Inhibitory effect on the tobacco mosaic virus infection by a plant RING finger protein.

In the yeast two-hybrid screening of plant factors interacting with tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp), we found a protein containing a RING finger motif in tobacco (Nicotiana tabacum) and designated it as TARF (TMV-associated RING finger protein). TARF is a homologue of a Lotus japonicus RING finger protein (LjnsRING) involved in the symbiotic interaction between L. japonicus and Mesorhizobium loti. When TARF was silenced by virus-induced gene silencing (VIGS) method, TMV RNA accumulation as well as the number of foci formed by GFP-tagged TMV increased drastically. Transient overexpression of TARF reduced the accumulation of TMV. Moreover, TARF transcription was rapidly upregulated by the inoculation of TMV in tobacco plants. These results indicated that TARF is a RING finger protein that inhibits the accumulation of TMV via the interaction of TMV RdRp.

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.

Mstu1, an APSES transcription factor, is required for appressorium-mediated infection in Magnaporthe grisea.

The APSES protein family includes important transcriptional regulators of morphological processes in ascomycetes. We identified a deletion mutant of the APSES protein Mstu1 in Magnaporthe grisea that showed reduced conidiation and mycelial growth. Mstu1 formed a number of appressoria comparable to the wild type, although appressorium formation was delayed. In M. grisea, rapid transfer of conidial glycogen and lipid droplets to incipient appressoria is required for appressorial turgor generation, which the fungus uses to penetrate plant cuticles. Appressorial turgor was low in mstu1 and the mutant was deficient in appressorium-mediated invasion of rice leaves. The transfer of conidial glycogen and lipid droplets was remarkably delayed in mstu1, and a consequent delay in degradation of these conidial reserves was observed. Our results indicate that Mstu1 is required for appressorium-mediated infection due to its involvement in the mobilization of lipids and glycogen.

Identification of a novel tobacco DnaJ-like protein that interacts with the movement protein of tobacco mosaic virus.

The movement protein (MP) of tobacco mosaic virus (TMV) mediates the transport of viral RNA from infected cells to neighboring uninfected cells via plasmodesmata by interacting with putative host factors. To find such host factors, we screened tobacco proteins using the yeast two-hybrid system. NtMPIP1, a novel subset of DnaJ-like proteins, was identified from a tobacco cDNA library, and its specific interaction with TMV MP was confirmed with an in vitro filter-binding assay. In a deletion analysis, using a series of truncated TMV MPs and NtMPIP1s, at least two regions of TMV MP, amino acid residues 65-86 and 120-185, conferred the ability to interact with the C-terminal domain of NtMPIP1, which is thought to be involved in substrate binding. Virus-induced gene silencing of NtMPIP1 significantly inhibited the spread of TMV. Therefore, it is reasonable to consider that endogenous NtMPIP1 is a host factor involved in virus cell-to-cell spread by interacting with TMV MP.

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco.

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.

In vivo interaction between Tobacco mosaic virus RNA-dependent RNA polymerase and host translation elongation factor 1A.

Several host translation elongation factors have been suggested to play essential roles in the replication and translation of viral RNAs in plants, animals and bacteria. Here, we show the interaction between eukaryotic translation elongation factor 1A (eEF1A) and Tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp) in vivo by immunoprecipitation. The tobacco eEF1A interacted not only with 3'-untranslated region (3'-UTR) of TMV RNA but also directly with RdRp without mediation by the 3'-UTR. The methyltransferase domain of TMV RdRp was indicated to be responsible for the interaction with eEF1A in vitro and in yeast. These results suggest that eEF1A is a component of the virus replication complex of TMV.

Self-interaction of ORF II protein through the leucine zipper is essential for Soybean chlorotic mottle virus infectivity.

The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII-PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154-201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII-PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection.

Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene.

We introduced the rice chitinase (Cht-2; RCC2) gene into calli of Italian ryegrass (Lolium multiflorum Lam.), with a hygromycin phosphotransferase (HPT) gene as a selectable marker, by particle bombardment. Hygromycin-resistant calli were selected and transferred to regeneration medium for shoot formation. Polymerase chain reaction (PCR) analysis revealed regenerants containing the HPT gene. The RCC2 gene was detected in 65.5% of those regenerants. Southern hybridization detected both HPT and RCC2 genes and indicated that the transgenic plants were independently transformed. Expression of the RCC2 gene in the transgenic plants was confirmed by Northern hybridization, reverse transcription-PCR and Western blotting. Bioassay of detached leaves indicated increased resistance to crown rust (Puccinia coronata) in transgenic plants, which exhibited higher chitinase activity than a nontransgenic plant.

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas.

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.

In planta dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission.

