Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana.

The Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf. Prf is absolutely required for specific recognition of both effectors. Here, using stable transgenic lines, we show that expression of Pto from its genomic promoter in susceptible tomatoes was sufficient to complement recognition of Pseudomonas syringae pv. tomato (Pst) bacteria expressing either avrPto or avrPtoB. Pto kinase activity was absolutely required for specific immunity. Expression of the Pto N-myristoylation mutant, pto(G2A), conferred recognition of Pst (avrPtoB), but not Pst (avrPto), although bacterial growth in these lines was intermediate between resistant and susceptible lines. Overexpression of pto(G2A) complemented recognition of avrPto. Transgenic tomato plants overexpressing wild-type Pto exhibited constitutive growth phenotypes, but these were absent in lines overexpressing pto(G2A). Therefore, Pto myristoylation is a quantitative factor for effector recognition in tomato, but is absolutely required for overexpression phenotypes. Native expression of Pto in the heterologous species Nicotiana benthamiana did not confer resistance to P. syringae pv. tabaci (Pta) expressing avrPto or avrPtoB, but recognition of both effectors was complemented by Prf co-expression. Thus, specific resistance conferred solely by Pto in N. benthamiana is an artefact of overexpression. Finally, pto(G2A) did not confer recognition of either avrPto or avrPtoB in N. benthamiana, regardless of the presence of Prf. Thus, co-expression of Prf in N. benthamiana complements many but not all aspects of normal Pto function.