Figure 3. PgmLs are essential during autogamy and interact with Pgm in cell extracts.

(A) Effect of PGML KDs on the recovery of post-autogamous progeny with functional new MACs. For PGML1, PGML2 and PGML3c, only the results obtained using IF1 RNAi constructs (Figure 3—figure supplement 1) are shown. For groups of duplicated paralogs, individual gene KDs were performed using gene-specific IF2 constructs (Figure 3—figure supplement 1), while double KDs were performed using either IF2 or cross-hybridizing IF1 (*) constructs. Error bars represent standard deviations (n = 2 to 14, see Supplementary file 6) (B) Pull down of HA-PgmL fusions with MBP-Pgm using recombinant proteins expressed in insect cells. In each panel, the HA-tagged protein that was co-expressed with MBP or MBP-Pgm is indicated on the left and the band revealed on western blots (WB) using anti-HA antibodies is indicated on the right. The full-size blot with molecular weight marker is shown in Figure 3—figure supplement 2.

Figure 3—figure supplement 1. Map and coordinates of PGML feeding inserts.

Two feeding inserts (IF1 and IF2) were designed for each gene. Within multigenic PGML groups, gene-specific inserts are in red and inserts that are able to cross-silence other genes of the same family (* constructs in the main text) are in blue (the cross indicates that blue inserts can target the two paralogs simultaneously). The coordinates of each fragment refer to their 5’ and 3’ nucleotide positions, with +1 corresponding to the first base of the ATG start codon.

Figure 3—figure supplement 2. Co-precipitation of MBP-Pgm with HA-PgmL fusions.

(A) Control DRaCALA DNA binding assay (Differential Radial Capillary Action of Ligand Assay, see [Donaldson et al., 2012]). MBP-Pgm was purified from insect cells. Purified MBP-Pgm (400 nM final concentration) was mixed with a ³²P-radiolabeled 80 bp double-strand DNA fragment carrying IES 51A1835 from the surface antigen A gene (25 nM final concentration) in 25 mM HEPES pH7.5, 0.1 mg/ml BSA, 0.5 mM DTT and 50 mM NaCl-containing buffer. NaCl was then added to reach 50, 100, 250 or 500 mM final concentration and complexes were loaded in duplicate onto a Nitrocellulose Hybond ECL membrane. DNA binding is detected at 50 and 100 mM NaCl, while complexes are destabilized at higher salt concentrations (250 mM NaCl and above). (B) Pull down of each HA-PgmL fusion with MBP-Pgm. The full-size western blot is identical to the one used for Figure 3. Recombinant MBP-Pgm was co-expressed in insect cells with each indicated HA-tagged protein, cell extracts were prepared as indicated in Materials and methods and MBP-Pgm was pulled down using amylose beads. HA-tagged proteins were revealed using monoclonal α-HA antibodies (HA-7 from Sigma Aldrich). Expected sizes: 128 kDa (Pgm-HA), 69 kDa (HA-PgmL1), 76 kDa (HA-PgmL2), 88 kDa (HA-PgmL3a), 127 kDa (HA-PgmL4a) and 116 k Da (HA-PgmL5a). ‘Precision Plus Protein Standards’ from Bio-Rad were used as molecular weight markers. (C) Co-immunoprecipitation of MBP-Pgm with each HA-PgmL fusion. For each lane, 1.5 μg of monoclonal α-HA antibodies were incubated overnight at 4°C on a rotating wheel with 10 μL of protein A sepharose beads (GE Healthcare). The coated beads were incubated with the same cell extracts as in (B) for 2 hr at 4°C, then washed 3 times with 1 mL of lysis buffer A and re-suspended in Laemmli buffer (Laemmli, 1970) before electrophoresis in SDS-polyacrylamide gels. MBP-Pgm was detected using HRP-coupled α-MBP antibodies according to the manufacturer’s instructions (New England Biolabs).