Characterization and analysis of the MYB98 gene regulatory network in Arabidopsis

The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA-binding properties. We show that MYB98 binds to a specific DNA sequence and that a MYB98-GFP fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid cell-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We show that five of the downstream genes encode proteins that are secreted into the filiform apparatus and diffuse away from the synergid cells, suggesting that they play a role in cell signaling. To identify direct targets of MYB98, we examined the promoters of seven of the downstream genes, DD2, DD4, DD11, DD12, DD18, DD32, and DD34. We show that two of these genes contain a MYB98-binding site required for synergid cell expression and that multimerization of the MYB98 binding site is sufficient for synergid cell expression, suggesting that these two genes are directly regulated by MYB98. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and controls the expression of genes required for pollen tube guidance and filiform apparatus formation.