Table 3. Phenotype of fbl17 pollen at anther dehiscence. Normal (%) Abnormal (%) Col-0 fbl17-1 fbl17-2 97 56 55 3 44 45 n 1,355 1,483 1,492 Pollen from dehiscent anthers of WT and fbl17 +/2 plants was stained with DAPI and observed under UV illumination. n = total number. Normal pollen all showed two sperm cells. Abnormal pollen showed a single sperm-like cell. doi:10.1371/journal.pone.0004780.t003 and allowed the formation of viable seeds with only a diploid endosperm [37]. To test whether flb17 fertilized ovules behave in a similar manner, we crossed female fie+/2 mutant with pollen from the heterozygous fbl17-1 and fbl17-2 mutants. The F1 generation was then genotyped to assess the transmission of both the fbl17 and fie mutant alleles (Table 4). In contrast with Col-0 fertilized by fbl17 mutants that only allowed 6–8% of transmission efficiency of the fbl17 mutant allele, the fie mutation raised the transmission efficiency of fbl17 to 27–32%. At the same time, the transmission efficiency of the fie mutant allele through the female gametophyte increased from 8% in crosses with wild-type pollen to 28–32% indicating a mutual rescue of fbl17 and fie similar to what has been previously reported for fis mutants and cdka;1. Taken together, we conclude that the single fb17 generative-like cell is able to some degree to function as a sperm cell leading to the production of viable seed when sufficiently developed endosperm is provided. FBL17 interacts with specific ASKs to form SCF-like complexes Since FBL17 carries an F-box motif, we next examined whether this protein forms an SCF-type complex. Yeast two hybrid assays were conducted with the different A. thaliana SKP1-like proteins, called ASKs [38]. In yeast, several ASKs were able to interact with FBL17 when fused to the GAL4 DNA binding domain while failing to allow yeast growth on selective medium when fused to the activating domain (Figure 4A). In contrast, ASK11 displayed a robust interaction with FBL17 in both fusions. Interestingly, ASK11 is among the members of the A. thaliana ASK family that are strongly expressed in pollen [39,40] and constitutes therefore a strong candidate for participating in an SCFFBL17 complex. It is noteworthy that the most predominant ASK, ASK1, was not found to interact with FBL17 in our yeast-two hybrid experiment. To further test a possible interaction between FBL17 and ASK11, we performed bimolecular fluorescence complementation (BiFC) experiments. As a control we fused to the N- and C-terminal half of YFP to the C-terminus of FBL17 and GUS, respectively. After co-injection of these fusion constructs into Nicotiana benthamiana leaf cells no YFP fluorescence could be found (data not shown). In contrast, when split-YFP fusions with FBL17 and ASK11 were co- injected, a strong fluorescence signal was recovered corroborating an interaction between these two components of an SCF-type complex in vivo (Figure 4B–G). Moreover transient expression of YFP fusions revealed that the FBL17 protein is essentially nuclear, whereas ASK11 localizes both to the cytoplasm and the nucleus (Figure 4B–E). Consequently, an interaction between ASK11 and FBL17 in the BiFC assays was restricted to nuclei (Figure 4F,G). FBL17 interacts with KRP7 in vivo The FBL17 loss-of-function pollen phenotype is similar, if not identical, to the recently described cdka;1 mutant [25] strongly SCF in Male Gametogenesis Figure 3. Fertilization with fbl17 mutant pollen leads to embryo developmental arrest and seed abortion. Embryo development in wild type plants fertilized with fbl17-1 mutant (A, E) and wild type (B) pollen, 3 days after pollination. Embryo development in wild type plants fertilized with fbl17-1 mutant (C, F) and wild type (D) pollen, 4 days after pollination. Early fertilization-independent development of the endosperm is visible in fbl17-1-fertilized seeds (E) as evidenced by the presence of multiple nuclei (asterisks). Arrested globular embryo in fbl17-1 mutant seeds with degenerated endosperm (F). Bars: 50 mm. doi:10.1371/journal.pone.0004780.g003 supporting a cell cycle function of this F-box protein. An interesting hypothesis is that an SCFFBL17 E3 ligase targets one or more members of the plant cell cycle-dependent kinase inhibitors, called ICK/KRPs (reviewed in [19,41]). In such a scenario, ICK/KRPs would accumulate in fbl17 mutant pollen and consequently would inhibit the kinase activity mimicking the cdka;1 mutant phenotype. To test this possibility, we first carried out yeast two-hybrid experiments, with binding and activation domain fusions of both FBL17 and all seven KRPs being tested. However, no strong interaction between FBL17 and KRPs were PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4780