the faster component (190S-2) contained p88 and p78. Previous peptide-mapping studies have shown the three capsid proteins to be closely related. Analysis by SDS-PAGE of in vivo-radiolabeled Hv190S virions indicated that 32P was predominantly incorporated in p88. Significantly less was detected in p83 and none in p78. Similar results were obtained in in vitro phosphorylation studies using [gamma-32P]ATP and purified 190S-1 and 190S-2. The in vitro results suggest that the Hv190S virions copurify with a protein kinase activity that catalyzes the transfer of gamma-phosphate from ATP to a target protein, presumably p78 in the 190S-2 virions and p83 in the 190S-1 component. Selective chemical cleavage at tryptophan residues of in vitro 32P-labeled capsid proteins revealed four labeled peptides among the cleavage products of both p83 and p88. Radioiodination studies with intact Hv190S virions indicated that p88 and p83, but not the nonphosphorylated p78, were accessible to iodination, suggesting that capsid protein phosphorylation entailed conformational changes.

Backbone Trace of Partitivirus Capsid Protein from Electron Cryomicroscopy and Homology Modeling

Most dsRNA viruses have a genome-enclosing capsid that comprises 120 copies of a single coat protein (CP). These 120 CP subunits are arranged as asymmetrical dimers that surround the icosahedral fivefold axes, forming pentamers of dimers that are thought to be assembly intermediates. This scheme is violated, however, in recent structures of two dsRNA viruses, a fungal virus from family Partitiviridae and a rabbit virus from family Picobirnaviridae, both of which have 120 CP subunits organized as dimers of quasisymmetrical dimers. In this study, we report the CP backbone trace of a second fungal partitivirus, determined in this case by electron cryomicroscopy and homology modeling. This virus also exhibits quasisymmetrical CP dimers that are connected by prominent surface arches and stabilized by domain swapping between the two CP subunits. The CP fold is dominated by alpha-helices, although beta-strands mediate several important contacts. A dimer-of-dimers assembly intermediate is again implicated. The disordered N-terminal tail of each CP subunit protrudes into the particle interior and likely interacts with the genome during packaging and/or transcription. These results broaden our understanding of conserved and variable aspects of partitivirus structure and reflect the growing use of electron cryomicroscopy for atomic modeling of protein folds.

Disease Phenotype of Virus-Infected Helminthosporium victoriae Is Independent of Overexpression of the Cellular Alcohol Oxidase/RNA-Binding Protein Hv-p68

ABSTRACT The cellular protein Hv-p68 is a novel alcohol oxidase/RNA-binding protein that is overexpressed in virus-infected isolates of the plant-pathogenic fungus Helminthosporium victoriae (teleomorph: Cochliobolus victoriae). Overproduction of Hv-p68 has been hypothesized to lead to the accumulation of toxic aldehydes and to induce the disease phenotype associated with the virus-infected isolates. We overexpressed the Hv-p68 gene in virus-free isolates and evaluated the morphology of the resulting colonies. We cloned and sequenced the Hv-p68 genomic DNA, which contains five introns and the complete Hv-p68 coding sequence. Vectors for overexpression of the Hv-p68 gene were constructed with either Hv-p68 cDNA or the intron-containing Hv-p68 genomic DNA. Expression of Hv-p68 was significantly higher if the genomic sequence was used for transformation than if the cDNA sequence was used. The virus-free fungal transformants that overexpressed Hv-p68 gene did not exhibit the disease phenotype. In contrast, these transformants showed enhanced growth rates when compared with the nontransformed and empty vector controls. Interestingly, overexpression of Hv-p68 in a fungal isolate infected with both the totivirus Helminthosporium victoriae 190S virus (Hv190SV) and the chrysovirus Helminthosporium victoriae 145S virus (Hv145S) showed enhanced accumulation of the Hv145SV double-stranded (ds)RNA, but not of the Hv190SV. These results are consistent with an earlier report that Hv-p68 co-purified with viral dsRNA, mainly that of the Hv145SV. Elucidation of the role of Hv-p68 in disease induction is important for an understanding of host-virus interactions in this fungus-virus system.