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PubMed1993

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean.

A new disease resistance locus in Arabidopsis, RPS3, was identified using a previously cloned avirulence gene from a non-Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis, and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB, and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F2 plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3. Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and < or = 1 cM from another disease resistance locus, RPM1. In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1. Thus, RPG1 and RPS3 both confer avrB-specific disease resistance, suggesting that these genes may be homologs.

Arabidopsis↗
PubMed2005

Behavioural responses of diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) to extracts derived from Melia azedarach and Azadirachta indica.

The impact of three different doses of botanical insecticide derived from the syringa tree, Melia azedarach and the neem tree, Azadirachta indica was tested on the behaviour of the diamondback moth, Plutella xylostella (Linnaeus). Both botanical insecticides had a significant impact on larval behaviour. At higher doses the extracts showed feeding deterrent activity, with larvae preferring the untreated sides of cabbage leaves and consuming less of the treated half of cabbage leaves. The botanical insecticides had less of an effect on the oviposition behaviour of P. xylostella moths. In laboratory and glasshouse trials, significantly fewer eggs were oviposited on the plants that had been treated with syringa extracts. Therefore, the syringa extracts appear to have a repellent effect. In contrast, when exposed to the neem extracts the moths did not discriminate between control plants and treated plants. Behavioural observation indicated that, despite the lower number of eggs oviposited on cabbage treated with syringa extracts, the moths chose cabbage treated with the highest dose of syringa more often than they chose control cabbage plants. Similar observations were found in cabbage plants treated with neem, moths chose the medium dose more often than they chose the control. Oviposition and feeding deterrent properties are important factors in pest control, and results from this study indicate that botanical insecticides have the potential to be incorporated into control programmes for P. xylostella in South Africa.

Animals↗
PubMed2002

Pseudomonas lipodepsipeptides and fungal cell wall-degrading enzymes act synergistically in biological control.

Pseudomonas syringae pv. syringae strain B359 secreted two main lipodepsipeptides (LDPs), syringomycin E (SRE) and syringopeptin 25A (SP25A), together with at least four types of cell wall-degrading enzymes (CWDEs). In antifungal bioassays, the purified toxins SRE and SP25A interacted synergistically with chitinolytic and glucanolytic enzymes purified from the same bacterial strain or from the biocontrol fungus Trichoderma atroviride strain P1. The synergism between LDPs and CWDEs occurred against all seven different fungal species tested and P. syringae itself, with a level dependent on the enzyme used to permeabilize the microbial cell wall. The antifungal activity of SP25A was much more increased by the CWDE action than was that of the smaller SRE, suggesting a stronger antifungal role for SP25A. In vivo biocontrol assays were performed by using P. syringae alone or in combination with T. atroviride, including a Trichoderma endochitinase knock-out mutant in place of the wild type and a chitinase-specific enzyme inhibitor. These experiments clearly indicate that the synergistic interaction LDPs-CWDEs is involved in the antagonistic mechanism of P. syringae, and they support the concept that a more effective disease control is given by the combined action of the two agents.

Cell Wall↗
PubMed1990

Effect of aerosolization on subsequent bacterial survival.

To determine whether aerosolization could impair bacterial survival, Pseudomonas syringae and Erwinia herbicola were aerosolized in a greenhouse, the aerosol was sampled at various distances from the site of release by using all-glass impingers, and bacterial survival was followed in the impingers for 6 h. Bacterial survival subsequent to aerosolization of P. syringae and E. herbicola was not impaired 1 m from the site of release. P. syringae aerosolized at 3 to 15 m from the site of release at a temperature of 12 degrees C and a relative humidity of 80% survived 35- to 65-fold better than P. syringae released at 27 degrees C and a relative humidity of 40%. No difference was observed in the survival of P. syringae and E. herbicola following aerosolization at the same temperature and relative humidity. Bacteria sprayed directly onto bean and oat plants established stable populations at comparable numbers on both plants over an 8-day period following inoculation. Bacteria that inoculated adjacent plants by drifting downwind up to 5 m were detectable at an initial population of 10(2) CFU/g on oats and 10(5) CFU/g on beans 2 h after the spray. However, bacterial populations on both plants were undetectable within 48 h.

