Efficient foreign gene expression in planta using a plantago asiatica mosaic virus-based vector achieved by the strong RNA-silencing suppressor activity of TGBp1.

Plant virus expression vectors provide a powerful tool for basic research as well as for practical applications. Here, we report the construction of an expression vector based on plantago asiatica mosaic virus (PlAMV), a member of the genus Potexvirus. Modification of a vector to enhance the expression of a foreign gene, combined with the use of the foot-and-mouth disease virus 2A peptide, allowed efficient expression of the foreign gene in two model plant species, Arabidopsis thaliana and Nicotiana benthamiana. Comparison with the widely used potato virus X (PVX) vector demonstrated that the PlAMV vector retains an inserted foreign gene for a longer period than PVX. Moreover, our results showed that the GFP expression construct PlAMV-GFP exhibits stronger RNA silencing suppression activity than PVX-GFP, which is likely to contribute to the stability of the PlAMV vector.

Lectin-mediated resistance impairs plant virus infection at the cellular level.

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.

Genetic heterogeneity found in the replicase gene of poinsettia mosaic virus isolates.

The complete nucleotide sequences of five isolates of poinsettia mosaic virus (PnMV) from Japan (JN, JO1, JO2, JO4, and JO5) were determined. These isolates contained a single large open reading frame in their genomes and shared 96.6-97.8% identity at the nucleotide level and 91.3-98.1% identity at the amino acid level with two previously reported European isolates. Interestingly, the JO isolates were found to possess eight common translational frameshift sites in the interdomain region between the methyltransferase and protease domains, resulting in considerable variation in the interdomain region compared to the other isolates. This suggests that PnMV might have evolved by creating variations in its genome by such translational frameshifts.

Viral-induced systemic necrosis in plants involves both programmed cell death and the inhibition of viral multiplication, which are regulated by independent pathways.

Resistant plants respond rapidly to invading avirulent plant viruses by triggering a hypersensitive response (HR). An HR is accompanied by a restraint of virus multiplication and programmed cell death (PCD), both of which have been observed in systemic necrosis triggered by a successful viral infection. Here, we analyzed signaling pathways underlying the HR in resistance genotype plants and those leading to systemic necrosis. We show that systemic necrosis in Nicotiana benthamiana, induced by Plantago asiatica mosaic virus (PlAMV) infection, was associated with PCD, biochemical features, and gene expression patterns that are characteristic of HR. The induction of necrosis caused by PlAMV infection was dependent on SGT1, RAR1, and the downstream mitogen-activated protein kinase (MAPK) cascade involving MAPKKKalpha and MEK2. However, although SGT1 and RAR1 silencing led to an increased accumulation of PlAMV, silencing of the MAPKKKalpha-MEK2 cascade did not. This observation indicates that viral multiplication is partly restrained even in systemic necrosis induced by viral infection, and that this restraint requires SGT1 and RAR1 but not the MAPKKKalpha-MEK2 cascade. Similarly, although both SGT1 and MAPKKKalpha were essential for the Rx-mediated HR to Potato virus X (PVX), SGT1 but not MAPKKKalpha was involved in the restraint of PVX multiplication. These results suggest that systemic necrosis and HR consist of PCD and a restraint of virus multiplication, and that the latter is induced through unknown pathways independent from the former.

Significance of eukaryotic translation elongation factor 1A in tobacco mosaic virus infection.

Eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with both the viral RNA-dependent RNA polymerase and the 3'-terminal genomic RNA of tobacco mosaic virus (TMV). In this study, we demonstrated that the down-regulation of eEF1A mRNA levels by virus-induced gene silencing using potato virus X vector dramatically reduced the accumulation of TMV RNA and the spread of TMV infection. The translation activity of the eEF1A-silenced Nicotiana benthamiana leaves was not severely affected. Collectively, these results suggest an essential role of eEF1A in TMV infection.

Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome.

The role of RNA silencing as an antiviral defence has been well elucidated in plants and invertebrates, but not in filamentous fungi. We have previously determined the complete genome sequence of Magnaporthe oryzae virus 2 (MoV2), a dsRNA virus that infects the rice blast fungus Magnaporthe oryzae. In this study, we detected small interfering RNAs (siRNAs) from both positive- and negative-strand MoV2 viral RNA, suggesting that the RNA silencing machinery in M. oryzae functions against the mycovirus. Cloning and characterisation of MoV2 siRNAs indicated that, in MoV2, the ratio of virus-derived siRNAs to total small RNA is significantly lower than that in either plant viruses or Cryphonectria hypovirus 1 (CHV1), another mycovirus. Nevertheless, any MoV2-encoded proteins did not exhibit RNA silencing suppressor activity in both the plant and fungal systems. Our study suggests the existence of a novel viral strategy employed to evade host RNA silencing.

