Figure 4

The optimized interaction-mating protocol for screening a cDNA library of A. thaliana. (a) Multi-parallel yeast two-hybrid screens were carried out using the optimized method with yeast cells preincubated at low pH and high cell density. Mating efficiency in terms of numbers of zygotes versus total cell number was determined for every single screening by propagating aliquots of the mating mixture on SD-LW medium and counting cells in a hemocytometer. (b) Comparison of library screenings using the optimized protocol (grey columns) or the standard protocol (white columns), respectively. Mean values of three parallel experiments are shown. The bait proteins were (followed by GenBank accession numbers): (1) Rho-like GTP-binding protein (G15V), AAC78242; (2) Rho-like GTP-binding protein (T20N), AAC78242; (3) Rac-like protein (G60V), AAB68394; (4) Rac-like protein (T65N), AAB68394; (5) Rac GTP-binding protein Arac10, AAC63014; (6) putative serine/threonine protein kinase, AAF27020; (7) putative protein kinase, AAD29828; (8) Pto kinase interactor-like protein, CAB89391; (9) receptor protein kinase, BAB01743; (10) nonphototropic hypocotyl 1-like, AAC27293; (11) nonphototropic hypocotyl 1, AAC01753; (12) N. tabacum nonphototropic hypocotyl 1-like_1a, unpublished; (13) N. tabacum nonphototropic hypocotyl 1-like_1b, unpublished; (14) N. tabacum nonphototropic hypocotyl 1-like_2, unpublished; (15) FH protein interacting protein FIP2, AAF14550; (16) N. tabacum FH protein interacting protein FIP2, unpublished; (17) N. tabacum jacalin-like protein, unpublished; (18) unknown protein, AAF13095; (19) unknown protein, BAA96996; (20) putative LEA (late embryogenesis abundant) protein, AAC23428. If not otherwise specified, proteins originate from A. thaliana.