OBIR1 and Cf-4. Cf-4 GFP and SlSOBIR1 yc have been transiently coexpressed with Avr4 or the nonrecognized effector Avr9 infiltrated at two various optical densities. Interaction in between Cf-4 and SlSOBIR1 was still observed inside the presence of Avr4 and Avr9, indicating that the Cf-4/ SlSOBIR1 complex does not dissociate upon recognition of Avr4 by Cf-4 (Fig. S3C). We further studied whether SlSOBIR1 forms homodimers and/or heterodimerizes with SlSOBIR1-like or AtSOBIR1. For this experiment, SlSOBIR1 GFP was coexpressed with SlSOBIR1 yc, SlSOBIR1-like yc, or AtSOBIR1?Myc, whereas coexpression with Cf-4 yc was used as a handle. Upon pull-down of SlSOBIR1 GFP, Cf-4 yc strongly copurified with the RLK. Nevertheless, we did not observe copurification of SlSOBIR1 yc, SlSOBIR1-like yc, or AtSOBIR1 yc,Fig. 1. Tomato SlSOBIR1 interacts with Cf-4 and Ve1, but not with various RLKs. Tagged versions of Cf-4, Ve1, AtCLV1, SlSERK1, SlSERK3a/BAK1, and SlFLS2 (all fused to eGFP, except for SlFLS2, which was fused to GFP) had been coexpressed with SlSOBIR1 yc in N. benthamiana. Total protein extracts of transiently transformed leaf tissue had been subjected to immunopurification by using GFP-affinity beads. Total proteins (Input) and immunopurified proteins (IP) have been subjected to SDS/PAGE and blotted. Blots have been incubated with -GFP antibody to detect the immunopurified (e)GFP fusion proteins and incubated with -Myc antibody to detect coimmunopurifying SOBIR1 yc proteins.Formula of 1015610-39-5 Coomassie-stained blots showing the 50-kDa Rubisco band present in the input samples confirm equal loading. Representative results for 3 independent experiments are shown.PNAS | June 11, 2013 | vol. 110 | no. 24 |PLANT BIOLOGYindicating that SOBIR1 does not type homo- or heterodimers with SlSOBIR1-like or AtSOBIR1 (Fig.Buy2300099-98-1 S3D).PMID:23329319 SlSOBIR1 Localizes for the Plasma Membrane and Cytoplasmic Vesicles.It has been reported that AtSOBIR1 FP, when expressed beneath control of its own promoter in Arabidopsis, localizes for the plasma membrane and internal membrane compartments of epidermal leaf petiole cells and epidermal root cells (33). Confocal-laser scanning microscopy performed on N. benthamiana epidermal leaf cells transiently expressing SlSOBIR1 GFP beneath manage of the 35S promoter revealed that SlSOBIR1 mostly localizes to the plasma membrane (Fig. S4A). In addition, fluorescence signals had been observed in mobile cytoplasmic vesicles (Fig. S4A). As previously shown, the GFP A manage protein localizes to the cytoplasm and nucleus, whereas SlFLS2 FP localizes towards the plasma membrane (37) (Fig. S4 B ).Targeting SOBIR1 Compromises the Cf-4/Avr4 nduced and Ve1/Ave1Induced HR. The observation that the two SOBIR1 homologs fromtomato and N. benthamiana interact with Cf-4 and Ve1 (Fig. 1, Fig. S1C, and Tables S1 3) suggests that each proteins play a role in Cf-4?and Ve1-mediated defense signaling in Solanaceous plants. As a result, recombinant tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) constructs were generated to target expression of the NbSOBIR1 homologs, either individually or simultaneously (Fig. S2C), and transgenic N. benthamiana expressing Cf-4 was inoculated with all the unique TRV constructs. Three weeks right after viral inoculations, plants were transiently transformed to express Avr4 (40). Inoculation with TRV:NbSOBIR1/ NbSOBIR1-like resulted within a severely compromised Avr4-triggered HR, comparable to inoculation having a TRV construct targeting Cf-4 itself (TRV:Cf-4) (Fig. 2). The Avr4-triggered.