Function, structure and evolution of the RXLR effector AVR3a of Phytophthora infestans

Plant pathogens secrete effector proteins that modulate the defense circuitry of their host cells, thereby enabling parasitic colonization. The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete effectors, AVR3a carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The objective of my thesis research was to characterize the AVR3a effector to obtain a better understanding of the molecular mechanisms underlying P. infestans infection as well as host resistance. I investigated the structure and function of AVR3a by using the Nicotiana benthamiana experimental system. Co-expression assays showed that in addition to its avirulence activity, AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans pointing to a possible virulence function for this effector. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which does not include the RXLR region, is sufficient for avirulence and suppression functions. To gain further insight in the structural basis of AVR3a effector function I performed high-throughput mutagenesis screens and targeted mutagenesis of both forms of AVR3a. I identified 136 AVR3a EM gain of function mutants that activated R3a but only 13 AVR3a KI loss of function mutants. Interestingly, most AVR3aEM gain of function mutants did not suppress cell death. Thus, distinct amino acid residues of AVR3a condition R3a hypersensitivity and cell death suppression. Our results point to a model wherein R3a-mediated recognition of AVR3a most likely involves the interaction with one ore more host protein rather than an enzymatic activity. I investigated how AVR3a effector functions evolved in Phytophthora using functional analyses of Avr3a homologs from P. infestans, P. sojae and P. capsici. R3a activation was conserved among homologs from different Phytophthora species, P. infestans and P. sojae. These homologs did not suppress INF1 cell death, providing independent evidence that the two AVR3a effector functions are not tightly linked at the structural level. Also, these data show that recognition by R3a is a more widespread activity of the AVR3a family in Phytophthora than cell death suppression. Future studies, aimed at obtaining the three-dimensional structure of AVR3a and identification of plant interacting proteins will provide further insights on the molecular mechanisms underlying AVR3a effector functions.