A novel toti-like virus from a plant pathogenic oomycete Globisporangium splendens.

We investigated virus infection in the plant pathogenic oomycete Globisporangium splendens, formerly classified as Pythium splendens, in Japan. From 12 strains investigated, three strains contained virus-like double-stranded (dsRNA). Next-generation sequencing revealed that the G. splendens strain MAFF 425508 and MAFF 305867 contained a virus related to toti-like viruses, that we named Pythium splendens RNA virus 1 (PsRV1). PsRV1 has a ca. 5700 nt-length genome encoding two overlapping open reading frames (ORFs). The ORF2 encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis with deduced RdRp amino acid sequences indicated that PsRV1 was closely related to Pythium polare RNA virus 1 (PpRV1) from G. polare infecting mosses in the Arctic. PsRV1 was vertically transmitted through the hyphal swellings, vegetative organs of G. splendens, in a temperature-dependent manner. Also, we showed that PsRV1 infected in a symptomless manner.

A novel non-segmented double-stranded RNA virus from an Arctic isolate of Pythium polare.

We investigated virus infection in the oomycete Pythium polare from the Arctic. From 39 isolates investigated, 14 contained virus-like double-stranded RNA (dsRNA). Next generation sequencing revealed that the P. polare isolate OPU1176 contained three different virus-like sequences. We determined the full-length genome sequence of one of them. The 5397 nt-length genome had two overlapped open reading frames (ORFs) consistent with a toti and toti-like viruses, that we named Pythium polare RNA virus 1 (PpRV1). The ORF2 encoded an RNA-dependent RNA polymerase (RdRp). The shifty heptamer motif and RNA pseudoknot were predicted near the stop codon of ORF1, implying that the RdRp could be translated as a fusion protein with the ORF1 protein. Phylogenetic analysis with deduced RdRp amino acid sequences indicated that oomycete virus PpRV1 was closely related to the unclassified arthropod toti-like viruses. The comparison of PpRV1-free and -infected lines suggested that PpRV1 infected in a symptomless manner.

The entry of cucumber mosaic virus into cucumber xylem is facilitated by co-infection with zucchini yellow mosaic virus.

We investigated the synergistic effects of co-infection by zucchini yellow mosaic virus (ZYMV) and cucumber mosaic virus (CMV) on viral distribution in the vascular tissues of cucumber. Immunohistochemical observations indicated that ZYMV was present in both the phloem and xylem tissues. ZYMV-RNA was detected in both the xylem wash and guttation fluid of ZYMV-inoculated cucumber. Steam treatment at a stem internode indicated that ZYMV enters the xylem vessels and moves through them but does not cause systemic infection in the plant. CMV distribution in singly infected cucumbers was restricted to phloem tissue. By contrast, CMV was detected in the xylem tissue of cotyledons in plants co-infected with CMV and ZYMV. Although both ZYMV-RNA and CMV-RNA were detected in the xylem wash and upper internodes of steam-treated, co-infected cucumbers grown at 24 °C, neither virus was detected in the upper leaves using an ELISA assay. Genetically modified CMV harboring the ZYMV HC-Pro gene was distributed in the xylem and phloem tissues of singly inoculated cucumber cotyledons. These results indicate that the ZYMV HC-Pro gene facilitates CMV entry into the xylem vessels of co-infected cucumbers.

Detection of plant virus in meristem by immunohistochemistry and in situ hybridization.

Most plant viruses do not infect the shoot apical meristem (SAM) of a host plant, and this virus-free region of meristem tissue has been used to obtain virus-free clones by meristem tip culture. Thus, the validation of viral distribution in meristem tissues is important for ensuring the appropriate excision of virus-free meristem tips. Although immunohistochemical microscopy and in situ hybridization are classical techniques, they allow us to determine the presence or absence of plant viruses in the shoot meristem tissues of a host plant. Briefly, meristem tissues are excised from infected plants, fixed, embedded in paraffin medium, and prepared in semithin sections (10-15 µm). By treating these sections with an antibody against viral protein or with a probe complementary to viral RNA, the viral distribution in the meristem tissue can be clearly observed. Importantly, these procedures are broadly applicable to most virus (and viroid) and host plant combinations.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Coat protein mutations in an attenuated Cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants.

In tobacco plants, the Cucumber mosaic virus (CMV) pepo strain induces mosaic symptoms, including pale green chlorosis and malformed tissues. Here, we characterized the involvement of 2b protein and coat protein (CP) in the development of mosaic symptoms. A 2b mutant (R46C) that lacks viral suppressor of RNA silencing (VSR) activity showed an asymptomatic phenotype with low levels of virus accumulation. Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C, although it did restore high-level virus accumulation. However, R46C mutants expressing mutated CP in which the amino acid P129 was mutated to A, E, C, Q, or S induced chlorosis that was associated with reduced expression of chloroplast and photosynthesis related genes (CPRGs) and abnormal chloroplasts with fewer thylakoid membranes. These results suggest that the CP of the CMV pepo strain acquires virulence by amino acid mutations, which causes CPRG repression and chloroplast abnormalities.

Quantitative transcriptional changes associated with chlorosis severity in mosaic leaves of tobacco plants infected with Cucumber mosaic virus.

