AFFINITY-SELECTION PHAGE DISPLAY AND PAIRED-END PHAGE SEQUENCING IDENTIFY A SMALL REPERTOIRE OF CLIENT PROTEINS COMMON TO ORTHOLOGOUS ARABIDOPSIS AND SOYBEAN DEHYDRIN PROTEINS
Unêda-Trevisoli, S.H.1, Dirk, L.M.A.2, Hao, G.3, Barrios, L.L.3, Chakrabarti, M.3, Hunt, A.G.3 and Downie A.B.2
1Dept. Crop Science, UNESP, University of São Paulo State, Jaboticabal, SP, Brazil
2Department of Horticulture, University of Kentucky, Lexington, Kentucky, USA
3Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky, USA
Contact: Lynnette M.A. Dirk, firstname.lastname@example.org
The seed’s tolerance to desiccation is the result of synergistic interaction of several mechanisms, many of which involve the hydrophilic Late Embryogenesis Abundant (LEA) proteins. The total complement of LEA proteins has been cataloged, and orthologous pairs identified, for Arabidopsis (Arabidopsis thaliana) and soybean (Glycine max). Aiming to expand our knowledge of LEA function in protecting cells from lethal desiccation damage, we focused on the seed stored proteome, one seed component requiring protection during maturation desiccation. Our objective is to identify preferred client proteins (CP) should they exist, for LEA orthologs in Arabidopsis and soybean. Recombinant dehydrin orthologs from these species, At2g21490 (Arabidopsis) and Glyma04g01180 (soybean), were used as bait for affinity-selection phage display. A T7 phage cDNA library, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed Arabidopsis seeds, was used in triplicate for each immobilized LEA dehydrin, along with triplicate wells containing BSA as a random, unrelated control protein, to assess protein binding preferences. Phage titer increased considerably over four rounds of biopanning in the three replicate wells for both LEA dehydrin proteins and for BSA. Phage was PEG purified from each of the final round sub-libraries. Two limited-round, PCR reactions were performed which attached regions complementary to the Illumina flow cell oligomers, including Illumina sequencing primer sites, and a bar code unique to each microtiter plate well (replication), on the tags, allowing analysis by Paired-end PhageSeq (PEPS). PEPS exhaustively canvassed the recovered CPs, which are being analyzed using a translated tag processing procedure focused on proteins in-frame with the phage coat protein and represented in the Arabidopsis proteome. PEPS greatly increased the depth of CP coverage allowing greater refinement of the LEA-bound CP regions assisting in our understanding of CP motifs aiding binding by the intrinsically disordered LEAs.