Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: RSA1, RSB1, and RSL1. Infection with RSA1 and RSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with RSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with RSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All RSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. RSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active RSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as RSL1, instead of a phage cocktail with highly virulent phages. Using this method, RSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting.

Four kinds of bacteriophage (wRSL, wRSA, wRSM and wRSS) were isolated from Ralstonia solanacearum, a soil-borne Gram-negative bacterium that is the causative agent of bacterial wilt in many important crops. The Myovirus-type phages wRSL1 and wRSA1 contained dsDNA genomes of 240 kbp and 39 kbp, respectively. These phages have relatively wide host ranges and gave large clear plaques with various host strains; especially wRSA1 was able to infect all 15 R. solanacearum strains of different races or different biovars tested in this study. Three host strains contained wRSA1-related sequences in their genomic DNAs, suggesting a lysogenic cycle of wRSA1. Two phages, wRSM1 and wRSS1, were characterized as Ff-type phages (Inovirus) based on their particle morphology, genomic ssDNA and infection cycle. However, despite their similar fibrous morphology, their genome size (9.0 kb for wRSM1 and 6.6 kb for wRSS1) and genome sequence were different. Strains of R. solanacearum that were sensitive to wRSM1 were resistant to wRSS1 and vice versa. Several R. solanacearum strains contained wRSM1-related sequences and at least one strain produced wRSM1 particles, indicating the lysogenic state of this phage. These phages may be useful as a tool not only for molecular biological studies of R. solanacearum pathogenicity but also for specific and efficient detection (wRSM1 and wRSS1) and control of harmful pathogens (wRSL and wRSA) in cropping ecosystems as well as growing crops.

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

Six copies of insertion elements accumulate in the subtelomeric region immediately proximal to the telomeric repeats on Chlorella chromosome I. The elements, designated Zepps, bear the characteristic features of non‐viral (LINE‐like) retrotransposons, including a poly(A) tail, 5′‐truncations, a retroviral reverse transcriptase‐like ORF and flanking target duplications. Detailed sequence analysis of the Chlorella subtelomeric region revealed a novel mechanism of Zepp transposition; successive insertions of each Zepp element into another Zepp as a target, leaving a tandem array of their 3′‐regions with poly(A) tracts facing toward the centromere. Only the most distal Zepp copy was inverted to connect its poly(A) tail with the telomeric repeats. A similar Zepp cluster but without the telomeric repeats was also found at the terminus of another Chlorella chromosome. These structures contrast with that proposed for the addition of HeT‐A and TART elements to Drosophila telomeres. Expression of Zepp elements is induced by heat shock treatment. Possible roles of the subtelomeric retrotransposons in formation and maintenance of telomeres are discussed.

Abstract. The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4'-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2'-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-l,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.Abbreviations: BrdU=5-bromo-2'-deoxyuridine; DAPI=4'-6-diamidino-2-phenylindole; DiOC 6 = 3,3'-dihexyloxacarbocyanine; mtDNA=mitochondrial DNA; mt-nucleoid=mitochondrial nucleoid; ptDNA = plastid DNA; pt-nucleoid = plastid nucleoid; Rubisco = ribulose-l,5-bisphosphate carboxylase/oxygenase Correspondence to: M. Fuije; FAX: 81(3)38141408; Tel.: 81(3)38122111, Ext. 4471

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ϕRSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ϕRSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ϕRSS1, resulting in early wilting. Tomato plants inoculated with ϕRSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ϕRSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ϕRSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ϕRSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.

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.

phiRSL1 is a jumbo myovirus stably and lytically infecting the phytopathogenic bacterium Ralstonia solanacearum. In this study, we investigate the infection cycle of varphiRSL1 and provide a genomic, proteomic and transcriptomic view of this phage. Its 231-kbp genome sequence showed many genes lacking detectable homologs in the current databases and was vastly different from previously studied phage genomes. In addition to these orphan proteins, varphiRSL1 was found to encode several enzymes that are unique among known viruses. These include enzymes for the salvage pathway of NAD(+) and for the biosynthetic pathways of lipid, carbohydrate and homospermidine. A chitinase-like protein was found to be a potential lysis enzyme. Our proteomics analysis suggests that varphiRSL1 virions contain at least 25 distinct proteins. We identified six of them including a tail sheath protein and a topoisomerase IB by N-terminal sequencing. Based on a DNA microarray analysis, we identified two transcription patterns.