The Wheat NLR Protein PM3b Localises to Endoplasmic Reticulum–Plasma Membrane Contact Sites and Interacts With AVRPM3^b2/c2 Through Its LRR Domain

Molecular Plant Pathology, Journal Year: 2025, Volume and Issue: 26(2)

Published: Feb. 1, 2025

ABSTRACT Plant nucleotide‐binding leucine‐rich repeat (NLR) proteins are intracellular immune receptors that directly or indirectly perceive pathogen‐derived effector to induce an response. NLRs display diverse subcellular localisations, which associated with the capacity of receptor confer disease resistance and recognise its corresponding avirulence effector. In wheat, NLR PM3b recognises wheat powdery mildew AVRPM3 b2/c2 we examined molecular mechanism underlying this recognition. We show other PM3 variants localise endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (EPCS), while localises nucleocytoplasmic space. Additionally, found interacts in planta through LRR domain. further demonstrate full‐length interaction is considerably weaker than for isolated domain susceptible variant PM3CS, indicating activation leads dissociation complex. line this, observed a strong between P‐loop mutant was unable initiate cell death response, when inactive used. propose transiently residues conserved among variants, amino acids necessary full signalling unique PM3b. Our data suggests localisation differ from well‐studied highlights mechanistic diversity NLR‐mediated responses against pathogens plants.

Language: Английский

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Structure‐guided insights into the biology of fungal effectors

New Phytologist, Journal Year: 2025, Volume and Issue: unknown

Published: March 25, 2025

Phytopathogenic fungi cause enormous yield losses in many crops, threatening both agricultural production and global food security. To infect plants, they secrete effectors targeting various cellular processes the host. Putative effector genes are numerous fungal genomes, generally encode proteins with no sequence homology to each other or known domains. Recent studies have elucidated predicted three-dimensional structures of from a wide diversity plant pathogenic fungi, revealing limited number conserved folds. Effectors very diverse amino acid sequences can thereby be grouped into families based on structural homology. Some different some expanded specific taxa. Here, we describe features these discuss recent advances predicting new families. We highlight contribution analyses deepen our understanding function evolution effectors. also prospects offered by modeling for studying virulence targets plants.

Language: Английский

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Engineering the plant intracellular immune receptor Sr50 to restore recognition of the AvrSr50 escape mutant

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 9, 2024

Abstract Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici . The recognizes effector AvrSr50 through its C-terminal domain, initiating a localized cell death immune response. However, this immunity is compromised mutations in effector, as escape mutant QCMJC , which evades Sr50 detection. In study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering to gain recognition Following initial hypothesis driven molecular docking, identified K711D single mutant, induces intermediate response without losing AvrSr50. Increasing gene expression with stronger promoter enabled elicit robust response, indicating weak can be complemented enhanced expression. Further refinements led creation five double mutants two triple dual greater intensities than mutant. All effective required substitution, that multiple solutions exist recognition, but path reach these may confined. Furthermore, substitution alters prediction AlphaFold 2, allowing it model complex structure match our final hypothesis. Collectively, study outlines framework NLR systems overcome provides datasets future models resurrection.

Language: Английский

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A versatile protocol for purifying recombinant proteins fromNicotiana benthamianafor structural studies

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 13, 2024

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

Language: Английский

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Identification of a key gain-of-function residue for effector binding byin vitroshuffling of barleyMla NLRgenes

Published: Oct. 29, 2024

Abstract Natural plant populations maintain high resistance ( R ) gene diversities that provide effective pathogen resistance; however, such diversity has been reduced significantly by the genetic bottlenecks associated with domestication and breeding. Agricultural crops typically contain limited so is often short-lived as pathogens evolve rapidly to evade recognition. The mildew locus A Mla family of barley wheat represents a rich source natural variation ideal for mining disease specificities. genes encode immune receptor proteins nucleotide-binding leucine-rich repeat (NLR) class recognise unrelated binding secreted virulence termed effectors. NLRs MLA13 MLA7 confer different strains powdery through direct interaction effectors AVR A13 A7 respectively. Using DNA shuffling, we generated variant library recombining Mla7 Mla13 in vitro . was cloned into yeast generating ∼4,000 independent clones screened using yeast-two-hybrid (Y2H) assay. This yielded number NLR interacted Sequence analysis showed interacting MLA can be clustered three groups, all which critical residue from MLA13. While differ 30 residues across LRR domain, replacement leucine serine at this position facilitated -dependent signalling planta We have established pipeline evolves MLAs distinct without requirement protein structural knowledge use rationale design. suggest these findings represent step towards evolving novel recognition capabilities

Language: Английский

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A barley MLA immune receptor is activated by a fungal nonribosomal peptide effector for disease susceptibility

New Phytologist, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 6, 2024

Summary The barley Mla locus contains functionally diversified genes that encode intracellular nucleotide‐binding leucine‐rich repeat receptors (NLRs) and confer strain‐specific immunity to biotrophic hemibiotrophic fungal pathogens. In this study, we isolated a gene Scs6 , which is an allelic variant of but confers susceptibility the isolate ND90Pr ( Bs ) necrotrophic fungus Bipolaris sorokiniana . We generated transgenic lines showed sufficient in genotypes naturally lacking receptor. Scs6‐ encoded NLR (SCS6) activated by nonribosomal peptide (NRP) effector produced induce cell death Nicotiana benthamiana Domain swaps between MLAs SCS6 reveal domain specificity determinant for receptor activation NRP effector. maintained both wild domesticated populations. Our phylogenetic analysis suggests Hordeum ‐specific innovation. infer bona fide immune likely directly disease barley. study provides stepping stone future development synthetic crops are less vulnerable modification

Language: Английский

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