Figure 11.

RHA2a interacts with ANAC019 and ANAC055. A, RHA2a interacts with ANAC019 and ANAC055 in yeast. Full-length ANAC019 or ANAC055 was cloned into pGADT7 to generate pAD-ANAC019 or pAD-ANAC055. Full-length RHA2a was cloned into pGBKT7 to generate pBD-RHA2a. Yeast cells were cotransformed with a combination of the indicated plasmids and streaked on plates lacking Leu and Trp or Leu, Trp, and His. To corroborate the interactions between the two ANACs and RHA2a, β-galactosidase (β-gal) activity was assayed on replica filters. SD, Synthetic dextrose. B, In vitro pull-down assay. Purified MBP and the MBP-RHA2a fusion proteins were incubated with in vitro-translated [³⁵S]ANAC019 and [³⁵S]ANAC055. Bound proteins were pulled down with amylose resin, separated by 10% SDS-PAGE, and analyzed by autoradiography. Input proteins [³⁵S]ANAC019 and [³⁵S]ANAC055 exhibited the same size, and only [³⁵S]ANAC019 is shown. C, In vitro pull-down assay. Purified MBP and MBP-RHA2a fusion proteins were incubated with the 6myc-ANAC019 and 6myc-ANAC055 expressed in N. benthamiana leaves. Bound proteins were pulled down with amylose resin, separated by 10% SDS-PAGE, and probed with anti-c-myc antibody. Input proteins 6myc-ANAC019 and 6myc-ANAC055 exhibited the same size, and only 6myc-ANAC019 is shown. [See online article for color version of this figure.]