Statement of Work.pdf

Fragmentation and Sequencing Statement of Work

1.0 Scope of Work:

• 192 Yeast-Two-Hybrid (Y2H) samples resulting from a screen of 51 baits will be sequenced on an Novaseq6000 S2 300 cycle with 150 cycle sequencing. To align with previous experimental materials from the project, all samples will undergo fragmentation to the target size of 350 bp in preparation for making sequence libraries. To create the sequencing libraries, targets will be 1) converted to double-stranded DNA and 2) have oligonucleotide adapters (with unique dual indexes) attached to their ends. Final libraries will be run on Novaseq6000 S2 300 cycle flow cells to generate 10-20 million reads per sample.

2.0 Background:

• Pathogen effectors are ideal probes to dissect host immunity as well as basic plant cell function. For example, effectors expressed at early stages of infection may disrupt function of host immune receptors, while effectors expressed at later stages might manipulate membrane transporters for nutrient acquisition. We leveraged previous work to discover new protein-protein interactions (PPI) between a fungal pathogen and its barley host. To establish a framework of barley-fungus PPI, selection of effector probes was based on: 1) high level of expression (>75th percentile); 2) peaks of expression at 16-20 hours (penetration) and/or 32-48 hours (development of haustoria); 3) DESeq2 FDR < 0.05 & |Log2 fold change| > 2 relative to wildtype; 4) expressed uniquely on immune signaling mutants; and 5) contribution to fungal infection. We also targeted effectors that possessed corresponding orthologues across powdery mildews that infect wheat, barley, oat, Lolium and Poa. Thus, to identify host targets with major roles in immunity we focused on conserved effectors that are dynamically expressed during infection and/or with documented contributions to pathogen virulence.

Recent advances in Y2H analyses, collectively termed next-generation interaction screening (NGIS) (Suter et al. 2015), use deep sequencing to score the output from Y2H screens. These approaches result in a massive increase in throughput and facilitate a quantitative measure of which preys interact with each bait protein, identifying reproducible PPI with 70-90% accuracy. We optimized the NGIS DEEPN protocol (Pashkova et al. 2016), where a single individual can complete 36 screens/month. This methodology has distinct advantages over other NGIS protocols: 1) it is compatible with using a cDNA prey library (comprehensive ORF libraries are not currently available in most crop plants, including barley); 2) it facilitates the in silico reconstruction of the entire PPI domain of each prey instead of relying on short sequence tags; 3) it allows the determination of whether the preys are in-frame with the GAL4 activation domain (AD);

and 4) it is truly quantitative in that it not only identifies candidate PPI, but also measures their degree of enrichment under selection.

Fifty fungal effectors were used to identify and validate bona-fide host interactors, with the goal of finding central hubs targeted by multiple effectors. We employed an established Gateway-compatible CEN/ARS GAL4system to screen a 3-frame, normalized cDNA library that captures the transcriptional landscape of the barley host + fungal pathogen over the first 48 hours of infection. For the bait constructs, fungal effectors were synthesized and tested for auto-activation of the HIS3 reporter prior to screening. A negative control bait, firefly luciferase, was also used to identify preys that interact non-specifically or auto-activate the reporter gene. To initiate screening, mating of the effector bait strain with the prey cDNA library was performed in liquid yeast media. Diploids were enriched in SC-Leu-Trp liquid media and sub-cultured under two conditions: 1) non-selected diploid growth (SC-Leu-Trp) and 2) selected for reporter activation (SC-Leu-Trp-His). Diploids expressing a positive PPI activated the HIS3 reporter and multiplied in SC-Leu-Trp-His media whereas diploids expressing two non-interacting proteins were unable to grow under this selection. Yeast populations were sub-cultured twice and cells were collected once the populations reached saturation.

Plasmids were isolated, and prey cDNA pools were amplified using low-cycle PCR to maintain the ratio of high-confidence to low-confidence interactions. All baits were subjected to three independent biological replicates, which is key to determining reproducible PPI. One hundred ninety-two libraries from the prey amplicons will be multiplex sequenced on an Novaseq6000 platform resulting 10 – 20 million reads per sample. Using the non-selected population as a baseline, we can detect which preys are significantly enriched under reporter selection, thus identifying candidate PPI (Pashkova et al. 2016).

CONTRACTOR REQUIREMENTS

3.0 Technical Requirements/Tasks:

• Contractor must have experience working with frozen plant tissue and large cereal genomes.

• Contractor must use / implement the following to align with previously sequenced samples. This will allow for direct comparison between the new and previously sequenced samples.

o Must sonicate samples to an average size of 350 base pairs for fragmentation o Must use unique dual indexes.

o Must be able to complete the work by November 2021.

4.0 Government Furnished:

• Government will furnish PCR amplicon sample pools generated from barley prey found to interact with baits of interest during a Y2H screen.

5.0 Deliverables / Schedule:

Item No.

Deliverable

Objective

Due

1 DNA fragmentation All samples will undergo fragmentation to the target size of 350 bp

2 weeks after contract award

2 Library Construction

Targets will be converted to double-stranded DNA and have oligonucleotide adapters (with unique dual indexes) attached to their ends.

6 weeks after contract award

3 NGIS Sequencing Final libraries will be run on Novaseq6000 S2 300 cycle flowcells to generate 10-20 million reads per sample.

8 weeks after contract award

• Contractor should consult with USDA-ARS client and provide expert technical and bioinformatic analysis to ensure sample read ratios are not masked during library construction and amplification. This is critical to ensure proper back-end interpretation of the data and comparison between samples.

• Results to be provided to Location Monitor listed below.

6.0 Travel:

• No travel required.

7.0 Contractor’s Key Personnel:

• Contractor’s personnel must have knowledge of Yeast-two-Hybrid (Y2H), Next Generation Interaction Screening (NGIS) DNA sequencing

• Contractor should offer expertise in sonicated DNA fragmentation and subsequent DNA library construction for sequencing.

• Contractor must ensure read ratios are not masked during library construction and amplification. This is critical to ensure proper back-end interpretation of the data and comparison between samples.

8.0 Security Requirements:

• No security requirements.

9.0 Data Rights:

• All materials and data are the sole property of USDA-ARS project 5030-21000-067-00D.