Fig 4. Effector-dependent oligomerization of full-length RPP1 is generally unaffected by mutations in the NB subdomain.

(A) RPP1_NdA oligomerization is induced by co-expression with ATR1_Emoy2 (E) but not ATR1_Cala2 (C). Constructs were transiently expressed in Nicotiana benthamiana and samples were collected at 36 hours post-infiltration (hpi) for co-immunoprecipitation using α-Flag agarose beads. The expression of ATR1:citrine was detected with an α-GFP antibody. Staining of RuBisCO with Ponceau S provides a loading control. (B) Site-directed mutagenesis of the NB subdomain compromises effector-dependent HR induction by RPP1_NdA. Constructs were transiently co-expressed with ATR1_Emoy2 in N. tabacum and images were captured at 48 hpi. The presence or absence of HR is indicated by a “+” or “-“, respectively. Protein expression for these constructs is documented in S9 Fig. (C) Detection of self-association of RPP1_NdA NB subdomain mutants by co-immunoprecipitation, tested as in (A). Experiments were performed at least three times with similar results.