Functional profiling of candidate RXLR effector candidates for response in Solanum.

Mining of RXLR effectors in P. infestans resulted in 54 predicted extracellular (Pex) candidate effectors containing an RXLR-DEER (RD) motif. Some of these PexRD belong to known gene families, others represent unknown genes or gene families of up to 5 members or alleles of candidate effectors. All candidates were cloned into the Potato virus X (PVX) expression vector pGR106 [42] in A. tumefaciens enabling functional profiling for hypersensitivity on Solanum[13] (1). The strains were toothpick-inoculated in leaves of ten resistant wild Solanum genotypes of wide taxonomic diversity [16], R3a-resistant Desiree [41] and the susceptible RH89-039-16 and potato cultivar Bintje (Table 1). The empty pGR106-empty vector was included as a negative control, and pGR106-Crn2 which induces non-specific necrosis [32], [40] as a positive control. At least 8 replicates were inoculated, and necrotic responses occurring at frequencies higher (+) or lower (?) than 30% of the inoculated sites are presented. Candidate effectors are classified in specific vs. non-specific, representing candidates that showed response specifically in resistant plants or in both resistant and susceptible plants, respectively.1PVX agroinfection was developed as a sensitive screening system [13], yet PVX may cause overproduction of the effector compared to the natural situation. Therefore, candidates identified in this PVX essay need subsequent confirmation using different methods such as agroinfiltration [20], [21] (Figure 2). For example, the observed cell death in Sto17605-4 and Blb8005-8 to pGR106-IpiO4 (PexRD6-3) might reflect such oversensitive reaction: no response to pGR106-IpiO4 was detected neither in the Sto��RH population using the PVX essay (Table 3), nor in N. benthamiana co-infiltrated with A. tumefaciens strains expressing IpiO4 and Rpi-sto1 (Figure S2).2Specific R3a-Avr3a interaction3nd, not determined