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Next-generation interaction screening to discover protein-protein interactions regulating barley powdery mildew disease immunity and susceptibility.

dataset
posted on 2024-02-06, 23:57 authored by CICGRU, USDA/ARS
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. Overall design: Y2H screening of a cDNA library was coupled to next-generation sequencing to identify and rank positive protein-protein interactions. For each bait, screens were conducted in triplicate 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. Two constructs of luciferase were used as bait negative controls. Samples were run on three lanes of Illumina's HiSeq 3000.

History

Data contact name

BioProject Curation Staff

Publisher

National Center for Biotechnology Information

Temporal Extent Start Date

2020-05-12

Theme

  • Non-geospatial

ISO Topic Category

  • biota

National Agricultural Library Thesaurus terms

genetics

Pending citation

  • No

Public Access Level

  • Public

Accession Number

PRJNA631974

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