A Novel High-throughput Technology Allows the Identification of Thousands of New Pairs of Interacting Proteins in Plants
Millions of protein-protein interactions can be screened using this new system, advancing towards a genome-scale understanding of bioenergy crops.
Protein-protein interactions are an important component of cellular regulatory networks and therefore, the simultaneous and large-scale analysis of those interactions is necessary to gain a deeper understanding of important biological functions. Using a modified yeast two-hybrid system, researchers at the Salk Institute for Biological Studies developed a technique that allows genome-scale analysis of pairs of interacting proteins that are identified by high-throughput sequencing of their coding DNA.
This new technique that couples yeast two-hybrid and high-throughput sequencing approaches allowed the research team to screen 36 million pairs of interacting proteins, reaching a scale not previously achievable. Testing the technology in the model plant Arabidopsis, they managed to identify a network of thousands of previously unknown interactions among the plant’s transcription factors. Further optimization of the technique will make it possible to map complete networks of interacting proteins in crops and microbes relevant for bioenergy production.
The technique called Cre-reporter-mediated yeast two-hybrid coupled with next-generation sequencing (CrY2H-seq) was developed by taking advantage of the Cre recombinase to physically link the coding sequences of pairs of proteins that physically interact within a cell. The identities of the interacting protein pairs are determined by sequencing the linked DNA fragments in a highly parallel manner. A proof of concept all-by-all massively multiplexed screening carried out with 1,453 Arabidopsis transcription factors uncovered 8,577 interactions, more than 90 percent of which had not been previously reported. These results nearly triple the number of known Arabidopsis transcription factor interactions. CrY2H-seq can be optimized to potentially discover all protein-protein interactions within an organism in multiple conditions. Moreover, due to the method’s large scale and low cost it is now possible to analyze the cellular interaction maps of different phenotypes or tissue types at a genomic scale, providing new insights into the genotype-phenotype relationships of DOE-relevant plants and microbes.
The Salk Institute for Biological Studies
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DE-SC0007078. The authors also acknowledge support from the National Science Foundation.
Trigg, Shelly, Renee M. Garza, Andrew MacWilliams, Joseph R. Nery, Anna Bartlett, Rosa Castanon, Adeline Goubil, Joseph Feeney, Ronan O’Malley, Shao-shan C. Huang, Zhuzhu Z. Zhang, Mary Galli, and Joseph R. Ecker. 2017. “CrY2H-Seq: A Massively Multiplexed Assay for Deep-Coverage Interactome Mapping,” Nature Methods 14, 819-825. DOI:10.1038/nmeth.4343