Aerosols↗
PubMed1996

Utility of microcosm studies for predicting phylloplane bacterium population sizes in the field.

Population sizes of two ice nucleation-active strains of Pseudomonas syringae were compared on leaves in controlled environments and in the field to determine the ability of microcosm studies to predict plant habitat preferences in the field. The P. syringae strains investigated were the parental strains of recombinant deletion mutant strains deficient in ice nucleation activity that had been field tested for their ability to control plant frost injury. The population size of the P. syringae strains was measured after inoculation at three field locations on up to 40 of the same plant species that were studied in the growth chamber. There was seldom a significant relationship between the mean population size of a given P. syringae strain incubated under either wet or dry conditions in microcosms and the mean population size which could be recovered from the same species when inoculated in the field. Specifically, on some plant species, the population size recovered from leaves in the field was substantially greater than from that species in a controlled environment, while for other plant species field populations were significantly smaller than those observed under controlled conditions. Population sizes of inoculated P. syringae strains, however, were frequently highly positively correlated with the indigenous bacterial population size on the same plant species in the field, suggesting that the ability of a particular plant species to support introduced bacterial strains is correlated with its ability to support large bacterial populations or that indigenous bacteria enhance the survival of introduced strains. Microcosm studies therefore seem most effective at assessing possible differences between parental and recombinant strains under a given environmental regime but are limited in their ability to predict the specific population sizes or plant habitat preferences of bacteria on leaves under field conditions.

Journal Article↗
PubMed2006

Comparative analysis of induction pattern of programmed cell death and defense-related responses during hypersensitive cell death and development of bacterial necrotic leaf spots in eggplant.

Pseudomonas cichorii causes necrotic leaf spots (NLS), while Pseudomonas syringae pv. tabaci induces a hypersensitive response (HR) in eggplant. P. cichorii induced cell death at 9 h after inoculation (HAI), reaching a maximum of around 24-30 HAI. On the other hand, cell death was induced 6 HAI with P. syringae pv. tabaci, reaching a maximum of around 12-18 HAI. Superoxide generation was observed in eggplant inoculated with both bacteria. DNA fragmentation, cytochrome c release into the cytosol and expression of defense-related genes such as PR-1 and hsr203J was also induced by inoculation with both bacteria, but these plant reactions were more rapidly induced in eggplant inoculated with P. syringae pv. tabaci rather than those with P. cichorii. Lipid peroxidation and induction of lipoxygenase (LOX) was drastically induced in eggplant inoculated with P. syringae pv. tabaci compared to P. cichorii-inoculated eggplant. Pharmacological studies showed that induction of the cell death, and the NLS or the HR in response to both bacteria was commonly associated with de novo protein synthesis, reactive oxygen species and caspase III-like protease. Interestingly, involvement of lipid peroxidation, LOX, serine protease, and DNase differed between induction of NLS and HR. These results suggest that programmed cell death might be closely associated not only with the HR but also NLS. However, there may be differences not only in the induction kinetics and level of plant responses but also in the infection-related responses between HR and NLS.

Cell Death↗
PubMed2006

Specific bacterial suppressors of MAMP signaling upstream of MAPKKK in Arabidopsis innate immunity.

Plants and animals possess innate immune systems to prevent infections and are effectively "nonhosts" for most potential pathogens. The molecular mechanisms underlying nonhost immunity in plants remain obscure. In Arabidopsis, nonhost/nonpathogenic Pseudomonas syringae sustains but pathogenic P. syringae suppresses early MAMP (microbe-associated molecular pattern) marker-gene activation. We performed a cell-based genetic screen of virulence factors and identified AvrPto and AvrPtoB as potent and unique suppressors of early-defense gene transcription and MAP kinase (MAPK) signaling. Unlike effectors of mammalian pathogens, AvrPto and AvrPtoB intercept multiple MAMP-mediated signaling upstream of MAPKKK at the plasma membrane linked to the receptor. In transgenic Arabidopsis, AvrPto blocks early MAMP signaling and enables nonhost P. syringae growth. Deletions of avrPto and avrPtoB from pathogenic P. syringae reduce its virulence. The studies reveal a fundamental role of MAMP signaling in nonhost immunity, and a novel action of type III effectors from pathogenic bacteria.

