Shoji Usami

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

SummaryT-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.

Genomic Characterization of the Filamentous Integrative Bacteriophages φRSS1 and φRSM1, Which Infect Ralstonia solanacearum

The genomic DNA sequences were determined for two filamentous integrative bacteriophages, φRSS1 and φRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of φRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within φRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the φRSS1 intergenic (IG) region. The 9,004-base sequence of φRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the φRSM1 replication module. Genomic DNA fragments containing the φRSM integration junctions were cloned and sequenced from φRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5′-TGGCGGAGAGGGT-3′, corresponding to the 3′ end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the φRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of φRSS1 is within a sequence element of the IG region.

Genomic Characterization of Ralstonia solanacearum Phage φRSB1, a T7-Like Wide-Host-Range Phage

PhiRSB1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The phiRSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of phiRSB1 were demonstrated.

Plant-inducible recombination between the 25 bp border sequences of T-DNA in Agrobacterium tumefaciens

SummaryWe developed a model system for detecting and assaying the circular forms of T-DNA which may be generated in Agrobacterium by intramolecular recombination between the 25 bp border repeats of T-DNA. We demonstrated using this system that the DNA region flanked by the 25 bp direct repeats is in fact circularized by recombination between these repeats in cells of Agrobacterium cocultured with tobacco protoplasts. Furthermore, quantitative analysis of the recombination revealed the following: (1) the recombination is also induced when the agrobacterial cells are incubated in protoplast-free conditioned medium prepared by filtering the protoplast culture. The conditioned medium is effective, even after it has been heated at 100°C. (2) The DNA region encompassing the virulence region of the Ti-plasmid is required for recombination. (3) The recombination takes place only between 25 bp repeats with the same orientation. On the basis of these results, we conclude that the circular form of T-DNA is generated by homologous recombination between the border repeats which is mediated by gene product(s) encoded by the virulence region of the Ti-plasmid. Either the recombination itself, or the expression of the virulence gene(s) responsible for the recombination, is induced by diffusible and heatstable factor(s) secreted by plant cells.

Host recognition and integration of filamentous phage ϕRSM in the phytopathogen, Ralstonia solanacearum

Two prophages, called varphiRSM3 and varphiRSM4, that are closely related to, but differ from, filamentous phage varphiRSM1, have been detected in strains of the Ralstonia solanacearum species complex. The prophage varphiRSM3, found in host strain MAFF730139, could be converted to infectious phage by means of PCR and transfection. The nucleotide sequence of varphiRSM3 is highly conserved relative to varphiRSM1 except for open reading frame 2 (ORF2), encoding an unknown protein, and ORF9 encoding the presumed adsorption protein that determines host range. The two host ranges differ dramatically and correlate closely with different gel electrophoresis banding patterns for cell surface fimbriae. Infections by varphiRSM1 and varphiRSM3 enhance bacterial cell aggregation and reduce the bacterial host virulence in tomato plants. Database searches in the R. solanacearum strains of known genomic sequence revealed two inovirus prophages, one designated varphiRSM4 that is homologous to varphiRSM1 and varphiRSM3, and one homologues to RSS1, in the genome of strain UW551.

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2

Cell wall materials isolated from Chlorella cells were degraded by the polysaccharide‐degrading enzyme vAL‐1 encoded by chlorovirus CVK2. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analyses of the degradation products (oligosaccharides) revealed major oligosaccharides contain unsaturated GlcA at the reducing terminus, and a side chain attached at C2 or C3 of GlcA(C4=C5), which mainly consisted of Ara, GlcNAc and Gal. The results indicated that vAL‐1 is a novel polysaccharide lyase, cleaving chains of β‐ or α‐1,4‐linked GlcAs. The unique structures of Chlorella cell wall were also revealed. Studies on the complicated structures of naturally occurring polysaccharides will be greatly facilitated by using vAL‐1 as a tool in structural analysis.

Monitoring of Phytopathogenic Ralstonia solanacearum Cells Using Green Fluorescent Protein-Expressing Plasmid Derived from Bacteriophage ϕRSS1

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

Two catalytic domains of Chlorella virus CVK2 chitinase

VChti-1 chitinase encoded by the Chlorella virus CVK2 contained two catalytic domains belonging to family 18 glycosyl hydrolases. The first catalytic domain on a C-terminal-truncated derivative of vChti-1 generated exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin in the enzyme reaction, a typical characteristic of an exochitinase. In contrast, N-acetylglucosamine was produced from chitobiose as well as from chitooligosaccharides by the second catalytic domain on an N-terminal-truncated derivative of vChti-1. Therefore, the second domain possessed N-acetylglucosaminidase activity as well as endochitinase activity. The presence of two catalytic domains with different enzymatic properties in the viral enzyme seems to be necessary for hydrolyzing natural substrates in a cooperative fashion.

Isolation of high-quality RNA from high-phenolic tissues of eelgrass (Zostera marina L.) by keeping temperature low

We describe a modified LiC1 method for isolating good-quality RNA from leaves, rhizomes and roots of eelgrass (Zostera marina L.) which are rich in phenolic compounds. In ordinary protocols, RNA extraction was strongly inhibited by contamination with the large amounts of polyphenol compounds. Only by keeping the temperature low during extraction could RNA be successfully isolated. The resulting high-quality RNA was suitable for reverse transcription-polymerase chain reaction, reverse transcription followed by quantitative real-time PCR, cDNA library construction and Northern blot analysis. The total RNA extracted from leaves was much higher than that from rhizomes and roots. Using this method, 20–40 μg of total RNA was routinely obtained from 1 g of fresh tissue. This is the first report of low temperature RNA extraction from plant tissues rich in phenolic compounds.