Progress 08/15/06 to 08/14/09
Outputs
OUTPUTS: The rate of swarming motility is inversely proportional to the concentration of available iron in Pseudomonas syringae. The rate of swarming motility is related directly to the number of lesions formed by P. syringae in susceptible hosts such as bean. The incidence of infection of bean under field conditions is reduced by up to 11-fold by topical applications of iron salts. Isogenic strains differing in swarming motility incited lesion numbers after topical application to plants that was directly related to their rate of swarming movement in culture. Pyoverdine siderophores produced by all P. syringae strains appear to be necessary for fitness on leaves of high tannin plants; pyoverdine siderophore mutants of P. syringae are incapable of growth on plants high in tannin, while those blocked only in achromobactin can still multiple well. The genome sequence of strain B728a reveals a gene encoding a putative tanninase. Mutants blocked in this enzyme differ somewhat in their ability to grown on tannin-containing culture media. The hypothesis is being tested that two different acyl homoserine lactone (AHL) acylases of P. syringae degrade the AHLs nessessary for antibiotic production by neighboring cells on plants. Only a very small subset epiphytic antibiotic-producing strains also produced AHLs and only a small subset of these appeared to use AHL-mediated signaling to control antibiotic production. In addition to degrading AHLs the AHL acylases produced by P. syringae could also degrade several of the antibiotics produced by other epiphytic bacteria. Thus, the substrates of AHL acylases may be broader than that of AHLs alone, and they may have a more direct role in detoxifying molecules in or on plants as well. The super-regulation of AHL biosynthesis by iron in P. syringae has also been extensively examined. A mutant screen revealed that PtsP mutants over-expressed AHLs, even under conditions of low iron availability. Subsequent work has now shown that this regulator appears to suppress AHL production under a range of iron conditions. Thus this super-regulator of quorum sensing is not specifically linked to iron homeostasis, but instead has a role in suppressing quorum sensing under a range of conditions. Fur and two small regulatory RNAs under the control of Fur (prrF1 and prrF2) also have now been shown to be required for iron stimulation of quorum sensing. Likewise a gene we call OqsR, a luxR-type regulator that is located in the genome away form any apparent genes encoding AHL synthases, also serves to negatively regulate quorum sensing in P. syringae. Studies are underway to determine if this super-regulation is directly at the level of transcription, or whether OqsR and AhlR may interact directly to alter their functions as transcriptional activators. Transcriptional profiling of a wild-type and AhlR mutant deficient in quorum sensing has now been completed. The size of the AHL-dependent regulon is surprisingly small, being less than about 20 genes. Most of the genes regulated in an AHL-dependent fashion and dependent on ahlR are in the vicinity of ahlR. PARTICIPANTS: Ryan Shepherd, Postdoctoral Scientist, performed studies that determined the role of iron in regulating the production of AHLs by P. syringae as well as iron regulation of acyl homoserine lactone acylases produced by P. syringae and the role of acyl homosreine lactone aclases on defense against antibiotic-producing neighbors on plants. Kavitha Mattaparti , undergraduate student performed studies of the antibiotic production by epiphytic bacteria and the dependence of antibiotic production on AHL-mediated signaling. Audrey Parangan, Graduate student, performed studies of regulation of genes in P. syringae that are influenced by accumulation of AHLs. Eugenia Ramos, Graduate student, performed studies of intercellular growth of various mutants of P. syringae in various plant species to determine the role of quorum sensing on endophytic fitness. Juliana Cho, graduate student studied the process of movement of P. syringae within plants and its dependence on iron levels and signal molecules. Russell Scott, graduate student studied the genes mediating the iron regulation of quorum sensing. TARGET AUDIENCES: The target audience includes researchers studying plant-microbe interactions. In addition, researchers interested in biological control of plant diseases will find the results highly relevant to their efforts to develop new methods to identify biological control agents. Growers of a variety of vegetable and fruit crops also will find the results to be useful in that new strategies of disease control are demonstrated. The fundamental process of iron regulation of quorum sensing will be of importance to the larger field of microbiology, particularly those many researchers who study pathogenesis of animals and humans by bacteria. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Plant pathogenic bacteria build up inoculum by growing on the surface of plants as epiphytes, thus increasing the risk of plant disease which would necessitate control measures. This project has studied the process whereby bacteria that live in large cell aggregates on leaves become tolerant of environmental stresses on plants and thus build up large populations by expressing sets of genes only found in such aggregates. The work has also shown that the accumulation of acyl homoserine lactone signal molecules within tissues during the infection process limits the extent of lesion formation. This observation suggests that elevation of signal molecules may be an effective means of disease control by altering pathogen behavior within plants. The identification of traits controlled by quorum sensing in Pseudomonas syringae, an important plant pathogen, should enable considerable insight into its interaction with the plant and how cells in cellular aggregates differ in their interaction with the plant. Iron also plays an important role in modulating quorum sensing. As low iron conditions iron suppresses quorum sensing, and thus the fitness of bacterial plant pathogens on plants, modulation of iron levels such as by topical application of iron chelators, the fitness of bacterial pathogens might be decreased. Such a strategy should reduce the incidence of frost damage which is incited by epiphytic populations of bacteria such as P. syringae. Conversely, as iron suppresses movement of bacteria its application might reduce the invasion of plants and hence be used for disease control.
