SCARN a Novel Class of SCAR Protein That Is Required for Root-Hair Infection during Legume Nodulation
Fig 7
Assays of interaction between the WA domain of SCARN and ARPC1 and ARPC3.
(A) Yeast-two-hybrid assay of interactions. The SCARN-WA domain was cloned in frame with the yeast Gal4 activation domain (AD). ARPC1 or ARPC3 were cloned in frame with the Gal4 binding domain (BD) and S. cerevisiae AH109 was co-transformed with the AD and BD plasmids and also transformed with each construct separately. As a positive control, SV40 fused to the activation domain was co-transformed with p53 fused to the binding domain. SV40 and Lam were negative controls. Potential interactions were assayed by comparing growth in the presence (-LW) or absence of histidine (-LWH) and in the absence of histidine in medium containing 10 mM 3-amino-1,2,4-triazole (+ 10 mM 3-AT, -LHW*). Quantification of β-galactosidase assays is shown. ND, not determined. (B) Pull-down experiments to test interactions in vitro. The WA domain of SCARN fused to glutathione transferase (GST-SCARN-WA) or the glutathione transferase (GST) were purified separately from E. coli and incubated with His-tagged ARPC3 also purified from E. coli. The GST-SCARN-WA and GST proteins were adsorbed onto glutathione sepharose beads, washed extensively and eluted. The eluents along with the input His-tagged ARPC3 were separated by SDS-PAGE and transferred to a membrane which was stained for His-tagged ARPC3 using antiserum to poly histidine.