Progress 09/15/01 to 09/14/05
Outputs
Whole-cell bacterial biosensors of Pantoea agglomerans, a common epiphytic species, were used to measure the water potential sensed by bacteria on leaves in a growth chamber under conditions favorable for epiphytic growth. These cells sensed water potentials of at least -0.3 to -0.5 MPa, which were water potentials that permitted strong growth of this species in culture. Inoculation of the plants at 3 relative humidity levels showed that the relative humidity influenced the rate of decrease in the water potential sensed by the cells, but not the water potential sensed once steady-state conditions were achieved. When whole-cell P. agglomerans biosensors were introduced onto leaves under field conditions, their ability to estimate water potential was severely limited by GFP photobleaching in sunlight and by attenuated induction of the transcriptional fusion. Such attenuation probably resulted from the physiological demands of responding to a highly stressful environment,
and indicates a potential limitation of transcription-based bioreporters. Endophytic populations of the foliar bacterial pathogen Pseudomonas syringae pv. tomato and its avirulent derivatives were negatively correlated with the water potential of the apoplast, suggesting that water availability within leaves is a major determinant of endophytic population size. Furthermore, after introduction of avirulent P. syringae cells into endophytic sites, the water potential in these sites decreased rapidly to -1.6 to -2.2 MPa; these levels were low enough to prevent cell division in the majority of cells and to significantly delay division in those few that could divide, based on their growth at these water potentials in culture. These results indicate that water potential reduction may be at least one means by which a plant restricts the growth of bacterial cells during a hypersensitive defense response. In the absence of such a defense response, endophytic P. syringae pv. tomato cells
encountered water potentials of -0.4 to -0.9 MPa, which permitted strong growth in culture. Moreover, nonpathogenic bacterial species encountered water potentials of only -0.3 to -0.4 MPa after their introduction into endophytic sites, indicating that the subsequent lack of growth of these bacteria was not related to limited water availability. When the impact of low water availability on the growth and culturability of 3 P. syringae strains was examined, P. syringae pv. syringae strain B728a, which is considered to be a strong epiphyte, exhibited significantly more tolerance to water stress than did P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A. All three strains were found to produce the same compatible solutes, although the relative amounts were strain-specific, and to derive protection from similar exogenously provided solutes, although the dynamics of this protection differed among the strains; thus, the physiological basis for the distinct levels of
tolerance to water stress was not identified. This was the first characterization of the detailed adaptative response of the common leaf-associated species, P. syringae, to low water potentials.
Impacts
These findings improve our mechanistic understanding of how plants defend themselves against pathogens by indicating that plants may actively modulate water availability to limit the growth of bacterial invaders. This mechanistic understanding is important to efforts to improve disease resistance in plants, to plant disease management, and to plant disease forecasting, which often uses water status measurements as key input parameters. Furthermore, by improving our understanding of the ecology of plant-associated microorganisms, these findings strengthen the foundation of ecological knowledge needed for the successful introduction and manipulation of agonomically beneficial microorganisms.
Publications
- Wright, C. A. 2003. Construction and use of bacterial sensors to investigate the role of water deprivation in bacterial-plant interactions. Ph.D. Thesis, Iowa State University.
- Wright, C. A. and G. A. Beattie. 2004. Bacterial species specificity in proU osmoinducibility and nptII and lacZ expression. Journal of Molecular Microbiology and Biotechnology 8:201-208
- Beattie, G. A. and C. A. Axtell. 2002. Role of water availability during colonization of leaf surfaces. Phytopathology 92:S97.
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Progress 01/01/04 to 12/31/04
Outputs
We previously reoriented our focus in objective I to identify how bacterial exposure to water stress affects bacterial growth and death within leaves rather than on leaves. Having demonstrated that after infection of a resistant plant, pathogenic Pseudomonas syringae cells experienced water stress severe enough to restrict their growth but after infection of susceptible plants they did not, we introduced various saprophytic bacteria into leaves and evaluated their exposure to water stress. Based on the low induction of a water stress-responsive transcriptional fusion, our results indicate that the lack of growth of these bacteria within leaves was not related to increased exposure to water stress following their introduction. We previously characterized the impact of water stress on the growth characteristics of the saprophyte Pantoea agglomerans for objective II. We have now extended these studies to include several Pseudomonas syringae strains: the epiphytically
adapted P. syringae pv. syringae strain B728a and the pathogens P. syringae pv. tomato DC3000 and pv. phaseolicola 1488A. We have found a surprising amount of diversity in the impact of various water stress levels on their growth dynamics and survival. We have been characterizing the physiological responses of these strains to water stress in order to identify physiological and molecular targets that, when altered, will lower their water stress tolerance. The resulting sensitive strains will be used to identify how water stress affects patterns of bacterial growth and death within and on leaves.
Impacts
This study is continuing to improve our mechanistic understanding of how plants defend themselves against invasion by bacteria, including pathogens and nonpathogens, and thus should contribute to efforts to improve disease resistance. Knowledge of how water stress affects the growth, physiology and survival of plant-associated bacteria is important to our understanding of the ecology of these organisms, and should contribute to improved strategies for pathogen control and successful deployment of agriculturally beneficial bacteria onto plants.
Publications
- Wright, C. A. and G. A. Beattie. 2004. Pseudomonas syringae pv. tomato cells encounter inhibitory levels of water stress during the hypersensitive response of Arabidopsis thaliana. Proceedings of the National Academy of Sciences USA 101:3269-3274.
- Chen, C. and G. A. Beattie. 2004. Osmosensitivity and osmoprotection of selected Pseudomonas syringae strains. Phytopathology 94:S17.