Publications
- Shepherd, R.W., and S.E. Lindow. 2009. Two dissimilar N-acyl-homoserine lactone acylases of Pseudomonas syringae influence colony and biofilm morphology. Appl. Environ. Microbiol. 75:45-53.
|
Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Iron is essential in regulation of plant growth and development, and thus iron levels are thought to be tightly controlled in the plant., however, the status of iron in and on plants is unknown. The availability of iron also significant effects bacterial movement, and profoundly affects the virulence activity of bacteria. Preliminary data suggests that local iron concentrations greatly affect the virulence behavior of P. syringae on plants via its effect on quorum sensing control of motility and other traits. An ice nucleation-based iron biosensor was developed and used to detect iron levels within and on the surface of bean and other plants. The external levels of iron averaged around 5 uM, while internal levels of iron available averaged around 8.2 uM . Thus there is more iron inside the plant apoplast when compared to the leaf surface. This level is intermediate to that which confers a switch form low to high affinity iron uptake systems in bacteria; studies on high affinity uptake systems in P. syringae reveals the threshold iron level necessary to switch from a high affinity to low affinity uptake systems occur around a 5 uM threshold. P. syringae may be utilizing different iron sequestration strategies, depending on whether the bacteria are on the plant surface, or have made their way inside the plant apoplast. Motility is a factor that is highly suppressed by quorum sensing (QS), and appears crucial to formation of stress tolerant surface cell aggregates as well as for the spread of bacteria after invasion of the apoplast. However, the high population sizes of P. syringae in intercellular locations during lesion development would be expected to lead to quorum sensing and hence blockage of motility and hence stoppage of lesion expansion. We thus simultaneously monitored the process of growth and QS to understand lesion formation by this pathogen. The movement of P. syringae cells of a non-flagellated mutant, a mutant blocked in QS, or with a wild-type strain.inoculated into bean pods were measured by fluorescne microscopy as a function of time. Lesion sizes were recorded with the Lumar Stereoscope microscope as the area exhibiting watersoaking. After 48 hours, watersoaked lesions are clearly visible in both wild-type B728a and the QS mutant but not in pods inoculated with the non-motile mutant indicating that active motility is required for lesion formation. The rate of expansion of the QS mutant was 3- fold faster than that of the wild-type strain indicating the QS suppresses movement of P. syringae in the apoplast. Regulatory genes involved in iron dependent enhancement of QS in P. syringae have been identified analysis of random trnasposon mutants that expressed QS on a culture medium containing an iron chelator. Genes homologous to SigX and PtsP in other bacterial species were identified that were required for the blockage of QS in P. syringae under low iron conditions. In contrast, two genes with homology to ahlR (OqsR and Psyr1858) appear to play no role in iron-regulated QS since accumulation of acyl homoserine lactone is not altered when they are disrupted. PARTICIPANTS: Ryan Shepherd, Postdoctoral Scientist, performed studies that determined the role of iron in regulating the production of AHLs by P. syringae as wellas iron regulation of acyl homoserine lactone acylases produced by P. syringae. Audrey Parangan, Graduate student, performed studies of regulation of genes in P. syringae that are influenced by accumulation of AHLs. Eugenia Ramos, Graduate student, performed studies of intercellular growth of various mutants of P. syringae in various plant species to determine the role of quorum sensing on endophytic fitness. Juliana Cho, graduate student studied the process of movement of P. syringae within plants and its dependence on iron levels and signal molecules. Russell Scott, graduate student studied the genes mediating the iron regulation of quorum sensing. TARGET AUDIENCES: The target audience includes researchers studying plant-microbe interactions. In addition, researchers interested in biological control of plant diseases will find the results highly relevant to their efforts to develop new methods to identify biological control agents. Growers of a variety of vegetable and fruit crops also will find the results to be useful in that new strategies of disease control are demonstrated. The fundamental process of iron regulation of quorum sensing will be of importance to the larger field of microbiology, particularly those many researchers who study pathogenesis of animals and humans by bacteria. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Plant pathogenic bacteria build up inoculum by growing on the surface of plants as epiphytes, thus increasing the risk of plant disease which would necessitate control measures. This project has studied the process whereby bacteria that live in large cell aggregates on leaves become tolerant of environmental stresses on plants and thus build up large populations by expressing sets of genes only found in such aggregates. The work has also shown that the accumulation of acyl homoserine lactone signal molecules within tissues during the infection process limits the extent of lesion formation. This observation suggests that elevation of signal molecules may be an effective means of disease control by altering pathogen behavior within plants.
