Ontology highlight
ABSTRACT:
PROVIDER: PRJNA422048 | ENA |
REPOSITORIES: ENA
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Project description:This luciferase (construct # 1) 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 # 1) 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.
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Project description:The goals of this study were 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. Y2H baits consisted of Blumeria graminis effector proteins and different domains of the barley MLA6 powdery mildew resistance protein. These baits were 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. We demonstrate a high rate of confirmations for top-ranked candidates. Altogether, this study establishes a high-throughput method for protein-protein interaction discovery in non-model organisms where ORF libraries are not available.
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