UC Riverside

The Shoot Apical Meristem of plants harbors a small population of undifferentiated stem cells that is responsible for propagating all of the post-embryonic organ development through the differentiation of a subset of these stem cell daughters. WUSCHEL is a homeodomain transcription factor that moves from its domain of expression to act in a non-cell autonomous manner to promote stem cell maintenance and repress differentiation promoting factors in the stem cell domain. The mechanisms regulating this non cell autonomous behavior are poorly understood. Protein interactions are important in modulating the function of target proteins, and this study sought to identify factors that regulate this WUSCHEL activity by uncovering the WUSCHEL-protein interaction network through a yeast two-hybrid interaction screen in tandem with a high throughput sequencing approach. A total of 119 putative WUS-interactors were identified, and a subset of 62 was functionally characterized through a RNAi reverse genetics suppressor screen in the clv3-2 pWUS::eGFP-WUS background. The four interactors TCTP, FLOR1, AT3G12300, and AT1G67035 were shown to suppress the clv3-2 phenotype, and all of these except FLOR1 reduced the accumulation of WUS in the SAM. SUMO, a regulator of protein localization, transcriptional activity, and stability via post-translational modifications and non-covalent protein interactions was also identified in the yeast two-hybrid screen. Molecular analysis revealed an interaction between WUS and SUMO1/SUMO2 occurs across a broad sequence that includes a homodimerization motif, a transcriptional enhancer motif, and a motif that regulates both target gene repression and nuclear accumulation. Genetic analysis with sumo1/2 lines revealed smaller meristems and reduced accumulation of eGFP-WUS. Additionally, an inducible form of WUS lacked the ability to strongly activate expression of CLV3 in the sumo1/2 background.

These data reveal a potential network of WUS interactors that could facilitate new understanding in the modulation of WUS function. The interaction between WUS and SUMO links an essential highly conserved protein regulator to WUS-mediated stem cell maintenance, and provides a fascinating avenue for future study of protein complex modulation of WUS function.