Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

Acibenzolar-S-methyl-mediated restriction of loading of plantago asiatica mosaic virus into vascular tissues of Nicotiana benthamiana.

Long-distance movement via vascular tissues is an essential step for systemic infection by plant viruses. We previously reported that pre-treatment of Nicotiana benthamiana with acibenzolar-S-methyl (ASM) both suppressed the accumulation of plantago asiatica mosaic virus (PlAMV) in inoculated leaves and delayed the long-distance movement to uninoculated upper leaves. These two effects occurred independently of each other. However, it remained unclear where and when the viral long-distance movement is inhibited upon ASM treatment. In this study, we found that ASM treatment restricted the loading of GFP-expressing PlAMV (PlAMV-GFP) into vascular tissues in the inoculated leaves. This led to delays in viral translocation to the petiole and the main stem, and to untreated upper leaves. We used cryohistological fluorescence imaging to show that ASM treatment affected the viral localization and reduced its accumulation in the phloem, xylem, and mesophyll tissues. A stem girdling experiment, which blocked viral movement downward through phloem tissues, demonstrated that ASM treatment could inhibit viral systemic infection to upper leaves, which occurred even with viral downward movement restricted. Taken together, our results showed that ASM treatment affects the loading of PlAMV-GFP into the vascular system in the inoculated leaf, and that this plays a key role in the ASM-mediated delay of viral long-distance movement.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Two Novel Endornaviruses Co-infecting a Phytophthora Pathogen of Asparagus officinalis Modulate the Developmental Stages and Fungicide Sensitivities of the Host Oomycete.

Two novel endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) were found in isolates of a Phytophthora pathogen of asparagus collected in Japan. A molecular phylogenetic analysis indicated that PEV2 and PEV3 belong to the genus Alphaendornavirus. The PEV2 and PEV3 genomes consist of 14,345 and 13,810 bp, and they contain single open reading frames of 4,640 and 4,603 codons, respectively. Their polyproteins contain the conserved domains of an RNA helicase, a UDP-glycosyltransferase, and an RNA-dependent RNA polymerase, which are conserved in other alphaendornaviruses. PEV2 is closely related to Brown algae endornavirus 2, whereas PEV3 is closely related to Phytophthora endornavirus 1 (PEV1), which infects a Phytophthora sp. specific to Douglas fir. PEV2 and PEV3 were detected at high titers in two original Phytophthora sp. isolates, and we found a sub-isolate with low titers of the viruses during subculture. We used the high- and low-titer isolates to evaluate the effects of the viruses on the growth, development, and fungicide sensitivities of the Phytophthora sp. host. The high-titer isolates produced smaller mycelial colonies and much higher numbers of zoosporangia than the low-titer isolate. These results suggest that PEV2 and PEV3 inhibited hyphal growth and stimulated zoosporangium formation. The high-titer isolates were more sensitive than the low-titer isolate to the fungicides benthiavalicarb-isopropyl, famoxadone, and chlorothalonil. In contrast, the high-titer isolates displayed lower sensitivity to the fungicide metalaxyl (an inhibitor of RNA polymerase I) when compared with the low-titer isolate. These results indicate that persistent infection with PEV2 and PEV3 may potentially affect the fungicide sensitivities of the host oomycete.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Acibenzolar-S-Methyl Restricts Infection of Nicotiana benthamiana by Plantago Asiatica Mosaic Virus at Two Distinct Stages.

Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PlAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator-mediated virus restriction.

Magnaporthe oryzae chrysovirus 1 strain D confers growth inhibition to the host fungus and exhibits multiform viral structural proteins.

A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1-4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis.

N-terminal region of cysteine-rich protein (CRP) in carlaviruses is involved in the determination of symptom types.

Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine-rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N-terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole-plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX-expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N-terminal region was replaced with the corresponding region of another CRP, suggested that the N-terminal region of carlavirus CRPs determined the exhibited symptom types. The up-regulation of a plant gene upp-L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.

Characterization of Magnaporthe oryzae chrysovirus 1 structural proteins and their expression in Saccharomyces cerevisiae.

Magnaporthe oryzae chrysovirus 1 (MoCV1), which is associated with an impaired growth phenotype of its host fungus, harbors four major proteins: P130 (130 kDa), P70 (70 kDa), P65 (65 kDa), and P58 (58 kDa). N-terminal sequence analysis of each protein revealed that P130 was encoded by double-stranded RNA1 (dsRNA1) (open reading frame 1 [ORF1] 1,127 amino acids [aa]), P70 by dsRNA4 (ORF4; 812 aa), and P58 by dsRNA3 (ORF3; 799 aa), although the molecular masses of P58 and P70 were significantly smaller than those deduced for ORF3 and ORF4, respectively. P65 was a degraded form of P70. Full-size proteins of ORF3 (84 kDa) and ORF4 (85 kDa) were produced in Escherichia coli. Antisera against these recombinant proteins detected full-size proteins encoded by ORF3 and ORF4 in mycelia cultured for 9, 15, and 28 days, and the antisera also detected smaller degraded proteins, namely, P58, P70, and P65, in mycelia cultured for 28 days. These full-size proteins and P58 and P70 were also components of viral particles, indicating that MoCV1 particles might have at least two forms during vegetative growth of the host fungus. Expression of the ORF4 protein in Saccharomyces cerevisiae resulted in cytological changes, with a large central vacuole associated with these growth defects. MoCV1 has five dsRNA segments, as do two Fusarium graminearum viruses (FgV-ch9 and FgV2), and forms a separate clade with FgV-ch9, FgV2, Aspergillus mycovirus 1816 (AsV1816), and Agaricus bisporus virus 1 (AbV1) in the Chrysoviridae family on the basis of their RdRp protein sequences.

Resistant and susceptible responses in tomato to cyst nematode are differentially regulated by salicylic acid.

To understand the machinery underlying a tomato cultivar harboring the Hero A gene against cyst nematode using microarrays, we first analyzed tomato gene expression in response to potato cyst nematode (PCN; Globodera rostochiensis) during the early incompatible and compatible interactions at 3 and 7 days post-inoculation (dpi). Transcript levels of the phenylalanine ammonia lyase (PAL) and Myb-related genes were up-regulated at 3 dpi in the incompatible interaction. Transcription of the genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) was also up-regulated at 3 dpi in the incompatible interaction. On the other hand, the four genes (PAL, Myb, PDC and ADH) were down-regulated in the compatible interaction at 3 dpi. When the expression levels of several pathogenesis-related (PR) protein genes in tomato roots were compared between the incompatible and compatible interactions, the salicylic acid (SA)-dependent PR genes were found to be induced in the incompatible interaction at 3 dpi. The PR-1(P4) transcript increased to an exceptionally high level at 3 dpi in the cyst nematode-infected resistant plants compared with the uninoculated controls. The free SA levels were elevated to similar levels in both incompatible and compatible interactions. We then confirmed that PR-1(P4) was not significantly induced in the NahG tomato harboring the Hero A gene, compared with the resistant cultivar. We thus found that PR-1(P4) was a hallmark for the cultivar resistance conferred by Hero A against PCN and that nematode parasitism resulted in the inhibition of the SA signaling pathway in the susceptible cultivars.