The N-terminal region of the Plantago asiatica mosaic virus coat protein is required for cell-to-cell movement but is dispensable for virion assembly.

Potexvirus cell-to-cell movement requires coat protein (CP) and movement proteins. In this study, mutations in two conserved in-frame AUG codons in the 5' region of the CP open reading frame of Plantago asiatica mosaic virus (PlAMV) were introduced, and virus accumulation of these mutants was analyzed in inoculated and upper noninoculated leaves. When CP was translated only from the second AUG codon, virus accumulation in inoculated leaves was lower than that of wild-type PlAMV, and the viral spread was impaired. Trans-complementation analysis showed that the leucine residue at the third position (Leu-3) of CP is important for cell-to-cell movement of PlAMV. The 14-amino-acid N-terminal region of CP was dispensable for virion formation. Immunoprecipitation assays conducted with an anti-TGBp1 antibody indicated that PlAMV CP interacts with TGBp1 in vivo and that this interaction is not affected by alanine substitution at Leu-3. These results support the concept that the N-terminal region of potexvirus CP can be separated into two distinct functional domains.

Variability in the level of RNA silencing suppression caused by triple gene block protein 1 (TGBp1) from various potexviruses during infection.

RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode RNA silencing suppressors as a counter defence. In this study, we analysed the RNA silencing suppression ability of multiple virus species of the genus Potexvirus. Nicotiana benthamiana plants exhibiting RNA silencing of a green fluorescent protein (GFP) transgene showed reversal of GFP fluorescence when systemically infected with potexviruses. However, the degree of GFP fluorescence varied among potexviruses. Agrobacterium-mediated transient expression assay in N. benthamiana leaves demonstrated that the triple gene block protein 1 (TGBp1) encoded by these potexviruses has drastically different levels of silencing suppressor activity, and these differences were directly related to variations in the silencing suppression ability during virus infection. These results suggest that suppressor activities differ even among homologous proteins encoded by viruses of the same genus, and that TGBp1 contributes to the variation in the level of RNA silencing suppression by potexviruses. Moreover, we investigated the effect of TGBp1 encoded by Plantago asiatica mosaic virus (PlAMV), which exhibited a strong suppressor activity, on the accumulation of microRNA, virus genomic RNA and virus-derived small interfering RNAs.

A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium.

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.

The efficiency of interference of Potato virus X infection depends on the target gene.

RNA silencing is a natural defense response against viral infection. This phenomenon has been used to interfere with viral infections by exploiting fragments of viral genomes as sources of RNA silencing. Agrobacterium-mediated transient expression of a hairpin RNA derived from the TGBp1 gene of Potato virus X (PVX) induced RNA silencing of the TGBp1 gene and resulted in interference of PVX infection. The interference was induced in the infiltrated leaves but not in the upper non-infiltrated leaves. Transient expression of a CP hairpin RNA also induced interference of PVX. The TGBp1 hairpin RNA showed more efficient interference of PVX infection than the CP hairpin RNA.

Phylogenetic characteristics, genomic heterogeneity and symptomatic variation of five closely related Japanese strains of Potato virus X.

To elucidate the genomic determinants of Potato virus X (PVX) strains, which cause diverse responses in host plants, we determined the complete genomic RNA sequences of four Japanese PVX strains: PVX-BS, -BH, -OG, and -TO. These four strains, plus the previously sequenced PVX-OS strain, differ in their pathogenicity in wild potato (Solanum demissum) and tobacco (Nicotiana tabacum cv. Samsun NN). The genomic sequences of these five PVX strains were highly homologous (i.e., the nucleotide sequence identity ranged from 95.4 to 98.5%). Phylogenetic analysis indicated that the Japanese PVX strains originated from an ancestral PVX strain in the European group, and that the virulence of these strains in both S. demissum and tobacco is not correlated with their phylogenetic relationships, suggesting that the pathogenicity of each strain in these host plants is determined by a relatively small number of nucleotides and can easily be altered independent of phylogenetic relationships. Particularly, OS, BH, and BS, which respectively produce markedly contrasting ringspot, mosaic, and asymptomatic infections in tobacco leaves, were the most closely related, suggesting that these three strains are an attractive model for analyzing the genetic determinants causing these symptoms. A possible correlation between the genomic and biological differences of these strains is discussed.