Arabidopsis↗
PubMed2004

Arabidopsis DND2, a second cyclic nucleotide-gated ion channel gene for which mutation causes the "defense, no death" phenotype.

A previous mutant screen identified Arabidopsis dnd1 and dnd2 "defense, no death" mutants, which exhibit loss of hypersensitive response (HR) cell death without loss of gene-for-gene resistance. The dnd1 phenotype is caused by mutation of the gene encoding cyclic nucleotide-gated (CNG) ion channel AtCNGC2. This study characterizes dnd2 plants. Even in the presence of high titers of Pseudomonas syringae expressing avrRpt2, most leaf mesophyll cells in the dnd2 mutant exhibited no HR. These plants retained strong RPS2-, RPM1-, or RPS4-mediated restriction of P. syringae pathogen growth. Mutant dnd2 plants also exhibited enhanced broad-spectrum resistance against virulent P. syringae and constitutively elevated levels of salicylic acid, and pathogenesis-related (PR) gene expression. Unlike the wild type, dnd2 plants responding to virulent and avirulent P. syringae exhibited elevated expression of both salicylate-dependent PR-1 and jasmonate and ethylene-dependent PDF1.2. Introduction of nahG+ (salicylate hydroxylase) into the dnd2 background, which removes salicylic acid and causes other defense alterations, eliminated constitutive disease resistance and PR gene expression but only weakly impacted the HR- phenotype. Map-based cloning revealed that dnd2 phenotypes are caused by mutation of a second CNG ion channel gene, AtCNGC4. Hence, loss of either of two functionally nonredundant CNG ion channels can cause dnd phenotypes. The dnd mutants provide a unique genetic background for dissection of defense signaling.

Arabidopsis↗
PubMed2006

Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors.

Limited information is available about the roles of specific WRKY transcription factors in plant defense. We report physical and functional interactions between structurally related and pathogen-induced WRKY18, WRKY40, and WRKY60 transcription factors in Arabidopsis thaliana. The three WRKY proteins formed both homocomplexes and heterocomplexes and DNA binding activities were significantly shifted depending on which WRKY proteins were present in these complexes. Single WRKY mutants exhibited no or small alterations in response to the hemibiotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. However, wrky18 wrky40 and wrky18 wrky60 double mutants and the wrky18 wrky40 wrky60 triple mutant were substantially more resistant to P. syringae but more susceptible to B. cinerea than wild-type plants. Thus, the three WRKY proteins have partially redundant roles in plant responses to the two distinct types of pathogens, with WRKY18 playing a more important role than the other two. The contrasting responses of these WRKY mutants to the two pathogens correlated with opposite effects on pathogen-induced expression of salicylic acid-regulated PATHOGENESIS-RELATED1 and jasmonic acid-regulated PDF1.2. While constitutive expression of WRKY18 enhanced resistance to P. syringae, its coexpression with WRKY40 or WRKY60 made plants more susceptible to both P. syringae and B. cinerea. These results indicate that the three WRKY proteins interact both physically and functionally in a complex pattern of overlapping, antagonistic, and distinct roles in plant responses to different types of microbial pathogens.

Amino Acid Motifs↗
PubMed2006

NDR1 interaction with RIN4 mediates the differential activation of multiple disease resistance pathways in Arabidopsis.

Recognition of pathogens by plants involves the coordinated efforts of molecular chaperones, disease resistance (R) proteins, and components of disease resistance signaling pathways. Characterization of events associated with pathogen perception in Arabidopsis thaliana has advanced understanding of molecular genetic mechanisms associated with disease resistance and protein interactions critical for the activation of resistance signaling. Regulation of R protein-mediated signaling in response to the bacterial pathogen Pseudomonas syringae in Arabidopsis involves the physical association of at least two R proteins with the negative regulator RPM1 INTERACTING PROTEIN4 (RIN4). While the RIN4-RPS2 (for RESISTANCE TO P. SYRINGAE2) and RIN4-RPM1 (for RESISTANCE TO P. SYRINGAE PV MACULICOLA1) signaling pathways exhibit differential mechanisms of activation in terms of effector action, the requirement for NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) is shared. Using a yeast two-hybrid screen, followed by a series of coimmunoprecipitation experiments, we demonstrate that the RIN4-NDR1 interaction occurs on the cytoplasmically localized N-terminal portion of NDR1 and that this interaction is required for the activation of resistance signaling following infection by P. syringae expressing the Cys protease Type III effector protein AvrRpt2. We demonstrate that like RPS2 and RPM1, NDR1 also associates with RIN4 in planta. We suggest that this interaction serves to further regulate activation of disease resistance signaling following recognition of P. syringae DC3000-AvrRpt2 by Arabidopsis.

