The repetitive effector Rsp3
Rsp3 is predicted to be a non-processed repeat-containing protein lacking a GPI anchor. Downstream of the signal peptide, a glutamine and proline rich region is followed by a cysteine-rich region. The long C-terminal domain comprising more than half of the molecule consists of a complex array of several different repetitive units. In field isolates rsp3 shows strong length polymorphisms resulting from deletions and rearrangements in the C-terminal domain. rsp3 is highly expressed during the biotrophic stage and is required for virulence and anthocyanin accumulation. Compared to wild type infections rsp3 mutants accumulate only little fungal biomass in infected tissue. By immunolocalization we have shown that Rsp3 is attached to and decorates the surface of biotrophic hyphae. To elucidate the function of Rsp3 we have identified maize interactors of Rsp3 by co-immunoprecipitation. These studies have shown that Rsp3 interacts with at least two secreted maize DUF26 domain-family proteins designated AFP1 and AFP2. These proteins are related to a mannose-binding antifungal protein from Ginkgo biloba seeds. We have shown that the AFP1 maize protein also binds mannose and displays antifungal activity against the rsp3 mutant but not against a strain constitutively expressing rsp3 (Figure 2). Maize plants silenced for AFP1/2 partially rescue the virulence defect of rsp3 mutants, suggesting that blocking the antifungal activity of AFP1/2 by the Rsp3 effector is an important virulence function. Rsp3 orthologs are present in all smut fungi and the ortholog from Sporisorium reilianum can complement the rsp3 mutant of U. maydis, suggesting a novel widespread fungal protection mechanism. Interestingly, we could also demonstrate that AFP1/2 silenced plants become more susceptible to the unrelated fungus Colletotrichum graminicola. This reveals that the antifungal maize proteins we have discovered play a more general role in restricting growth of plant-infecting fungi.