Statement of Work.pdf

Wise Project V4: IP/Mass Spec Validation of Transcription Factor Ab (for use in ChIP-Seq)

GENERAL INFORMATION

1.0 Scope of Work:

• A predicted barley gene regulatory network (GRN) has provided a list of transcription factor (TF) candidates for validation of their role in disease resistance of barley to the powdery mildew pathogen, Blumeria hordei. Four of these TFs [a MADS box, a NAC domain protein, a homeobox (HB) protein, and a WRKY2 protein] were selected for further analysis, and affinity purified antibodies were created for each by PhytoAB. We utilized 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. We now need to perform Immunoprecipitation Mass Spectrometry (IP-MS) to validate the specific target(s) for each of the four Ab. Our goal is to validate the same condition for both CI 16151 (Mla6) and mla6-m18982.

• IP-MS will consist of 24 tests; 4 Transcription Factor Ab (MADS, NAC, HB, and WRKY2) x 2 genotypes (CI 16151 and mla6-m18982 mutant) x 3 biological replications.

• Vendor will be required to perform the following:

• Isolate nuclear protein extracts from six 40g sets of pooled barley (2 genotypes x

3 biological replications) harvested into liquid nitrogen. Each pooled sample will consist of 10 cm first leaves harvested at 0, 16, 20, and 32 hours after inoculation (HAI) with the powdery mildew pathogen, Blumeria hordei.

• Perform IP assay to capture the interacting partners for each of the 4 antibodies (MADS, NAC, WRKY and HB)

• Perform qualitative proteomics analysis via Mass Spectrometry.

• Bioinformatics analysis highlighting the TFs and TF-related fragments.

2.0 Background:

• Transcriptional reprogramming is a key component of plant disease resistance.

The MLA immune receptor interacts with several transcription factors (TFs) and thus, 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

3.0 Technical Requirements/Tasks:

• Contractor’s personnel must have knowledge in immunoprecipitation (IP) followed by mass spectrometry (MS) and bioinformatic analysis to identify and validate the specific target / binding site(s) of each transcription factor Ab.

• Analysis from frozen plant (barley) tissue, not fixed tissue. The contractor should offer expertise in isolating mass-spec quality nuclear protein from plant (barley) tissue. The protein isolation and immunoprecipitation are key steps in the success of the project and require demonstrated expertise. Required IP-MS service requirements include a full service starting with frozen barley leaves from which protein will be isolated and immunoprecipitated with each of the 4 transcription-factor antibodies. Contractor must be then able to use the IP to identify the specific identity of the target(s) by mass spectrometry, including bioinformatics analysis.

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

• Quote shall include documentation that personnel are trained to extract nuclear plant protein and analyze by immunoprecipitation, mass spectrometry and resulting bioinformatic analysis.

4.0 Government Furnished:

• Government will furnish six (2 genotypes x 3 biological replications), 40g sets of pooled barley (10 cm first leaves) harvested into liquid nitrogen. Each pooled sample will consist of tissue harvested at 0, 16, 20, and 32 hours after inoculation (HAI) with pathogen Blumeria hordei.

• Government will furnish antibodies for each of the corresponding transcription factors (MADS, NAC, WRKY and HB).

5.0 Deliverables / Schedule:

Item No. Deliverable Objective Due 1 Nuclear Protein Isolation IP-MS quality nuclear protein to be used as input for the following steps

2 weeks after contract award

2 Immunoprecipitation and mass spectrometry (IP-MS)

Identification of specific protein(s) that Ab binds to using mass spectrometry

4 weeks after contract award

3 Antibody Validation Antibodies must pull down target transcription factors

6 weeks after contract award

4 Report highlighting the Transcription Factors (TFs) and TF-related fragments

Required to compare the samples and find genomic regions of interest

No later than 10 weeks after contract award

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

6.0 Contractor’s Key Personnel:

Contractor’s personnel must have knowledge in immunoprecipitation (IP) followed by mass spectrometry (MS) and bioinformatic analysis to identify and validate the specific target(s) of each transcription factor Ab, including MS quality nuclear protein isolation from frozen plant (barley) tissue, not fixed tissue. The nuclear protein isolation and immunoprecipitation are key steps in the success of the project and require demonstrated expertise.

7.0 Data Rights:

All material and date are the sole property of USDA-ARS:

Project 5030-21220-068-000-D.

8.0 Technical Point of Contact and Location Monitor: Will be established after award.