Plant Pathogens: Agrobacterium-Plant Interactions

Agrobacterium tumefaciens is a non-sporulating, Gram-negative rod commonly found in soil. Through sequence analysis, Agrobacterium and Rhizobium have been found to be so highly similar that Agrobacterium has recently been placed into the genus Rhizobium and has been renamed Rhizobium radiobacter. We will refer to this bacterium as Agrobacterium for the purpose of this experiment and because the commercial sources of this bacterial strain still refer to it by the old name. Like rhizobia, this microorganism has the ability to form a symbiotic relationship with a variety of plants. Unlike rhizobia whose interaction provides a direct benefit to the plant, the Agrobacterium interaction is parasitic in nature, causing crop loss in plants such as grapes, beets and some fruit trees. The benefit that Agrobacterium gains is that it is able to force the plant to provide a niche that only it can successfully colonize. Agrobacterium induces tumors, or "galls", on infected plants. All virulent, or tumorigenic, strains of Agrobacterium harbor a large tumor-inducing (Ti) plasmid. During infection, a specific section of this Ti plasmid, called the T-DNA (transferred-DNA), is physically transferred from the bacterial cell into the plant cell, where it becomes stably integrated into the plant chromosomal DNA. This T-DNA is expressed in plant cells, resulting in the expression of several "bacterial" genes. Some of these T-DNA genes code for enzymes that catalyze the synthesis of plant hormones (phytohormones) auxin and cytokinin. It is the overproduction of these hormones within the transformed plant cells that results in the uncontrolled cell division characteristic of a tumor. Overproduction of both of these phytohormones results in an unorganized tumor. However, when the auxin gene is mutated, the resulting Agrobacterium strain induces "shooty" tumors due to the overproduction of cytokinin. Similarly, when the cytokinin gene is mutated, the resulting tumors often display a "rooty" morphology due to the overproduction of auxin. In addition to genetic factors present in the bacterium, it is also important to recognize that other factors influence tumor growth and morphology, such as the plant host and the site of infection. The T-DNA also codes for enzymes that synthesize tumor-specific compounds called opines. Opines are usually simple derivatives of amino acids that can be utilized by Agrobacterium as a sole source of carbon, nitrogen, and energy. Recall that we learned about other organisms able to utilize proteins and amino acids as their sole energy source when we studied urea hydrolysis. Thus, Agrobacterium has evolved a very sophisticated scheme to provide a niche for itself. First it recognizes a suitable plant host and transfers its T-DNA into the plant genome. This results in the overproduction of phytohormones, causing tumor formation. In addition, the plant converts its photosynthetic products into opines that the infecting bacterium has the unique ability to catabolize. The bacteria then proliferate in this tumorous mass by utilizing opines as a food source.

A second region of the Ti-plasmid, called the VIR region is responsible for carrying out the T-DNA transfer process from the bacterium into the plant cell. The VIR region contains many genes, called VIR A-VIR G, all of which are required for efficient T-DNA transfer, and subsequent tumor formation, to occur. Mutation of any of these genes can reduce or abolish tumorigenesis.

Agrobacterium as a genetic engineering tool for plants: Agrobacterium is often called "nature's genetic engineer" because it has the natural ability to genetically transform dicotyledonous plant hosts. Although humans have been genetically engineering plants for a few decades, this remarkable bacterium has been successfully transforming plants for millions of years. In fact, our ability to introduce foreign DNA into plant cells is largely dependent on our knowledge of the Agrobacterium-plant interaction. As scientists determined the mechanism of Agrobacterium tumorigenesis, they recognized the potential for easily transferring genes of interest into plants. Many genetically modified organisms (GMOs) are generated by using Agrobacterium to transfer a modified T-DNA into plants. The Ti plasmid is

modified by removing the genes in the T-DNA that code for cytokinin and auxin production and replacing them with a gene of interest. The modified Agrobacterium is no longer a pathogen, in that it will not form tumors on plants, but instead it is now able to function as a tool to insert specific genes into a host plant. GMOs are useful in a research setting for determining the expression and function of a variety of genes. Scientists interested in studying the function of a particular gene can clone it into bacteria, manipulate it in some way, and then use Agrobacterium to reinsert that gene into the plant and observe the effect of the modified gene. In agriculture, GMO plants carrying insect or herbicide resistance genes allow farmers to grow crops using far less pesticides and herbicides than traditionally needed. Earlier in the quarter when studying the ability of some microorganisms to sporulate, we learned about the simultaneous production of a crystal toxin protein in the spore former Bacillus thuringiensis. Scientists have cloned the gene for the crystal toxin protein and introduced it into crop plants that were frequently targeted by nematode pests. These plants are now resistant to the pests without the addition of pesticides, which would normally be needed to control the nematode infestations. Other targets of genetic engineering include crop plants, which have been modified to have an increased yield, an increased tolerance to a normally detrimental environmental condition such as temperature extremes, or some other added benefit. Golden rice is an example of genetic engineering in which two genes, inserted into the rice genome, confer the ability to produce -carotene, an essential nutrient normally missing in the diets of many people in developing countries. (Ye X, Al-Babili S, Klti A, Zhang J, Lucca P, Beyer P, Potrykus I
(2000) Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303-305..)

In this experiment we will observe the effect of Agrobacterium and the Ti plasmid in promoting tumor formation on carrot slices.

Materials
Cultures: TSA streak plates of Agrobacterium tumefaciens (grown at 28oC): Wild-type strain A348 Mutant strain A1022 (auxin mutant) Mutant strain A1070 (cytokinin mutant) Kalanchoe diagremontiana, one plant per section Sterile toothpicks, small Colored tape for labeling Sterile water

Supplies

Lab period 1: Inoculation Procedure

1. Label plant or pot to indicate locations at which various Agrobacterium strains or control will be inoculated. Choose leaf and stem locations for inoculation sites. 2. Using a sterile toothpick, scrape plant tissue to wound plant. You do not need to push toothpick through plant. 3. Using a new sterile toothpick, pick up some Agrobacterium from the agar plate and inoculate wounded site. 4. For the control, the TA will pipet 50 ul of sterile water onto a wounded site. 5. After completing inoculations, plants will be placed in a growth room until our next observation.

Lab period 2: Observations

(about 6 weeks after inoculations) Observe the wound sites on the Kalanchoe plant and score for tumor formation. Note the size and morphology of the tumor. (-) avirulent (no tumorous response) (+) tumorous responses: unorganized tumor rooty tumor shooty tumor