Cucumber mosaic virus (CMV) causes mosaic disease in inoculated tobacco plants. Coat protein (CP) is one of the major virulence determinants of CMV, and an amino acid substitution at residue 129 in CP alters the severity of chlorosis, such as pale green chlorosis and white chlorosis, in symptomatic tissues of mosaic leaves of infected tobacco. In this study, we compared the transcriptomes of chlorotic tissues infected with the wild-type pepo strain of CMV and two strains carrying CP mutants with diverse chlorosis severity. Differential gene expression analysis showed that CMV inoculation appeared to have similar effects on the transcriptional expression profiles of the symptomatic chlorotic tissues, and only the magnitude of expression differed among the different CMVs. Gene ontology analysis with biological process and cellular component terms revealed that many nuclear genes related to abiotic stress responses, including responses to cadmium, heat, cold and salt, were up-regulated, whereas chloroplast- and photosynthesis-related genes (CPRGs) were down-regulated, in the chlorotic tissues. Interestingly, the level of CPRG down-regulation was correlated with the severity of chlorosis. These results indicate that CP mutation governs the repression level and mRNA accumulation of CPRGs, which are closely associated with the induction of chlorosis.

Large-scale codon de-optimisation of the p29 replicase gene by synonymous substitutions causes a loss of infectivity of melon necrotic spot virus.

The effect of synonymous substitutions in the melon necrotic spot virus p29 replicase gene on viral pathogenicity was investigated. The codons in the p29 gene were replaced by the least frequently used synonymous codons in Arabidopsis thaliana or melons. Mechanical inoculation of melon with p29 variants resulted in a loss of viral infectivity when all, one-half, or one-quarter of the gene was de-optimised. The effect of the de-optimisation in one-sixth of the gene was different depending on the de-optimised region. These results demonstrate that large-scale codon bias de-optimisation without amino acid substitutions of the p29 gene alter viral infectivity.

Cucumber mosaic virus: viral genes as virulence determinants.

TAXONOMIC RELATIONSHIPS: Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%-65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5' untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%-98%. GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SYMPTOMATOLOGY: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. PHYSICAL PROPERTIES: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10(-4) and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at -20 °C. CMV particles are isometric, approximately 28-30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer-hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s(20) ,(w) ) is c. 98 S and the particle weight is (5.8-6.7) × 10(6) Da. The virions contain 18% RNA. The RNA-protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. GENOMIC PROPERTIES: The genomic RNAs are single-stranded messenger sense RNAs with 5' cap and 3' tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3' untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV.

Single amino acid substitutions at residue 129 in the coat protein of cucumber mosaic virus affect symptom expression and thylakoid structure.

The symptomatic effect of the amino acid type at residue 129 in the coat protein of cucumber mosaic virus was investigated in tobacco using coat protein mutants of the pepo strain in which proline 129 was substituted with 19 other amino acids. These mutants caused six types of symptoms: white mosaic, pale green mosaic, veinal chlorosis, veinal necrosis, systemic necrosis, and necrotic local lesions. Transmission electron microscopy revealed that the chloroplasts of plants showing the three former types of symptoms contained few thylakoid membranes. Cytopathic effects characteristic of cells from plants showing the three latter symptom types were not observed.

The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants.

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Delta 2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Delta 2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Delta 2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Delta 2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Delta 2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Delta 2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Delta 2b, whereas accumulation of Delta 2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

Cucumber mosaic virus 2b protein compensates for restricted systemic spread of Potato virus Y in doubly infected tobacco.

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) inoculated with a necrotic strain of Potato virus Y (PVY, T01 isolate) developed necrotic symptoms in some systemically infected leaves, but not in younger leaves. However, PVY expressed distinct symptoms not only in the older leaves, but also in the younger leaves, of plants that had been doubly inoculated with PVY and with Cucumber mosaic virus (CMV, strain Pepo). A tissue blot immunoassay of tissues from various positions of the stem detected PVY weakly in each stem, but not in the shoot apex, of singly infected plants, whereas PVY was detected at high levels in almost all sections of doubly infected plants. CMV was also detected at high levels in sections of singly and doubly infected plants. Immunohistochemistry of stem tissues showed that in singly infected plants, PVY was confined to external phloem cells and was not detected in internal phloem cells. However, in doubly infected plants, PVY was distributed uniformly throughout whole tissues, including the external phloem, xylem parenchyma and internal phloem cells. In plants that were doubly infected with PVY and Pepo Delta 2b, a modified CMV that cannot translate the 2b protein, the spread of PVY was restricted as in singly infected plants. These results suggested that the plant host has a counterdefence mechanism that restricts systemic spread of PVY T01, and that the 2b protein of CMV strain Pepo negates this restriction.

Cucumber mosaic virus Establishes Systemic Infection at Increased Temperature Following Viral Entrance Into the Phloem Pathway of Tetragonia expansa.

ABSTRACT A potential regulatory site for Cucumber mosaic virus (CMV, pepo strain) movement necessary to establish systemic infection was identified through immunological and hybridization studies on Tetragonia expansa, which was systemically infected by CMV at 36 degrees C but not at 24 degrees C. In inoculated leaves, cell-to-cell movement of CMV was enhanced at 36 degrees C compared with that observed at 24 degrees C. CMV was distributed in the phloem cells of minor veins as well as epidermal and mesophyll cells at both 36 and 24 degrees C. CMV was detected in the petioles of inoculated leaves, stems, and petioles of uninoculated upper leaves at 36 degrees C, whereas CMV was detected only in the petioles of inoculated leaves and in stems at 24 degrees C. CMV moved into the phloem and was transported to the stem within 24 h postinoculation (hpi) at 36 degrees C. However, it did not accumulate in the petioles of the upper leaves until 36 hpi. In petioles of inoculated leaves at 24 degrees C, CMV was detected in the external phloem but not in the internal phloem. From these results, we conclude that systemic infection is established after viral entrance into the phloem pathway in T. expansa at 36 degrees C.