WD40 proteins form β-propeller structures essential for plant development and signaling; however, their complete domain architecture is often missed due to high sequence divergence. Here, we reannotated 117 plant genomes and found that many WD40 genes contain fewer than the canonical seven domains, raising questions about their functionality as complete WD40 proteins. Structure-based modeling of 17,369 WD40-only genes without the other associated domains revealed 41,379 additional WD40 domains that were entirely missed by sequence-based annotation but required for forming stable β-propeller structures. Despite being annotated as partial, two rice genes with sequence-invisible domains formed complete β-propeller folds and were critical for pollen development. CRISPR-Cas9 knockouts showed that OsWD40-31 is required for pollen tube elongation, while OsWD40-169 is necessary for pollen germination. Disruption of the sequence-invisible domains reduced binding affinity to key reproductive regulators, PME1 and Lipase3, as confirmed by structural interaction modeling, yeast two-hybrid assays, and co-immunoprecipitation assays. Residue substitution further revealed that WD40 domain stability depends on hydrogen bonding residues not captured by sequence conservation, explaining why many functional domains elude sequence-based detection. These findings highlight a fundamental disconnect between sequence conservation and structural integrity, establishing a structure-guided framework for uncovering hidden domain architectures in complex, repeat-rich gene families across plant genomes.
Cho et al. (Sun,) studied this question.