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Dennis A. Halterman, USDA-ARS Vegetable Crops Research Unit, Madison, WI, Maya C. Hayslett, Victoria Kartanos, and Douglas I. Rouse, Department of Plant Pathology, University of Wisconsin, Madison

Instructor Notes

INSTRUCTOR'S NOTE #1

Discussion of learning goals

At the introductory level, we start by emphasizing the gene-for-gene model and the idea that host recognition of a pathogen effector is only required by one R gene product whether other effectors are recognized or not. In this experiment students will discover that two different effectors may interact with one R gene product to induce susceptibility. We do not tell the students of this possibility but allow them to discover for themselves this complication to the gene-for-gene model by doing the experiment and discussing the implications.

INSTRUCTOR'S NOTE #2

Preparation of materials

Timing:

• 7 weeks before: Start tobacco plants
• 5 weeks before: Transplant tobacco
• 3 days before: Start A. tumefaciens cultures on solid media
• 1 day before:  Start A. tumefaciens liquid cultures
• 1 to 4 h before: Make inoculum from liquid cultures
• 1 day after class: Make inoculum and reinoculate some treatments with IPI-O1 only
• 7 days after class: Observe plants for HR

How to obtain seed and pathogen:

Seeds and cultures are available from Dennis A. Halterman at the USDA/ARS Vegetable Crops Research Unit. An APHIS “notification” is required 10 days before the movement occurs for obtaining the transgenic tobacco plants. An APHIS permit is required for movement of the transformed A. tumefaciens.  Alternatively, you may obtain the DNA only without a permit and perform your own transformation. Contact Dennis A. Halterman, Dennis.Halterman@ ARS.USDA.GOV, for the necessary information to complete the permits, notifications, and ship material.

How to start tobacco plants:

Tobacco seeds are small and it is best to start many seeds at once and then transplant them to individual pots when they have a few leaves. Seven weeks before class, sprinkle some seeds in a small pot with some potting mix. It is important to keep the seeds moist but not wash them out of the pot when watering.  Until the plants have 2-3 leaves, water the pots from the bottom by placing them in a tray of water or cover them with lightly with clear plastic.  After two weeks, plants will be large enough to transplant to individual pots.

How to make inoculum:

Three days before class, start cultures of each A. tumefaciens strain on plates of LB agar + 50 µg/ml kanamycin and incubate at 28˚C. One day before class inoculate 10 ml of LB broth + 50 µg/ml kanamycin with a colony from the culture plate, one for each strain. Grow the liquid cultures with shaking at 28˚C overnight.

To make the inoculum from the liquid cultures, spin the culture for 5 min at 10,000 g to pellet cells. Resuspend cells with 2 ml of 10 mM MgCl₂ + 200 µM acetosyringone (acetosyringone induces expression of the Ti plasmid). Spin cells again for 5 min at 10,000 g. Resuspend cells with 2 ml 10 mM MgCl₂ + acetosyringone. Take an OD600 of suspension and dilute culture to an OD600 of 0.8. This can be used for inoculum. However, incubating cells at room temperature for 2 to 3 h is recommended (this gives A. tumefaciens time to express the effector).

INSTRUCTOR'S NOTE #3

Explanation of the expected results:

Since the RB gene is not present, there should be no reaction to any of the treatments on the non-transgenic plant. Also, there should be no response to the buffer (negative control). This treatment is used for comparison because there will be a small amount of damage to the leaf due to the inoculation method. The RB gene product recognizes the IPI-O1 effector, which leads to a resistance reaction visualized by a hypersensitive response. The RB gene product does not activate resistance in the presence of the IPI-O4 effector, so there is no visible response. The last treatment where leaves are inoculated with both effectors does not give the standard response for a gene-for-gene interaction. In a gene-for-gene interaction, you would expect the RB gene product to recognize the IPI-O1 effector, which would result in a hypersensitive response. The fact that IPI-O4 does not activate resistance should not matter. In a gene-for-gene interaction, the RB protein only needs to recognize one effector to elicit a response. However, in this system, IPI-O4 suppresses the resistance response. The response can be completely suppressed (no reaction) or partially suppressed (mixed reaction).