Arabidopsis↗
PubMed2005

Spatial organization of dual-species bacterial aggregates on leaf surfaces.

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% +/- 8.2%) than that in monospecific aggregates of these two strains (1.6% +/- 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.

Bacterial Adhesion↗
PubMed1999

The transcriptional activator CorR is involved in biosynthesis of the phytotoxin coronatine and binds to the cmaABT promoter region in a temperature-dependent manner.

A modified two-component regulatory system consisting of the histidine protein kinase CorS and two highly homologous response regulators, CorR and CorP, controls biosynthesis of the polyketide phytotoxin coronatine (COR) by Pseudomonas syringae pv. glycinea PG4180 in a temperature-dependent manner. COR synthesis is maximal at 18 degrees C but does not occur at 28 degrees C. Fusions of CorR and CorP to the maltose-binding protein (MBP) were overproduced in Escherichia coli and P. syringae PG4180, and tested for functionality by complementation of corR and corP mutants of PG4180, respectively. The cmaABT promoter region was defined by deletion mapping, and the DNA-binding capability of CorR and CorP was examined by gel retardation assays. When overproduced in P. syringae at 18 degrees C and purified, MBP-CorR was shown to bind specifically to a 218-bp DNA fragment corresponding to positions -841 to -623 bp upstream of the transcriptional start site of the cmaABT operon. In contrast, MBP-CorP and MBP itself, when overproduced in P. syringae and E. coli at 18 degrees C and 28 degrees C, respectively, did not bind to the 218-bp fragment or to any other DNA fragment analyzed. The CorP protein lacks typical DNA-binding motifs, suggesting that it might modulate the function of CorR. However, addition of purified MBP-CorP did not alter the DNA-binding activity of MBP-CorR. On the other hand, this activity was completely abolished when MBPCorR was overproduced at 28 degrees C or in a corS mutant, indicating that the binding of CorR depended on the growth temperature at which it was produced and was controlled by CorS. In addition, overproduction of MBP-CorR in a corP mutant of PG4180 also yielded inactive protein, underlining the importance of CorP for CorR activation. We propose that CorR is activated by CorS at low temperature and that CorP is required for this activation before CorR can bind to DNA.

ATP-Binding Cassette Transporters↗
PubMed2003

Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells.

Many bacterial pathogens cause disease by injecting virulence proteins (effectors) into host cells via the specialized type III secretion system. Recently, exceptional progress in identifying effectors was made in the phytopathogen Pseudomonas syringae using a novel genetic screen and bioinformatic approach. These studies, along with localization experiments, suggest that most P. syringae effectors function by targeting the plasma membrane, chloroplasts or mitochondria of host cells. The type III secretome of P. syringae is highly variable and dynamic, a lesson gleaned from a comparative genomic analysis. Variation in the effector repertoire is likely to facilitate the adaptation of P. syringae to different hosts.

Amino Acids↗
PubMed1995

Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression.

The roles of salicylic acid (SA) and H2O2 in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a-GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae, SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H2O2 and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H2O2 leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 microM. Leaf disks preincubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H2O2-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.

Amitrole↗
PubMed1998

The defense-related rice gene Pir7b encodes an alpha/beta hydrolase fold protein exhibiting esterase activity towards naphthol AS-esters.

Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance-inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS-acetate. Using Pir7b-specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.

Amino Acid Sequence↗
PubMed1994

Isolation of phytoalexin-deficient mutants of Arabidopsis thaliana and characterization of their interactions with bacterial pathogens.

