Analysis of Protein–DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip

Stevenson, C.E.M.; Lawson, David M.

doi:10.1007/978-1-0716-1197-5_17

Cited by 8 publications

(9 citation statements)

References 15 publications

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“…The double-stranded DNA was then diluted to a working concentration of 1 μM. SPR measurements were performed at 25 °C using the reusable DNA capture technique (ReDCaT) as described ( 59 , 60 ) and using a Biacore 8K System (Cytiva). Further details are provided in SI Appendix , Materials and Methods .…”

Section: Methodsmentioning

confidence: 99%

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Mukhi

Brown

Gorenkin

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4C–RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY–W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

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Section: Methodsmentioning

confidence: 99%

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Mukhi

Brown

Gorenkin

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…8). SPR enables high-sensitivity measurements of the binding of analyte species (here, RirA proteins) to an immobilised ligand ( fhuA IRO box DNA), yielding binding affinities, 34–36 and overcomes the main problems associated with determining DNA-binding affinities of Fe–S cluster-containing regulatory proteins by EMSA (see Fig. S6†).…”

Section: Resultsmentioning

confidence: 99%

Stabilisation of the RirA [4Fe–4S] cluster results in loss of iron-sensing function

Gray,

Stewart,

Hanwell

et al. 2023

Chem. Sci.

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“…Surface plasmon resonance (SPR)-based DNA-binding assays ( 42 ) were also used to compare binding of each protein with each of the identified operator sites (Figure 4C and Supplementary Table S5 ). At concentrations of 1 μM, 6H-QseC2 induced a SPR response 183–210% of the theoretical maximum ( R max ) expected for binding of a single DNA site, as would be expected for 6H-QseC2 dimers simultaneously binding both O2 L and O2 R .…”

Section: Resultsmentioning

confidence: 99%

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element

Ramsay

Bastholm

Verdonk

et al. 2021

Nucleic Acids Research

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Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

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Analysis of Protein–DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip

Cited by 8 publications

References 15 publications

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Stabilisation of the RirA [4Fe–4S] cluster results in loss of iron-sensing function

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element

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