Significance Release of outer membrane vesicles (OMVs) is a general feature of Gram-negative bacteria. Most studies have addressed the mechanisms of their formation or the cargo they can carry, but other roles remain to be explored further. Here we provide evidence for a novel role for OMVs in Xylella fastidiosa , a bacterial pathogen that colonizes the xylem of important crop plants. OMVs, whose production is suppressed by a quorum-sensing system, serve as an autoinhibitor of cell adhesion to surfaces, thereby blocking attachment-driven biofilm formation that would restrict movement within the xylem and thus colonization of plants. The ubiquity of OMV formation in the bacterial world suggests that these extracellular products may have alternative roles that might modulate movement and biofilm formation.
The rpfF gene from Xylella fastidiosa, encoding the synthase for diffusible signal factor (DSF), was expressed in 'Freedom' grape to reduce the pathogen's growth and mobility within the plant. Symptoms in such plants were restricted to near the point of inoculation and incidence of disease was two- to fivefold lower than in the parental line. Both the longitudinal and lateral movement of X. fastidiosa in the xylem was also much lower. DSF was detected in both leaves and xylem sap of RpfF-expressing plants using biological sensors, and both 2-Z-tetradecenoic acid, previously identified as a component of X. fastidiosa DSF, and cis-11-methyl-2-dodecenoic acid were detected in xylem sap using electrospray ionization mass spectrometry. A higher proportion of X. fastidiosa cells adhered to xylem vessels of the RpfF-expressing line than parental 'Freedom' plants, reflecting a higher adhesiveness of the pathogen in the presence of DSF. Disease incidence in RpfF-expressing plants in field trials in which plants were either mechanically inoculated with X. fastidiosa or subjected to natural inoculation by sharpshooter vectors was two- to fourfold lower in than that of the parental line. The number of symptomatic leaves on infected shoots was reduced proportionally more than the incidence of infection, reflecting a decreased ability of X. fastidiosa to move within DSF-producing plants.
of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation. Radiat ResWe have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation (IR). In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains following irradiation. Of the top 33 strains most sensitive to IR in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied in detail. Only one, that deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity both in the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to IR. Keywords:Saccharomyces cerevisiae, radiosensitive mutants, genome-wide, RAD61, ionizing radiation 3 INTRODUCTIONFor many years the budding yeast Saccharomyces cerevisiae has been an important model system for understanding DNA repair in eukaryotic cells (1). This is primarily a result of the ease of genetic manipulations of this organism and of the remarkable conservation of pathways and genes from yeast to man in the area of DNA repair (2-4).In mammalian cells the most important lesions leading to cell death by ionizing radiation (IR) are believed to be DNA double strand breaks (DSBs), and mutants defective in the repair of these lesions are highly sensitive to radiation-induced cell killing (5-7). Radiation-induced DSBs are also potentially lethal in yeast based on evidence that a single double strand break can be lethal in repair deficient mutants (8-10). Two major pathways for DNA double strand break repair have been identified in eukaryotic cells. The first, homologous recombination (HR), involves exchange of DNA between broken and non-broken DNA strands. This is the major pathway of repair in S. cerevisiae but plays a lesser role in mammalian cells. The second pathway, non-homologous end-joining (NHEJ), which involves direct ligation of the broken ends, is the major pathway for repair of IR-induced DSBs in mammalian cells but plays at most a minor role in repair of IR damage in S. cerevisiae. Despite the lesser importance of HR in repairing radiation induced DSBs in mammalian cells (11,12), it is clearly a pathway of major impo...
The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X rays, we are screening these mutants to identify additional genes that cause increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype cosegregates with the deletion allele and are obtaining multipoint survival-vs.-dose assays in at least one homozygous diploid and two haploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1, and VID21/EAF1 and discuss their potential roles in repair. Eight of these genes cause a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, results in at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultraviolet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino acids are also X-ray sensitive, which confirms that methylation of the lysine-79 residue is required for effective repair of radiation damage.
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