Distinct amino acids of the Phytophthora infestans effector AVR3a condition activation of R3a hypersensitivity and suppression of cell death.

The AVR3a protein of Phytophthora infestans is a polymorphic member of the RXLR class of cytoplasmic effectors with dual functions. AVR3a(KI) but not AVR3a(EM) activates innate immunity triggered by the potato resistance protein R3a and is a strong suppressor of the cell-death response induced by INF1 elicitin, a secreted P. infestans protein that has features of pathogen-associated molecular patterns. To gain insights into the molecular basis of AVR3a activities, we performed structure-function analyses of both AVR3a forms. We utilized saturated high-throughput mutant screens to identify amino acids important for R3a activation. Of 6,500 AVR3a(EM) clones tested, we identified 136 AVR3a(EM) mutant clones that gained the ability to induce R3a hypersensitivity. Fifteen amino-acid sites were affected in this set of mutant clones. Most of these mutants did not suppress cell death at a level similar to that of AVR3a(KI). A similar loss-of-function screen of 4,500 AVR3a(KI) clones identified only 13 mutants with altered activity. These results point to models in which AVR3a functions by interacting with one or more host proteins and are not consistent with the recognition of AVR3a through an enzymatic activity. The identification of mutants that gain R3a activation but not cell-death suppression activity suggests that distinct amino acids condition the two AVR3a effector activities.