Publications
- Dulla, G. and S. E. Lindow. 2008. Iron-dependent quorum sensing controls epiphytic fitness and virulence in Pseudomonas syringae. M. Loretto, S. Woo, and F. Scala (eds). Biology of Plant-Microbe Interactions, Vol. 6. Proceedings of the 13th International Congress on Molecular Plant-Microbe Interactions. International Society for Molecular Plant-Microbe Interactions. ISBN 978-0-9654625-5-6
|
Progress 08/15/06 to 08/14/07
Outputs
OUTPUTS: Leaf extracts from 52 plant species were tested for their ability to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron to determine if plants could alter the chemical environment on leaves with respect to iron availability. Leaf washings from 16 of the 52 plant species as well as tannic acid solutions stimulated pyoverdine synthesis in Pseudomonas syringae in a high-iron medium. These compounds also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not that of the wild-type bacterium. The stimulation of siderophore production and growth inhibition by plant extracts and purified tannins were both reversed by the addition of ferric chloride to culture media indicating that iron was made unavailable by the compounds released onto the leaf surface. The use of a whole cell biosensor also revealed that the levels of iron available on plant surfaces of plants was much lower on those plants in
which putative iron sequestering compounds were detected. These results strongly suggest that plants have evolved mechanism to alter iron levels in a way that alters the behavior of potential plant pathogens.
PARTICIPANTS: Glenn Dulla was a graduate student who performed studies of quorum sensing on leaves by Pseudomonas syringae. Ryan Shepherd is a postdoctoral scientist who has examined the role of n-acyl homoserine lactones on the behavior of Pseudomonas syringae on leaves and in culture. Renee Koutsoukis is a research technician who performed studies of iron-mediated changes in behavior of Pseudomonas syringae on leaves in greenhouse and field studies. Steven Lindow is PI of the project and provided guidance to all of the other participants and also performed field studies of iron-mediated changes in behavior of Pseudomonas syringae. All of the participants gained valuable opportunities for advancing their technical skills in molecular biology as well as in the microbiology of palnt-associated bacteria in the project. In addition, all participated in weekly group discussions of the results with the aim to stimulate problem solving skills and synthesis of new data.
TARGET AUDIENCES: The target audience for the project are other scientists who work in the area of plant microbiology and plant pathology. The aim is to show how the chemical environment of the plant is mediated by the plant itself and that it has a large effect on hte accociated microflora. This is an important concept that will be forwarded by the on-going wrok. Another audience is growers of a wide variety of pants that suffer from bacterial diseases, especially those cuased by Pseudomonas syringae. The goals are to provide new methods of disease control based on altering the chemical environment of the leaf surface before infection occurs.
Impacts
The paradigm of bacterial quorum sensing (QS), which mediates cell-density-dependent gene expression, usually has been studied in high-cell-density planktonic liquid cultures or in biofilms in which signal concentrations accumulate to sufficiently high levels to induce QS. Presumably under conditions with restricted diffusion of the signal molecule, smaller population sizes could achieve such a state of QS induction. The plant-pathogenic bacterium Pseudomonas syringae, in which QS controls traits involved in epiphytic fitness and virulence, occurs on leaf surfaces in aggregates of various sizes. Because leaves often harbor limited surface water, we investigated the size of aggregates that would permit QS in a nonsaturated environment. QS induction was visualized via dual fluorescence of P. syringae cells harboring a transcriptional fusion of mRFP1 with ahlI, which exhibits N-acyl homoserine lactone-dependent transcriptional activity, and a constitutive GFP marker to
account for all P. syringae cells on a leaf. Confocal microscopy revealed that, on wet leaves, no QS induction was evident within 2 days after inoculation, but it increased rapidly with increasing aggregate sizes >40 and 22 cells per aggregate by 3 and 4 days, respectively. In contrast, QS induction was common in aggregates >33 cells by 2 days after inoculation on dry leaves and increased rapidly with increasing aggregate sizes >35 and 13 cells after 3 and 4 days, respectively. These observations demonstrate that small groups of cells experience QS conditions on dry leaves where signal diffusion is restricted. Quorum size of bacteria in non-water-saturated environments such as on leaves is small, and QS induction may be commonly operative.
Publications
- Jones, A.M., S.E. Lindow, and M.C. Wildermuth. 2007. Salicylic acid, yersiniabactin, and pyoverdine production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: Synthesis, regulation, and impact on tomato and Arabidopsis host plants. J. Bacteriol. 189:6773-6786.
- DeAngelis, K.M., M.K. Firestone, and S.E. Lindow. 2007 A sensitive whole-cell biosensor for detecting a variety of n-acyl homoserine lactones in rhizosphere microbial communities. Appl. Environ. Microbiol. 73:3724-3727.
|
|