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. 2011 May 26;6(5):e20327. doi: 10.1371/journal.pone.0020327

Figure 3. Interactions of OsSpo11 and OsTopVIB proteins.

Figure 3

A and B, Yeast two-hybrid assay. Different dual-combinations of prey (AD-fusion) and bait (BD-fusion) constructs were cotransformed into yeast strain AH109. A, X-gal staining results and a comparison with the positive control (con) supplied by the Yeast Two Hybrid Kit. These colonies were first screened by growth on QDO (SD/-Ade/-His/-Leu/-Trp) medium lacking adenine, histidine, leucine and tryptophan, and then analyzed by X-gal staining for 7 h. B, Interaction evaluated according to X-gal staining results: strong (++), weak (+) or no (−) interaction. s5, OsSpo11-5; s5N, N-extension of 412 amino acids of OsSpo11-5; s5C, C-terminal 302AAs TopVIA region of OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, OsTopVIB. C and D, Pull-down assay. C, Purified pET-OsSpo11-4 or pET tag (from pET32a vector) expressed in E. coli incubated with GST-OsTopVIB-bound resin. The pulldowns were examined by western blot analysis with an antibody against pET-Spo11-4. Lane 1, purified pET tag alone. Lane 2, precleared pET tag using GST-binding resin. Lane 3, resin-bound GST-OsTopVIB incubated with precleared pET tag. Lane 4, purified pET-OsSpo11-4 fusion protein alone. Lane 5, precleared pET-OsSpo11-4 fusion protein using GST-binding resin. Lane 6, resin-bound GST-OsTopVIB incubated with precleared pET-OsSpo11-4 fusion protein. D, Proteins extracted from rice flowers at the male meiosis stage incubated with resin-bound GST (con) or GST-OsTopVIB (VIB). The pulldowns were subjected to SDS-PAGE and then Western blot analysis with an antibody against GST (GST Ab) or OsSpo11-4 (S4 Ab).