Biology subjects

Lawson, A. W.

Publications and source records attributed to Lawson, A. W..

5 recordsLinked to original sources

bioRxiv2024

A versatile protocol for purifying recombinant proteins from Nicotiana benthamiana for structural studies

Structural biology is an essential tool for understanding the molecular basis of biological processes. Although predicting protein structures by fold recognition algorithms has become increasingly powerful, especially with the integration of deep-learning approaches, experimentally resolved structures are indispensable for guiding structure-function studies and for improving modelling. However, experimental structural studies of protein complexes are still challenging, owing to, for example, the necessity for high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy (cryo-EM). The use of Nicotiana benthamiana leaves as a transient expression system for recombinant proteins has become an increasingly attractive approach as the plant is inexpensive to cultivate, grows rapidly, allows fast experimental turnaround and is easily scalable compared to other established systems such as insect cell cultures. Using N. benthamiana as an expression system, we present here a robust and versatile protocol for the purification of five heterocomplexes with sizes ranging from [~]140 kDa to [~]660 kDa consisting of immunoreceptors and their associated pathogen effectors, followed by electron microscopy. The plant-based protocol was applied to verify the structure of the insect cell-derived wheat Sr35 resistosome and to co-purify and co-resolve a [~]140 kDa homodimer of the AvrSr35 effector from the fungus Puccinia graminis f sp tritici (Pgt). In several cases, only a single epitope tag is needed for complex purification, reducing complications that come with multiple epitope tags and two-step affinity purifications. We identify codon usage, signal peptide fusion, epitope tag choice and detergents as critical factors for expression and purification of recombinant protein from N. benthamiana leaves.

biochemistry↗

bioRxiv2024

The barley MLA13-AVRA13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

Co-evolution between cereals and pathogenic grass powdery mildew fungi is exemplified by sequence diversification of an allelic series of barley resistance genes encoding Mildew Locus A (MLA) nucleotide-binding leucine-rich repeat (NLR) immunoreceptors with a N-terminal coiled-coil domain (CNLs). Each immunoreceptor recognises a matching, strain-specific powdery mildew effector encoded by an avirulence gene (AVRa). We present here the cryo-EM structure of barley MLA13 in complex with its cognate effector AVRA13-1. The effector adopts an RNase-like fold when bound to MLA13 in planta, similar to crystal structures of other RNase-like AVRA effectors purified from E. coli. AVRA13-1 interacts via its basal loops with MLA13 C-terminal leucine rich repeats (LRRs) and the central winged helix domain (WHD). Co-expression of structure-guided MLA13 and AVRA13-1 substitution variants show that the receptor-effector interface plays an essential role in mediating immunity-associated plant cell death. Furthermore, by combining structural information from the MLA13-AVRA13-1 heterocomplex with sequence alignments of other MLA receptors, we designed a single amino acid substitution in MLA7 that enables expanded effector detection of AVRA13-1 and the virulent variant AVRA13-V2. In contrast to the pentameric conformation of previously reported effector-activated CNL resistosomes, MLA13 was purified and resolved as a stable heterodimer from an in planta expression system. Our study suggests that the MLA13-AVRA13-1 heterodimer might represent a CNL output distinct from CNL resistosomes and highlights opportunities for the development of designer gain-of-function NLRs.

plant biology↗

bioRxiv2024

Species wide inventory of Arabidopsis thaliana organellar variation reveals ample phenotypic variation for photosynthetic performance

Efforts to improve photosynthetic performance are increasingly employing natural genetic variation. However, genetic variation in the organellar genomes (plasmotypes) is often disregarded due to the difficulty of studying the plasmotypes and the lack of evidence that this is a worthwhile investment. Here, we systematically phenotyped plasmotype diversity using Arabidopsis thaliana as a model species. A reanalysis of whole genome resequencing data of 1,531 representative accessions shows that the genetic diversity amongst the mitochondrial genomes is eight times lower than amongst the chloroplast genomes. Plasmotype diversity of the accessions divides the species into two major phylogenetic clusters, within which highly divergent subclusters are distinguished. We combined plasmotypes from 60 A. thaliana accessions with the nuclear genomes (nucleotypes) of four A. thaliana accessions to create a panel of 232 novel cytonuclear genotypes (cybrids). The cybrid plants were grown in a range of different light and temperature conditions and phenotyped using high-throughput phenotyping platforms. Analysis of the phenotypes showed that several plasmotypes alone or in interaction with the nucleotypes have significant effects on photosynthesis, and that the effects are highly dependent on the environment. Moreover, we introduce Plasmotype Association Studies (PAS) as a novel method to reveal plasmotypic effects. Within A. thaliana, several organellar variants can influence photosynthetic phenotypes, which emphasizes the valuable role this variation has on improving photosynthetic performance. The increasing feasibility of producing cybrids in various species calls for further research into how these phenotypes may support breeding goals in crop species. Significance statementPhotosynthesis is one of the few crop traits that has been largely unaddressed which can contribute to increasing crop yield potential. Exploiting genetic variation within organellar genomes presents a promising, yet untapped resource to improve photosynthesis. However, the extent of organellar variation and its impact on photosynthesis within a species remains largely unknown. Using Arabidopsis thaliana as a model species, we revealed highly divergent clusters of organellar variation. We constructed 232 novel combinations of species representative organellar and nuclear genomes, referred to as cybrids. High-throughput phenotyping of these cybrids revealed that organellar variants can substantially impact photosynthesis in different environments. These findings indicate that organellar genomes may be a valuable resource for improving photosynthesis in crops.

plant biology↗

bioRxiv2023

Structural polymorphisms within a common powdery mildew effector scaffold as a driver of co-evolution with cereal immune receptors

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLR) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7 and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22, and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the ribonuclease (RNase) T1/F1-family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1- family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.

plant biology↗

bioRxiv2022

The NDH complex reveals a trade-off that constrains maximising photosynthesis in Arabidopsis thaliana

The Green Revolution has resulted in major improvements in crop productivity, but left photosynthesis largely unimproved. Despite ample variation of photosynthetic performance in crops and their wild relatives, the photosynthetic capacity of elite breeding lines remains well below its theoretical maximum. As yield is often the primary selective trait, current plant breeding approaches result in photosynthetic trade-offs that prevent positive selection for photosynthetic performance itself. Currently, genetic variation for photosynthetic performance is seldomly validated at the genetic level, and as a result these photosynthetic trade-offs remain poorly understood. Here we reveal the physiological nature of a photosynthetic trade-off caused by the NAD(P)H dehydrogenase (NDH) complex. The use of an Arabidopsis thaliana cybrid panel revealed how a natural allele of the chloroplastic gene NAD(P)H-QUINONE OXIDOREDUCTASE SUBUNIT 6 - a subunit of the NDH complex - results in a faster recovery of photosystem II efficiency after a transition from high to low irradiances. This improvement is due to a reduction in NDH activity. Under low-light conditions this reduction in NDH activity has a neutral effect on biomass, while under highly fluctuating light conditions, including high irradiances, more NDH activity is favoured. This shows that while allelic variation in one gene can have beneficial effects on one aspect of photosynthesis, it can, depending on environmental conditions, have negative effects on other aspects of photosynthesis. As environmental conditions are hardly ever stable in agricultural systems, understanding photosynthetic trade-offs allows us to explore shifting photosynthetic performance closer to the theoretical maximum.

plant biology↗