Fig 4. Non-lethal fragments of AvrE1 associate with Arabidopsis PP2A B’ regulatory subunits homologous to the WtsE-interacting proteins, as well as maize WIPs.

(A) Diagram of non-lethal fragments of AvrE1 effector protein. WxxxE motifs and the putative ERMRS motif are indicated by blue lines. (B-C) AvrE1 fragments associate with specific Arabidopsis PP2A B’ subunits following Agro-transient expression in N. benthamiana. GFP-B’α, β, ξ, ι, η, or free GFP were co-expressed with AvrE1-HA fragments in N. benthamiana. Co-immunoprecipitation of GFP-B’α, β, ξ, ι, η, or free GFP was detected using anti-GFP antibody following HA-pull down of AvrE1 fragments (B). Reciprocally, PP2A B’ subunits were pulled-down using anti-GFP antibody and co-immunoprecipitation of AvrE1-HA fragments or HA-RIN4 was detected by immunoblotting with anti-HA antibody (C). Shown are representative blots from three biological replicates. GFP-B’ ξ in (C) was tested separately from the rest of the panel. (D) AvrE1 fragments associate with maize WIPs. Arabidopsis GFP-B’β, maize GFP-WIP1, GFP-WIP2, or free GFP were co-expressed with AvrE1-HA fragments in N. benthamiana. PP2A B’ subunits from Arabidopsis and maize were pulled down using anti-GFP antibody and co-immunoprecipitation of AvrE1-HA fragments was detected by immunoblotting with anti-HA antibody. Shown is a blot representative of three biological replicates, Arabidopsis GFP-B’β was included in two replicates. Arrows indicate predicted sizes of the GFP-PP2A B’ full-length constructs.