Biology subjects

Bentham, A. R.

Publications and source records attributed to Bentham, A. R..

3 recordsLinked to original sources

bioRxiv2021

pOPIN-GG: A resource for modular assembly in protein expression vectors

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials. HighlightsO_LIpOPIN-GG expression vectors allow for modular cloning enabling rapid screening of purification and solubility tags at no loss of expression compared to previous vectors. C_LIO_LICloning into the pOPIN-GG vectors can be performed from PCR products or from level 0 vectors containing the required parts. C_LIO_LISeveral vectors with different resistances and origins of replication have been generated allowing the effective co-expression and purification of protein complexes. C_LIO_LIAll pOPIN-GG vectors generated here are available on Addgene, as well as level 0 acceptors and tags. C_LI

biochemistry↗

bioRxiv2021

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

Plants use intracellular 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 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. We report here 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 {beta}2-{beta}3 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. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity. SignificanceThis study reveals a mechanism of effector recognition by a plant NLR immune receptor that carries an integrated domain (ID) which mimics an authentic pathogen effector target. An Arabidopsis immune receptor carrying RRS1 and RPS4 NLR proteins detects the Pseudomonas syringae pv. pisi secreted effector AvrRps4 via a WRKY ID in RRS1. We used structural biology to reveal the mechanisms of AvrRps4/WRKY interaction and demonstrated that this binding is essential for effector recognition in planta. Our analysis revealed distinctive features of the WRKY ID that mediate the recognition of structurally distinct effectors from different bacterial pathogens. These insights could enable engineering NLRs with novel recognition specificities, and enhances our understanding of how effectors interact with host proteins.

plant biology↗

bioRxiv2021

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grassspecific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

plant biology↗