- Chen, C. and G. A. Beattie. 2004. Critical evaluation of green fluorescent protein-based bioreporters deployed in stressful environments. Phytopathology 94:S17.
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Progress 01/01/03 to 12/31/03
Outputs
Transcriptional fusion-based bacterial biosensors have become increasingly popular for use in evaluating target characteristics of a microbe's environment, such as the nutritional conditions or the bioavailability of pollutants. Studies with water deprivation-responsive biosensors showed that water deprivation is likely a major mechanism by which resistant plants restrict the endophytic growth of bacteria (described below). However, their use also showed that bacterial exposure to environmental stresses that are severe enough to cause death in a portion of a population can negatively impact the physiology of the surviving cells and result in general reductions in transcriptional activity. Thus, although transcriptional fusion-based bacterial biosensors can be an effective measurement tool under some conditions, they are not effective under highly stressful conditions. This finding is significant to a broad range of studies in environmental microbiology that utilize
such sensors. It has important implications for our studies in that it restricts the use of our biosensors as tools for identifying water deprivation-driven population dynamics to conditions that do not cause significant death. During plant defense against bacterial pathogens, the hypersensitive response (HR) functions to restrict the growth and spread of the pathogen. Use of inaZ-based water deprivation-responsive biosensors showed that the water potential in the leaf apoplast significantly decreased during the HR and reached levels low enough to restrict bacterial growth. Specifically, these water potentials were low enough to prevent cell division in the majority of cells and to significantly delay division in those few cells that could divide. This water potential decrease occurred very rapidly (within 4 hr after bacterial infection). In contrast, the pathogen Pseudomonas syringae pv. tomato encountered apoplastic water potentials that were associated with optimal growth in
culture. Among the strains tested, the population sizes in the apoplast were significantly correlated with the apoplastic water potential, suggesting that water availability within leaves is a major determinant of bacterial population sizes in leaves, and that water potential reduction may be at least one means by which a plant restricts the growth of bacterial cells during the HR.
Impacts
This study is continuing to provide critical information linking knowledge of the water status of leaves to the physiology of bacteria associated with those leaves. This information is improving our mechanistic understanding of plant disease resistance, and thus should contribute to efforts to improve resistance, as well as to our understanding of the ecology of plant-associated microorganisms. This ecological foundation is needed for the successful introduction and manipulation of agronomically beneficial microorganisms.
Publications
- Beattie, G. A. and C. A. Axtell. 2002. The use of a proU-gfp transcriptional fusion to quantify water stress on the leaf surface, pp. 235-240. In: S. A. Leong, C. Allen, and E. W. Triplett (eds.), Biology of Plant-Microbe Interactions, vol. 3. International Society for Plant-Microbe Interactions, St. Paul, MN.
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Progress 01/01/02 to 12/31/02
Outputs
Sampling methods for field use of the water deprivation-responsive biosensors were optimized. This optimization included establishing suitable sample recovery and preservation methods. Initial field trials indicated an unexpected decrease in the fluorescence of the control strain that constitutively produced GFP. The lack of a similar decrease in the fluorescence of the biosensor cells provided evidence that the bacterial cells were exposed to water deprivation on leaves under the conditions tested; however, their level of exposure could not be accurately assessed. Three modified strategies for quantifying water deprivation were being explored. First, in vitro studies demonstrated that exposure to sunlight decreased GFP fluorescence, and promising results were observed with shaded plants in the field. Second, biosensor strains employing an ice nucleation reporter protein instead of GFP were demonstrated to report similar levels of water deprivation on leaves as the
GFP-based reporters. Third, the accumulation of compatible solutes in solute-amended bacterial suspensions is being evaluated as an independent quantification method for assessing water deprivation exposure levels.
Impacts
Microorganisms play a key role in agricultural productivity and environmental quality. Advances in our knowledge of the ecology of these organisms are closely tied to the research tools that are available. These findings illustrate a potential limitation to one of the most important research tools to become available for ecological studies this decade, fluorescent reporter proteins.
Publications
- No publications reported this period
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Progress 09/15/01 to 12/31/01
Outputs
The effect of water deprivation on the culturability and growth of the common leaf-associated species Pantoea agglomerans was investigated using cells grown on a solid minimal medium containing various concentrations of sodium chloride to impart low water potentials. Exposure of P. agglomerans cells to NaCl concentrations greater than 660 mM (-3 MPa) were toxic; exposure to 660 mM NaCl induced elevated EPS production and repressed pigment production; and exposure to NaCl concentrations between 300 and 660 mM reduced the rate of growth. A major effect of water deprivation on P. agglomerans growth was in the time required to detect growth on a solid medium. Specifically, with surface-grown cells, the lag time was linearly related to the NaCl concentration over the range 0 to 660 mM. We observed a similarly linear correlation between lag time and polyethylene glycol 8000 concentration; this compound confers water deprivation via desiccation rather than elevated
osmolarity. Using a bacterial biosensor that responds to water deprivation, we have found that P. agglomerans cells on bean leaves in a growth chamber are exposed, on average, to water potentials equivalent to those imposed by only 50 to 100 mM NaCl. Thus, bacteria on leaves under the conditions tested appear to be exposed to water deprivation levels that are sufficiently high to influence their lag time, i.e. their time before cell division, but are not sufficiently high to prevent growth or even reduce their growth rate.
Impacts
This study is providing critical information linking knowledge of the water status of leaves to the physiology of bacteria on those leaves. This information is important to plant disease forecasting, which often uses water status measurements as a key input parameters, plant disease management, and a general understanding of microbial ecology.
Publications
- No publications reported this period
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