ABSTRACT Due to the lack of a means to inoculate plants mechanically, the histological dynamics and in planta spread of phytoplasmas have been studied very little. We analyzed the dynamics of plant infection by phytoplasmas, using a technique to infect a limited area of a leaf, nested polymerase chain reaction (PCR), real-time PCR, and immunohistochemical visualization. Following localized inoculation of a leaf of garland chrysanthemum (Chrysanthemum coronarium) by the vector leafhopper Macrosteles striifrons, the onion yellows (OY) phytoplasma spread within the plant from the inoculated leaf to the main stem (1 day postinoculation [dpi]), to the roots and the top leaf (2 dpi), and to other leaves from top to bottom (from 7 to 21 dpi). The populations of the OY phytoplasmas in inoculated leaves and roots increased approximately sixfold each week from 14 to 28 dpi. At 14 dpi, the OY phytoplasmas colonized limited regions of the phloem tissue in both the root and stem and then spread throughout the phloem by 21 dpi. This information should form the basis for elucidating the mechanisms of phytoplasma multiplication and migration within a plant host.

"Candidatus Phytoplasma oryzae", a novel phytoplasma taxon associated with rice yellow dwarf disease.

In addition to rice yellow dwarf (RYD) phytoplasma, several phytoplasmas infect gramineous plants, including rice orange leaf, bermuda grass white leaf, brachiaria grass white leaf and sugarcane white leaf phytoplasmas. To investigate whether the RYD phytoplasma is a discrete, species-level taxon, several isolates of the aforementioned phytoplasmas were analysed using PCR-amplified 16S rDNA sequences. Two RYD isolates, RYD-J(T) and RYD-Th, were almost identical (99.2 %), but were distinct (similarities of 96.3-97.9 %) from other phytoplasma isolates of the RYD 16S-group. The notion that the RYD phytoplasma constitutes a unique taxon is also supported by its unique insect vector (Nephotettix sp.), its unique host plant in nature (rice) and its limited geographical distribution (Asia). In Southern blot analysis, chromosomal and extrachromosomal DNA probes of the RYD phytoplasma reportedly did not hybridize with those of closely related phytoplasmas. These properties of the RYD phytoplasma clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma oryzae'.

'Candidatus phytoplasma ziziphi', a novel phytoplasma taxon associated with jujube witches'-broom disease.

Phylogenetic relationships of five jujube witches'-broom (JWB) phytoplasma isolates from four different districts, and other phytoplasmas, were investigated by 16S rDNA PCR amplification and sequence analysis. The 16S rDNA sequences of any pair of the five isolates of JWB phytoplasmas were > 99.5% similar. The JWB phytoplasma 16S rDNA sequences were most closely related to that of the elm yellows (EY) phytoplasma in 16S-group VIII. Phylogenetic analysis of the 16S rDNA sequences from the JWB phytoplasma isolates, together with sequences from most of the phytoplasmas archived in GenBank, produced a tree in which the JWB isolates clustered as a discrete subgroup. The uniqueness of the JWB phytoplasma appears to be correlated with a specific insect vector (Hishimonus sellatus) and the host plant (Zizyphus jujuba), or with a specific geographical distribution. The unique properties of the JWB phytoplasma sequences clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma ziziphi'.

The nucleotide sequence and genome organization of Sclerophthora macrospora virus A.

Sclerophthora macrospora virus A (SmV A) found in S. macrospora, the pathogenic fungus responsible for downy mildew of gramineous plants, is a small icosahedral virus containing three segments (RNAs 1, 2, and 3) of the positive-strand ssRNA genome. In the present study we report the complete nucleotide sequence of the SmV A genome. The viral genome RNA 1 consists of 2928 nucleotides (nt) and has two open reading frames (ORFs 1a and 1b). ORF 1a contains the motifs of RNA-directed RNA polymerase (RdRp). The function of ORF 1b is unknown. RNA 2 consists of 1981 nt and single ORF (ORF 2). ORF 2 encodes a capsid protein. RNA 3 consists of 977 nt but not any ORFs, suggesting it as a satellite RNA. The deduced amino acid sequence of ORF 1a shows some similarity to those of RdRp of certain positive-strand RNA viruses, especially to the members of the family Nodaviridae, and that of ORF 2 to CP of the members in the family Tombusviridae. The nucleotide sequence of RNA 3 shows a 40-nucleotide length of partial similarity to S. macrospora virus B (SmV B) RNA. The capsid of SmV A is composed of two capsid proteins, CP 1 (p43) and CP 2 (p39), both encoded in ORF 2. CP 2 is apparently derived from CP 1 via proteolytic cleavage at the N-terminus. The genome organization of SmV A is characteristic and distinct from those of other known fungal RNA viruses, including SmV B. These results suggest that SmV A should be classified into a new group of mycoviruses.

First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome.

Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.

Interaction of replicase components between Cucumber mosaic virus and Peanut stunt virus.

Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) each have genomes consisting of three single-stranded RNA molecules: RNA 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome. Although RNA 3 is exchangeable between CMV and PSV, exchange of RNA 1 and 2 between the two viruses has been unsuccessful. In this study, reassortants containing PSV RNA 1 and CMV RNA 2 together with RNA 3 of CMV or PSV were shown to be able to replicate their genomic RNA, but not to transcribe subgenomic RNA 4 in tobacco protoplasts. Conversely, the reassortant consisting of CMV RNA 1 and PSV RNA 2 together with RNA 3 of CMV or PSV could not replicate. Subsequently, a yeast two-hybrid system was used to analyse the in vivo interaction between the 1a and 2a proteins. The C-terminal half of PSV-1a protein interacted with the N-terminal region of 2a protein of both PSV and CMV, but the C-terminal half of CMV-1a and the N-terminal region of PSV-2a did not interact. These results suggest that RNA replication in the interspecific reassortant between CMV and PSV requires compatibility between the C-terminal half of the 1a protein and the N-terminal region of the 2a protein, and this compatibility is insufficient for transcription of subgenomic RNA 4.

RNA recombination between cucumoviruses: possible role of predicted stem-loop structures and an internal subgenomic promoter-like motif.

We previously analyzed hybrids of Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) that contained CMV RNA2 with the 3'-terminal sequence from TAV RNA2. In this article, we scrutinized the RNA3 molecules in these hybrid viruses by Northern hybridization and RT-PCR and found some recombinant CMV RNA3 molecules and various recombinant RNA4 molecules whose 3'-termini were derived from TAV RNA1 or 2. Sequence analyses revealed that most of the crossover sites for recombination were located near putative stem-loop structures and an internal subgenomic promoter-like motif. We inoculated in vitro transcripts synthesized from cDNA clones of the recombinant RNA3 onto N. benthamiana along with either CMV RNA1 and 2 or TAV RNA1 and 2. Although all of the hybrids were infectious, many sequence deletions and nucleotide substitutions were found when RNA1 and 2 from TAV were used, which suggests that fidelity of TAV replicase was lower than that of CMV replicase. The possible role of secondary structures and an internal subgenomic promoter-like motif in RNA recombination is discussed.

Characterization of transgenic rice plants over-expressing the stress-inducible beta-glucanase gene Gns1.

The Gns1 gene of rice (Oryza sativa L. japonica) encodes 1,3;1,4-beta glucanase (EC 3.2.1.73), which hydrolyzes 1,3;1,4-beta-glucosidic linkages on 1,3;1,4-beta-glucan, an important component of cell walls in the Poaceae family. RNA and protein gel blot analyses demonstrated that blast disease or dark treatment induced the expression of the Gns1 gene. To assess the function of the Gns1 gene in disease resistance, we characterized transgenic rice plants constitutively expressing the Gns1 gene. The introduced Gns1 gene was driven by the CaMV 35S promoter and its products were found in the apoplast and accumulated in up to 0.1% of total soluble protein in leaves. Although transgenic plants showed stunted growth and impaired root formation, fertility, germination, and coleoptile elongation appeared unaffected compared to non-transgenic control plants, indicating that Gns1 does not play a crucial role in rice germination and coleoptile elongation. When transgenic plants were inoculated with virulent blast fungus (Magnaporthe grisea), they developed many resistant-type lesions on the inoculated leaf accompanying earlier activation of defense-related genes PR-1 and PBZ1 than in control plants. Transgenic plants spontaneously produced brown specks, similar in appearance to those reported for an initiation type of disease-lesion-mimic mutants, on the third and fourth leaves and occasionally on older leaves without inoculation of pathogens. Expression of the two defense-related genes was drastically increased after the emergence of the lesion-mimic phenotype.

'Candidatus Phytoplasma castaneae', a novel phytoplasma taxon associated with chestnut witches' broom disease.

In Korea, Japanese chestnut trees (Castanea crenata Sieb. and Zucc.) showing symptoms indicative of witches' broom disease, including abnormally small leaves and yellowing of young leaves, were examined. Since the symptoms were suggestive of a phytoplasma infection, tissues were assayed for phytoplasmas by PCR analysis using a pair of universal primers that amplify a 1.4-kbp phytoplasma 16S rDNA fragment. The phytoplasma-specific fragment was amplified from diseased plants, but not from healthy plants, indicating that a phytoplasma was the causal agent of the chestnut witches' broom (CnWB) disease. The phylogenetic relationship of the CnWB phytoplasma to other phytoplasmas was examined by sequence analysis of the 16S rDNA. A phylogenetic analysis of 16S rDNA sequences of the phytoplasmas placed the CnWB phytoplasma within a distinct subgroup in the phytoplasma clade of the class Mollicutes. The phylogenetic tree indicated that the CnWB phytoplasma is related most closely to coconut phytoplasmas and suggested that they share a common ancestor. The unique properties of the CnWB phytoplasma sequences clearly establish that it represents a novel taxon, 'Candidatus Phytoplasma castaneae'.

Genes Ia, II, III, IV and V of Soybean chlorotic mottle virus are essential but the gene Ib product is non-essential for systemic infection.

Soybean chlorotic mottle virus (SbCMV) is the type species of the genus 'Soybean chlorotic mottle-like viruses', within the family Caulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1.3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNA(Met) primer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case of Cauliflower mosaic virus.