Testing the Hypersensiti ye Response

Gene-for-gene resistance is usually accompanied by the hypersensitive response (HR), a form of localized cell death that occurs in response to pathogen challenge. P. syringae strains carrying one of the avirulence genes avrRpt2, avrRpml, avrB, avrPphB, and avrRps4 trigger the HR

in wild-type Columbia (for review, see Glazebrook et al. 1997). Other ecotypes may lack one or more of the R genes needed for recognition of these avirulence genes.

To test for the HR, it is best to use syringe inoculation and to inoculate only part of each leaf. This way, the lesion appears as a dry collapsed zone surrounded by healthy tissue and can be detected easily. The inoculation dose should be quite high; try an OD600 range between 0.01 and 0.1. Be sure to inoculate some plants with 10 mM MgS04 as a negative control. If the leaves are crushed with the syringe, the resulting lesions may be confused with the HR. The HR should appear after 6-24 hours, depending on environmental conditions and avirulence gene used. If the inoculation dose is very low, the HR will still occur, but the lesions will be very small. Microscopic lesions can be detected by staining with Trypan blue (see below, Trypan Blue Stain).

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