A genetic approach was used to assess the extent to which a particular plant defense response, phytoalexin biosynthesis, contributes to Arabidopsis thaliana resistance to Pseudomonas syringae pathogens. The A. thaliana phytoalexin, camalexin, accumulated in response to infection by various P. syringae strains. No correlation between pathogen avirulence and camalexin accumulation was observed. A biochemical screen was used to isolate three mutants of A. thaliana ecotype Columbia that were phytoalexin deficient (pad mutants). The mutations pad1, pad2, and pad3 were found to be recessive alleles of three different genes. pad1 and pad2 were mapped to chromosome IV and pad3 was mapped to chromosome III. Infection of pad mutant plants with strains carrying cloned avirulence genes revealed that the pad mutations did not affect the plants' ability to restrict the growth of these strains. This result strongly suggests that in A. thaliana, phytoalexin biosynthesis is not required for resistance to avirulent P. syringae pathogens. Two of the pad mutants displayed enhanced sensitivity to isogenic virulent P. syringae pathogens, suggesting that camalexin may serve to limit the growth of virulent bacteria.

Arabidopsis↗
PubMed2006

Suppression of defense response in plants by the avrBs3/pthA gene family of Xanthomonas spp.

Effector genes of some plant-pathogenic bacteria, including some members of the avrBs3/pthA effector gene family from Xanthomonas spp., confer not only genotype-specific disease resistance but also pathogen aggressiveness or virulence. In addition, some effector gene products suppress induction of a nonspecific (or general) hypersensitive response (HR). To determine whether the Xanthomonas avrBs3/pthA gene family members apl1, avrXa7, or avrXa10 also confer suppressor activity, we introduced constructs with each effector gene into Pseudomonas fluorescens 55 that expressed the entire hrp cluster from P. syringae pv. syringae in cosmid pHIR11. When inoculated to tobacco 'Bright Yellow', P fluorescens (pHIR11) induces the HR and expression of four tobacco defense response genes: HIN1, RbohB, PAL, and PR1. When P. fluorescens double transformants that contained pHIR11 and constructs with apl1, avrXa7, or avrXa10 were infiltrated into tobacco, the HR and expression of three defense response genes, RbohB, PAL, and PR1, were suppressed. The suppression of the HR and defense gene expression was more efficient in the transformants with the apl1 and avrXa7 than the transformant with avrXa10. Although expression of other defense genes was suppressed by the double transformants, HIN1 expression was the same level as was observed after infiltration with P. fluorescens (pHIR11), suggesting that HIN1 may not be involved directly in HR. Taken together, our data suggest that avrXa7, avrXa10, and apl1, when delivered to plant cells by the P. syringae pv. syringae hrp secretion system, can suppress nonhost HR and associated phenotypes.

Bacterial Proteins↗
PubMedDate not supplied

Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase in Pseudomonas viridiflava.

Four pleiotropic mutants of Pseudomonas viridiflava strain PJ-08-6A that were deficient in production of both pectate lyase (Pel) and protease (Prt) were isolated following transposon mutagenesis. Unlike secretion-defective (Out-) mutants, these four showed no accumulation of enzymes within the cells. Southern hybridization analysis revealed that each mutant had Tn5 inserted in one of two EcoRI genomic fragments. These EcoRI fragments (5.2- and 6.3-kb) appeared to contain two distinct gene loci, designated repA and repB, which were required for production of extracellular enzymes in this bacterium. Cosmid clones carrying the functional repA and repB DNA fragments were identified in a genomic library of strain PJ-08-6A. After analysis of repA+ plasmids by restriction mapping and marker-exchange mutagenesis, the repA gene was located in a joint region between the 1.8-kb EcoRI-HindIII and 2.8-kb EcoRI fragments cloned. Nucleotide sequence analysis of the repA region revealed the presence of an open reading frame consisting of 2,790 bases. The RepA protein predicted from the DNA sequence showed 93% similarity in amino acid sequence to the LemA protein of P. syringae pv. syringae, which was previously identified as a member of a two-component global regulatory system. A plasmid carrying the lemA gene of P. syringae pv. syringae was capable of complementing the RepA- mutation in P. viridiflava. The functions of the repA and lemA genes thus appear to be similar and interchangeable. Mutants of P. viridiflava strain SF312A deficient in production of Pel, Prt, and the exopolysaccharide alginate also were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Alginates↗