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HDX-MS of AcrIF9 and AcrIF2 binding to CRISPR IF Csy complex

ABSTRACT: CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.

INSTRUMENT(S): Bruker Daltonics micrOTOF series, Agilent instrument model

ORGANISM(S): Pseudomonas Aeruginosa N002

SUBMITTER: Angela Patterson

LAB HEAD: Brian Bothner

PROVIDER: PXD036446 | Pride | 2022-11-01

REPOSITORIES: Pride

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			Action	DRS
	005fd7b0-7f39-4920-a51b-baf217c29ca3_1.mcf	Other
	005fd7b0-7f39-4920-a51b-baf217c29ca3_1.mcf_idx	Other
	005fd7b0-7f39-4920-a51b-baf217c29ca3_2.mcf	Other
	005fd7b0-7f39-4920-a51b-baf217c29ca3_2.mcf_idx	Other
	0beba04c-71e1-485c-9c0e-3709df892be8_1.mcf	Other

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Project description:Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a destructive disease of wheat worldwide. Genetic resistance is the preferred method for controlling stripe rust, of which two major types are race-specific and race non-specific resistance. Race-specific resistance includes the qualitatively inherited all-stage resistance, controlled by single major resistance (R) genes. Conversely, adult-plant resistance is race non-specific, inherited quantitatively, and is durable. Previously, we characterized the gene expression signatures involved in Yr5-controlled all-stage resistance and Yr39-controlled adult-plant resistance using the Affymetrix Wheat GeneChip. For this study, we designed and constructed custom oligonucleotide microarrays containing probes for the 116 and 207 transcripts that we had found important for the Yr5 and Yr39 resistance responses, respectively. We used this custom microarray to profile the resistance responses of eight wheat genotypes with all-stage resistance (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15, and Yr17). The aim of this analysis was to identify common and unique gene expression signatures involved in race-specific resistance accross genotypes, which were used to infer information regarding the general pathways involved in all-stage resistance. Keywords: Stress response Eight wheat genotypes with defined single gene all-stage resistance to particular stripe rust (Pst) isolates were selected for this study, as well as the Pst susceptible genotype âAvocet Susceptibleâ (AVS). Near Isogenic Lines (NILs) for each of eight Yr resistance genes (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15 and Yr17) were developed at the Plant Breeding Institute, Sydney, Australia, by backcrossing the Yr gene donors with the recurrent susceptible spring wheat genotype (Triticum aestivum L.) AVS six times (AVS*6/Yr*) and selecting for the appropriate resistance at each generation. Both virulent and avirulent isolates of Pst were selected for each NIL, except for Yr15, for which no virulent isolate is currently known. Care was taken to select the fewest isolates needed to satisfy the spectrum of virulence/avirulence required, resulting in the use of six isolates identified as PST-17, PST-21, PST-43, PST-45, PST-78 and PST-AUS. Each isolate was selected and maintained on susceptible genotypes. For each of three biological replications, individual genotypes were planted in separate 25 X 42.5-cm flats using a potting mix (6 peat moss: 4 vermiculite with lime: 3 sand: 3 commercial potting mix: 2 perlite: 1.7 g/L lime: 3.3 g/L Osmocote: 2.2 g/L ammonium nitrate). Each flat consisted of three rows of six seedlings, with rows randomly assigned one of two harvest times (24 and 48 h post-inoculation). Seedlings from the 3rd row were used to monitor the expected disease responses to inoculation. Seedlings were grown to the second leaf stage (Feekes 1.2, emergence with second leaf unfolded, ~10 days after planting) in a greenhouse with a diurnal temperature cycle of 10ÂºC (2:00 am) to 25ÂºC (2:00 pm) and a 16 h light/8 h dark cycle. Inoculation was performed by misting the plants with sterile water and applying a 1:20 urediniospore:talc mixture to leaves with a sterile brush. Talc was used to aid in the spread and adhesion of spores over leaf surfaces. Control flats were treated the same way except for the absence of spores in the talc. All treatments for each biological replication were performed at 9 am Pacific Standard Time. To promote spore germination, all flats were transferred to a dew chamber (100% RH) operating at 10ÂºC in the dark for 24 h, before being placed in a growth chamber with a diurnal temperature cycle of 4ÂºC (2:00 am) to 20ÂºC (2:00 pm) and a 16 h light/8 h dark cycle. Rows of plants were harvested from all flats at the assigned times for RNA extraction.

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HDX-MS of AcrIF9 and AcrIF2 binding to CRISPR IF Csy complex

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