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Next-generation interaction screening to discover luciferase (construct #2) related protein-protein interactions regulating barley powdery mildew disease immunity and susceptibility

posted on 2024-06-11, 07:14 authored by CICGRU, USDA/ARS
This luciferase (construct # 2) dataset was part of a larger Y2H screen (GEO GSE150396) to identify novel proteins involved in barley immune responses. Yeast-two-hybrid (Y2H) screening was coupled with next-generation sequencing to identify and quantify interacting proteins. The luciferase (construct # 2) bait was mated to a cDNA prey library derived from a 0-48 hour time course of infected leaf tissue. Screens were performed in batch liquid culture to enrich yeast populations for cells expressing positive interactions. After two rounds of enrichment under selective (Histidine absent) and non-selective (Histidine present) conditions, yeast cells were collected. Y2H plasmids were extracted and prey cDNA amplicons were generated via low-cycle PCR. Fragmented amplicons were used as input to generate sequencing libraries and processed on the HiSeq 3000 platform. Reads were mapped to the barley and Blumeria graminis genomes and read counts were analyzed using a custom data processing and scoring pipeline. Putative interactors were cloned and binary Y2H was used to confirm interactions. Overall design: Y2H screening of a cDNA library was coupled to next-generation sequencing to identify and rank positive protein-protein interactions. Screening for the luciferase (construct # 2) bait was conducted in duplicate with yeast cells grown in selective and non-selective conditions. NuGEN’s AnyDeplete technology was used to deplete HORVU3Hr1G072260 (zinc finger protein) from the samples. This dataset represents one of two luciferase constructs that were used as bait negative controls. Samples were run on two lanes of Illumina’s HiSeq 3000.


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