Statement of Work.pdf

Statement of Work (SOW) Project: Transcription Factor ChIP-Seq

GENERAL INFORMATION

1.0 Scope of Work:

• A gene regulatory network (GRN) has provided a list of transcription factor (TF) candidates for validation of their role in disease resistance of barley to powdery mildew. We are interested in using ChIP-Seq to test the differential binding of these TFs in the gene promoters of two barley lines: CI 16151 (Mla6), which is resistant to the powdery mildew fungus, and it’s derived susceptible mutant, mla6-m18982. Four TFs have been selected including a MADS box, a NAC domain protein, a homeobox protein, and a WRKY protein. For the first experiment, we have selected the WRKY protein.

• Western blot analysis will be used to validate antibodies for ChIP-Seq. Our goal is to validate the same condition for both CI 16151 (Mla6) and mla6-m18982, for a total of two samples per TF.

• Following antibody validation, 1 Transcription Factor (TF) will be used for 2 genotypes: CI 16151 (Mla6) wildtype + mla6-m18982 mutant x 4 timepoints x 2 biological replications = 16 samples total to be sent for ChIP-Seq analysis.

2.0 Background:

• Transcriptional reprogramming is a key component of disease resistance. MLA as an immune receptor that interacts with several transcription factors (TFs) represents a model to explore the regulation of nucleotide binding, leucine rich repeat (NLR) receptors during defense. Previous work presented evidence that pointed to an interconnection between MLA-signaling and transcriptional reprogramming: first, two novel TFs were validated as interactors of MLA; second, the differentially resistant co-expressed interactome is associated with Mla through expression quantitative trait loci (eQTL) associations. Using time-series RNA-Seq data, a gene regulatory network (GRN) of barley immunity was reconstructed. Topological properties and gene ontology (GO) analysis were used to characterize the GRN, highlighting a hierarchical TF-structure of eight levels. A dynamic network of the regulation of targets during the immune response was found by calculating the enrichment of differentially expressed (DE) genes and eQTLs per timepoint. This analysis led to prediction of novel defense regulation mechanisms. Top TFs in the dynamic GRN regulate genes in the differentially co-expressed interactome. Two MLA interactors were predicted as top regulators of the GRN at early timepoints, including WRKY2 and a homeobox (HB) protein which was predicted to regulate Mla expression. Functional validation of these transcriptional relationships would reveal novel regulation mechanisms associated with the immune receptor.

Experimental approaches to validate these TF-target associations include ChIP-Seq that measures the physical binding of TFs in target promoters (Haque et al., 2019), and functional TF-target associations through virus-induced gene silencing (VIGS) and viral overexpression (VOX) (Lee et al., 2012). The validated TFs can be further studied for their effect in disease resistance and its maintenance when challenged with different powdery mildew strains. ChIP-Seq is key to understanding the mechanisms of how the TFs in the GRN are activated and how this process is affected by MLA.

This in turn will allow the engineering of NLR proteins with breeding purposes.

CONTRACTOR REQUIREMENTS

2.0 Technical Requirements/Tasks:

• Required ChIP-Seq service requirements include a full service starting with frozen barley leaves from which chromatin will be isolated and immunoprecipitated. These are the other required services:

o Chromatin shearing validation o Primer design and validation

• ChIP/Ab validation testing 1 Ab amounts per target of interest 1 Transcription Factor (TF) will be used for 2 genotypes: CI 16151 (Mla6) wildtype + mla6-m18982 mutant x 4 timepoints x 2 biological replications = 16 samples total.

o ChIP-Sequencing (Illumina platform) 16 libraries Paired-end reads, 2x 50 bp, 40 million raw reads per mark on average Quality check, alignment to reference barley genome, identification of enriched regions (peak calling).

o Identification of differential binding between samples based on previously identified ChIP-seq peaks.

o Differential binding analysis

Identification and annotation of differential binding between samples based on previously identified ChIP-Seq peaks.

• Contractor should consult with USDA-ARS client and provide expert bioinformatic analysis of differential TF binding between and within CI 16151 (Mla6), which is resistant to the powdery mildew fungus, and it’s susceptible derived mutant mla6-m18982.

3.0 Government Furnished:

• Government will furnish flash frozen 10 cm 1st leaves of barley (1.5 gram per sample) and antibodies for the corresponding transcription factors.

5.0 Deliverables / Schedule:

Item No.

Deliverable

Objective

Due

1 Chromatin Isolation This step assures the quality of the chromatin that will be used as input for the following steps

2 weeks after contract award

2 Chomatin Shearing Validation

Generating the appropriate size fragments to rescue the transcription binding sites

4 weeks after contract award

3 Primer Validation Primers will be tested looking for the genome regions of interest on

6 weeks after contract award the transcription binding sites 4 ChIP/Ab Validation Antibodies must pull down target transcription factors 10 weeks after contract award

5 ChIP-Sequencing 40 million raw reads per mark on average

No later than 16 weeks after contract award

6 Report Required to compare the samples and find genomic regions of interest

No later than 17 weeks after contract award

All results to be provided to Technical Point of Contact listed below.

6.0 Travel: N/A

7.0 Contractor’s Key Personnel:

Contractor’s personnel must have knowledge in ChIP Sequencing and analysis from frozen plant (barley) tissue. The contractor should offer expertise in isolating chromatin from plant (barley) tissue. The chromatin isolation and immunoprecipitation are key steps in the success of the project and require demonstrated expertise.

8.0 Security Requirements: N/A

9.0 Data Rights: All material and date are the sole property of USDA-ARS Project 5030-21000-067-00D.

10.0 Section 508 – Electronic and Information Technology Standards: N/A

11.0 Technical Point of Contact/Location Monitor: Gregory Fuerst 515-294-2893 gregory.fuerst@usda.gov