ABSTRACTPlants are essential to our existence and host to a variety of bacteria. As a pathogen, Pseudomonas syringae causes diseases on a variety of plants and can also cause ice nucleation (frost damage). The amount of disease or frost damage it causes depends on

how many P. syringae cells are on a plant, which in turn depends on its ability to survive environmental stresses. The goal of our research is to understand how P. syringae responds to stress in its environment and by identifying the genes expressed in response to stresses. This may lead to ways of modifying the pathogen’s behavior to use it as a biological control agent, and to a better understanding of its ecology so we can predict how changes in the climate or agricultural practices will affect disease outbreaks. Our

findings thus far have provided us with the knowledge of the conditions that promote growth and maximize stress gene expression. We also completed the first step in creating P. Syringae strains that will be used to find which stress genes are regulated by which regulators. Eventually, the expression of all 5,217 P. syringae will be examined using a micro array to identify the effects of environmental stresses on P. syringae gene regulons.

PROTOCOLS

•Designing Primers and PCR •Transformation/conjugation •Leaf/bacteria microscopy

and live/dead staining •Leaf bacteria identification•Ice nucleation assay•Monitoring bacterial growth•Monitoring gene expression

BACKGROUND Plants are found almost everywhere on Earth and are a vital part of our existence. Each plant contains a microscopic world of bacteria. Pseudomonas syringae is a plant pathogen and the subject of our study. P.syringae has been known to cause diseases in plants and plays a key role in ice nucleation. It is the topic of study because of its influence on agriculture. The goal of our research is to understand the bacterial responses to stresses in its environment and how it relates to gene expression. Low iron availability, low water availability, low nitrogen availability, and high oxidative stress are four environmental conditions found on plant leaves that produce significant stress on bacteria on leaves. The responses to these stresses will allow us to look at gene networks and genetic regulators. An understanding of the bacteria’s genetic responses to stress, and how its responses to known stresses are similar to its responses to the leaf environment, will help our understanding of its ecology and thus to the climatic and environmental conditions that promote or minimize P. syringae plant diseases. may lead to ways of changing the pathogens behavior having significant effects on agriculture as a biological control agent.

DISCUSSIONBacteria are an integral part in human and plant life cycles. Figure 1 displays functions of bacteria on leaves. The long term goal of our research is to minimize disease and frost injury P.syringae on plants. In order for this to take place we need a better understanding of what makes the bacteria survive well on leaves. Figure 2 displays some key regulators and genes of interest that affect the bacteria’s responses to environmental stresses shown at the top in blue that influence survival.

To evaluate the extent to which live bacteria are recovered from bean plants, we inoculated plants with P.syrigae cells labeled with the green fluorescent protein (GFP) and covered them in plastic bags to allow for bacterial growth. After 24 hours we removed the bags from half of the plants and allowed the leaves to dry. We removed the bacteria from both the wet and dry leaves in a sonicating water bath. Since the bacteria contain GFP we could use a fluorescent microscope to view the bacteria. We then stained the bacteria with a stain for dead cells, propidium iodide. Live bacteria glow green and dead bacteria glow red under the appropriate fluorescent wavelength (figures 3-6). The wet leaves had significantly more live bacteria present then the dry leaves, although surprisingly at least half of the cells on the dry leaves were alive and thus can be used to identify the genes expressed on leaves.

Bacteria require several factors for efficient growth. Culture media contain the nutrients required and incubators allow for the optimum growth temperature. To complete our growth study we needed to find a standard medium that would support growth of B728a and all 4 mutant types (ropN, ropS, ropE, and hrpL). After various growth studies we determined that Min A was the best growth medium, as it supported rapid growth and high cell yields (figure 7). However it did not support gene expression of some key genes, including genes for syringomycin and effectors (figure 2). We found that modifying a medium that supports this expression, HMM, by adding succinate allowed for ideal growth (figure 8).

To overexpress genes encoding key regulators, we amplified a promoter, PA1/03/04, to fuse to these genes. Future work will involve fusing the promoter to the rpo/hrp genes.

CLASSROOM CONNECTIONS•Scientific process•Leaf bacteria identification•Ice nucleation applications•What supports bacterial growth?

Figure 3: Dead bacterial cells from dry bean leaf

Figure 4: Live bacterial cells from dry bean leaf

Figure 5: Dead bacterial cells from wet bean leaf

Figure 6: Live bacterial cells from wet bean leaf

GacA/S

EPIPHYTIC FITNESS PATHOGENICITY

WaterAvailability

(+/- salt)

Oxidative Stress

(+/- H2O2)

IronAvailability

(low vs high FeCl3)

RpoE RpoS RpoN

AlginateSyringomycin,Syringopeptin

Effectors

Type III

AlhR, AefR

SalAAutoinducers

HrpL

Alternate N uptake

/utilization

Sigma factors

Leaf surface

Nitrogen Availability

(low vs high N)Leaf

interior

Alter plant growth by producing plant growth hormones

Cause diseases of plants

(phytopathogens)

Cause diseases of animals &

humans (foodborne pathogens)

Cause frost injury in

plants (ice nucleating bacteria)

Promote plant growth

Kill or excludephytopathogens

or ice nucleating bacteria(biological control)

Degradeairborne pollutants

Fix nitrogen?

Figure 1: Functions of bacteria in the phyllosphere

Figure 2:

Effect of environmental stresses on regulators and genes

Figure 8: Growth curves of B728a. Comparison between HMM media and Min A media

Figure 7: Growth curves of P. syrinea B728a. Comparison between MinA, MG, SRM, and ½ 21C media.

Fitness and Pathogenicity of Pseudomonas syringae Bacteria

Erin L. Yoder, Lance Maffin, and Gwyn A. BeattieMaple Valley Anthon-Oto High School, Bondurant-Farrar Junior/Senior High School, and Iowa State University Department of Plant Pathology

RESEARCH PROJECTS

•Evaluate methods for recovering bacteria from leaf surfaces

•Identify medium that best supports the growth of P.syringae growth and stress gene expression

•Make strains that overexpress key gene regulators

REFERENCES

Beattie, G., Nettleton, D., Gross, D., & Lindow, S. (2007). Functional Genomics of the Pathogenic and Epiphytic Lifestyles of the Bacterial Plant Pathogen Pseudomonas syringae. USDA grant.

Hirano, S. H., & Upper, C. D. (2000). Bacteria in the Leaf Ecosystem with Emphasis on Pseudomonas Syringae - a Pathogen, Ice Nucleus, and Epiphyte. Microbiology and Molecular Biology Reviews, 64(3), 624-653.

Lindeberg, M. (2008). Pseudomonas Syringae Genome Resources Home Page. Retrieved June 17, 2008 from http://pseudomonas-syringae.org/.

Lindow, S. E. (1995). The use of reporter genes in the study of microbial ecology. Molecular Ecology, 4, 557-565.

Louisiana State University (2008, February 29). Evidence Of 'Rain-making' Bacteria Discovered In Atmosphere And Snow. ScienceDaily.

ACKNOWLEDGEMENTSWe would like to thank the Plant Genomics Outreach Program including Adah Leshem-Ackerman and Gwyn Beattie for their support. We would also like to acknowledge the Biotechnology Outreach Education Center and the Office of Biotechnology for their generous assistance with equipment and facility support. Last but not least we would like to thank all those who work in the Beattie and Halverson Labs for answering our many questions and helping us with protocols.

•Structures of leaves•PCR•Gel electrophoresis•Inquiry / questioning

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MinAMGSRM1/2 21C