Characterization of growth regulatory protein complexes in plants via multi-functional TAP technology.

Proteins are one of the key components in the complex regulatory networks that underlie biological processes. Although some work independently, most proteins form interactions with other proteins or macromolecules, such as RNA and DNA, to perform their function. To aid functional annotation in plants in a system-wide manner, a wide variety of techniques have been constructed over the years
(Chapter 1), which often imply a genetically encoded tag to avoid the need of protein specific antibodies. Two of the most powerful approaches that use this tagging principle are tandem affinity purification (TAP) followed by mass spectrometry to identify protein-protein interactions, and chromatin immunoprecipitation coupled to next generation sequencing to assess proteinDNA interactions. A third application of protein tagging is found in the visualisation of localisation patterns of proteins by means of fluorescent protein tags. However, no tags have been constructed in plants that combine all three approaches. This makes studying complex biological processes, such as leaf growth and development, in which a myriad of proteins play a regulatory role (Chapter 2),
rather difficult. In this research, we constructed and evaluated a U+201CSwiss-knifeU+201D TAP tag, named
GSyellow, that allows the identification of interaction partners and localisation patterns of a protein under study (Chapter 3) in both Zea mays and Arabidopsis thaliana. By combining data gathered from TAP, ChIP and localisation experiments, we confirmed previously reported mechanisms of a set of bait proteins in their developmental contexts. Hence, the obtained data indicate that the GSyellow tag performs equally well as other tags in the analysis of protein functions in plants.
Furthermore, to preserve the endogenous regulatory sequences surrounding the gene of interest in Arabidopsis, an alternative workflow to produce genetically TAPtagged fusion proteins based on homologous recombination in combination with transformation-competent artificial chromosomes (TAC), also called recombineering, was successfully implemented (Chapter 4). Moreover, to provide
a deeper insight into the differences obtained with TAP and pull-down experiments, protein complexes purified from Arabidopsis and Zea mays with both techniques were compared (Chapter 5). This revealed that both experimental set-ups can detect different types of interactions, and that having both possibilities at hand provides a great flexibility for protein-protein interaction studies.
Finally, the applicability of the GSyellow TAP tag in the functional analysis of a protein complex in a developmental context was demonstrated in Zea mays leaves via the transcriptional co-activator ANGUSTIFOLIA3 (ZmAN3) that plays a known regulatory role in leaf growth (Chapter 6). Here, previous TAP data of ZmAN3 that indicated preferential binding with transcription factors of the GROWTH REGULATING FACTOR (GRF) family in the dividing tissues of the growth zone was
confirmed. Additionally, target gene identification through ChIP-seq on the full growth zone as well as on the division and expansion zone separately showed that ZmAN3 regulates the expression of genes involved in the maintenance of the proliferative state. Our data also indicated that the regulatory feedback loop that regulates GRF expression through binding of ZmAN3-GRF to its own promoter, is conserved between dicots and monocots.
In conclusion, the multifunctional GSyellow TAP tag that was evaluated here can be used to analyse the function of protein complexes in a developmental context in both monocots and dicots. By analysing the function of the growth regulatory ZmAN3 protein in leaf development, it was demonstrated how the GSyellow tag can be applied to gain insights into complex biological processes, which can aid to engineer the underlying pathways more precisely in the future.