Figure 4. FBL17 interacts with a subset of ASKs. (A) Yeast-two- hybrid analysis of the interaction between FBL17 and ASKs. Yeast were SCF in Male Gametogenesis grown for 3 days at 28uC. LTH- 3AT, low stringency selection, LTA, high stringency selection. Negative controls were done with empty bait vectors (pGBD) or empty prey vectors (pGAD). (B–G) Subcellular interaction of FBL17 with ASK11. Confocal laser-scanning micrographs of the abaxial surface of N. benthamiana leaves. (B, C) Transient expression of ASK11-YFP. The YFP signal is detected both in cytoplasm and nucleus. (D, E) Transient expression of FBL17-YFP. The signal is exclusively nuclear. (F–G) BiFC of FBL17-YN/ASK11-YC. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. (H–K) Subcellular interaction of FBL17 with KRP7. (H, I) Transient expression of KRP7-YFP. A weak YFP signal in the cytoplasm and a strong signal in the nucleus can be detected. (J, K) BiFC of FBL17- YN/KRP7-YC. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. (B, D, F, H, K, J) DIC images of the cells documented. (C, E, G, I, K) laser confocal micrograph of the YFP signal. Scale bars in B to K represent 45 mm. doi:10.1371/journal.pone.0004780.g004 Table 5. Rescue of the fbl17 pollen phenotype at anther dehiscence. Bicellular pollen (%) Tricellular pollen (%) n 100 51 100 65.3 237 235 255 2,352 Genotype Col-0 fbl17-1 krp6 0 fbl17-1+/2 krp6-1+/2 34.7 0 48.1 Pollen from dehiscent anthers of wild type and the indicated genotypes was stained with DAPI and observed under UV illumination. n = total number. doi:10.1371/journal.pone.0004780.t005 accumulation during gametogenesis would lead to CDKA;1 inactivation and as a consequence cell cycle arrest. Indeed, we observed a physical interaction between FBL17 and at least one members of the KRP family (e.g., KRP7). Moreover, KRP6 mutation suppressed, at least partially, the fbl17 pollen phenotype. After the completion of this work Kim et al. [47] reported a similar role of FBL17 during male gametogenesis. This study showed that both KRP6 and KRP7 are expressed in the male germ cells after asymmetric division, but disappear in sperm cells. Based on their in vitro degradation assays and the fact that a KRP6-GFP fusion protein remained stable in the fbl17 single germ cell, these authors also proposed that KRP6 and KRP7 are the main target this SCF E3 ligase [47]. Nevertheless, because a krp6 krp7 double loss-of- function mutant is not available, this model still awaits a definitive genetic prove. An interesting question, which has not yet been fully answered, is at which stage the cell cycle fbl17 mutant arrests? Based on our knowledge of the cell cycle in yeast and animals, a na¨ıve prediction was that fbl17 loss-of-function mutants would arrest at the G1/S transition. However, this was not the case because the mutant pollen had a higher DNA content than wild-type sperm and thus must have entered into S-phase ([47] and our work). In contrast, the DNA content of fbl17 was measured to be lower than 2C and thus, the mutant sperms appear to be not arrested at the entry into mitosis. However, as the fbl17 single germ cell fertilizes the egg cell, even allowing complete embryo development in a fie mutant background, we exclude that an aneuploid situation arose after fertilization and thus, it is likely that DNA replication was completed at the time of fertilization. Thus, we speculate that fbl17 mutants are arrested in the G1 phase and similarly to wild type would be still susceptible to an S- phase trigger late in development. This trigger would then be able to overwrite the FBL17 loss-of-function. It is noteworthy here that PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4780