NbMIP1.1a interacts with ToMV MP and Tm-2² in yeast and <i>in vitro</i>.

<p>(<b>A–D</b>) NbMIP1.1a interacts with ToMV MP, LRR domain of Tm-2² (Tm-2²-LRR) and full length Tm-2² (Tm-2²-FL) in yeast. (<b>A, C</b>) Yeast cells harboring BD-ToMV MP (A), BD-Tm-2²-LRR or BD-Tm-2²-FL (C) transformed with AD-NbMIP1.1a grew on Leu^- selection plates and turned blue on X-gal plate containing Gal/Raf but not on plates containing glucose. Yeast cells transformed with either BD or AD alone vector showed no growth on Leu⁻ selection plates and remained white on X-gal plates containing either Gal/Raf or glucose. For each experiment, yeast strains were maintained at 28°C for 5 days. (<b>B, D</b>) Quantification of β-galactosidase activities in yeast two-hybrid interactions. (<b>E, F</b>) GST pull-down assay for detection of <i>in vitro</i> interaction of NbMIP1.1a with ToMV MP (E) or Tm-2² LRR domain (F). For GST pull-down, GST-NbMIP1.1a or GST immobilized on glutathione-Sepharose beads was incubated with gradient dilutions of <i>E. coli</i>-expressed recombinant ToMV MP-3×Flag-6×His protein (E) and Tm-2²-LRR-3×Flag-6×His protein (F) at 1, 10⁻¹, 10⁻². Beads were washed and proteins were analyzed by SDS-PAGE and western blot assays using anti-Flag antibodies.