Publications from The Sainsbury Laboratory

Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI); yet the mechanisms driving elicitor-induced alkalinization and its role in immune signaling remain unclear. Here, we demonstrate that inhibition of autoinhibited H+-ATPases (AHAs) is required for elicitor-induced extracellular alkalinization. This alkalinization is essential for immune signaling mediated by diverse plasma membrane-localized receptor kinases (RKs) through modulation of ligand-receptor interactions. Notably, RKs transduce elicitor-triggered signaling via BOTRYTIS-INDUCED KINASE 1 (BIK1), which inhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF) interactions through a conserved phosphorylation event. Interestingly, this pathway is crucial for cell wall damage (CWD) responses involving the RK MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) and its ligand, SERINE RICH ENDOGENOUS PEPTIDE 18 (SCOOP18). Our findings reveal a conserved phospho-regulatory pathway that governs extracellular alkalinization to coordinate plant immune signaling, offering new insights into plant stress resilience.

Protein evolution is influenced by historical contingencies and functional constraints, but their combined impact on rapidly diversifying pathogen virulence effectors remains poorly understood. Here, we combined ancestral state reconstructions and functional assays to recapitulate the evolution of the MAX-fold effector protein APikL2 of the plant pathogenic blast fungus Magnaporthe (syn. Pyricularia) oryzae, focusing on the ancestral and functionally critical amino acid residue D66 (Asp, Codon: GAT). “Rewinding the tape” experiments based on ancestral sequence resurrection revealed that, out of the seven potential amino acid substitutions derived from single nucleotide polymorphisms, only the naturally occurring D66N (Asp to Asn, GAT to AAT) expanded the binding spectrum to host plant proteins of the heavy metal associated (HMA) family. In contrast, three of the nonsynonymous substitutions were deleterious resulting in loss of binding to HMA proteins. Additionally, we identified three cases of homoplasy in the APikL effector family, involving HMA-binding interfaces, indicating recurrent convergent evolution. Our findings suggest an experimental framework for predicting evolutionary outcomes of pathogen effector—host target interactions with implications for plant disease resistance breeding.

Small RNA-mediated gene silencing contributes to plant immunity. The secondary small interfering RNA (siRNA) pathway promotes defense by silencing target genes in invading fungal and oomycete pathogens. Many secondary siRNAs derive from transcripts potentially encoding pentatricopeptide repeat (PPR) proteins. Here, we report that siRNA production is an ancient function of an evolutionarily conserved clade of PPR genes that undergo extensive within-species diversification. In Arabidopsis thaliana, siRNA-source PPRs are physically clustered in one locus on Chromosome 1. These sequences are diversified through gene duplication followed by sequence diversification as well as accumulation of high-impact variations including pseudogenization. This diversity leads to the accumulation of a diverse PPR-siRNA pool, consistent with an engagement in a co-evolutionary arms race with the pathogens. This study defines siRNA-producing PPRs as a family of defense genes and highlights the potential of PPR-siRNA-based engineering for enhancing broad-spectrum disease resistance.

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) are known for their rapid evolution, even at the intraspecific level, yet the rates of evolution differ significantly across various NLR classes. Within the NRC (NLR Required for Cell Death) network, NLRs operate in complex sensor-helper configurations to confer immunity against a diverse array of pathogens, particularly in Asterids. While helper NLRs are typically conserved and evolve slowly, sensor NLRs tend to evolve more rapidly. However, the functional connections between slow and fast-evolving NLRs remain poorly understood, notably in important crop species. We conducted a comparative analysis of NLRs across 40 Solanales and 29 Asterales genomes to explore NRC network expansion and diversification within the less-studied Asterales order. Our findings reveal that the NRC network has expanded less in Asterales compared to Solanales. We functionally validated a minimal Asterales NRC network with 2 helpers and 9 sensors in common lettuce (Lactuca sativa). Through selection and diversification analysis and structural modeling of NRC helper and sensor subclades in the Lactuca genus, we found varying evolutionary diversification rates between NRC helpers and sensors. We found a correlation between sensor diversification rates and helper dependency, with sensors reliant on a phylogenetically conserved helpers experiencing limited diversification pressure. Our results highlight the lineage- and function-specific evolution of the NRC network, offering insights into the evolutionary pressures shaping plant immune receptor networks.

The Arabidopsis TIR-NLR immune receptors RPS4 and RRS1 function together to enable recognition of multiple effector proteins including AvrRps4 and PopP2. We show here that both in the presence and absence of effector, RPS4 and RRS1 form an oligomer that does not change in size upon effector provision. Oligomer formation involves interactions between the RPS4 and RRS1 TIR domains and requires nucleotide binding capacity in RPS4. RPS4 mutants that lose TIR domain NADase activity abrogate immune activation but retain oligomerization. A cysteine residue in the RPS4 LRR domain contributes to oligomer stabilization. We propose that upon effector recognition, conformational changes in the complex relieve inhibition of RPS4 TIR domains by RRS1 TIR domains, enabling proximity between RPS4 TIR domains to create NADase activity.

Plants constantly monitor their environment to adapt to potential threats to their health and fitness. This involves cell-surface receptors that can detect conserved microbe-associated molecular patterns (MAMPs) or endogenous immunogenic signals, initiating signaling pathways to induce broad-spectrum disease resistance, known as pattern-triggered immunity (PTI). In Arabidopsis thaliana, the leucine-rich repeat receptor kinase (LRR-RK) MIK2 is an exceptionally versatile receptor involved in the perception of the vast family of Brassicales-specific endogenous SCOOP peptides as well as potential MAMPs derived from Fusarium and related fungi. Although only plant species belonging to the order of Brassicales encode genes for SCOOP peptides and show SCOOP-responsiveness, the Fusarium-derived elicitor fraction also induces PTI responses in plants from other lineages. In this study, we demonstrate that Fusarium elicitor-responsiveness and proteins belonging to the MIK2-clade are widely conserved among seed plants. We identified a MIK2-clade protein from tomato, which shares properties of AtMIK2 in the perception of the Fusarium elicitor but not of SCOOP peptides. Tomato mutants lacking the receptor show compromised PTI responses to the fungal elicitor and enhanced susceptibility to infection by Fusarium oxysporum. Our data provide insights into the evolutionary trajectory of MIK2 as a multifunctional receptor involved in plant immunity.

Chitin triggers localised and systemic plant immune responses, making it a promising treatment for sustainable disease resistance. However, the precise molecular mechanisms underlying chitin-induced systemic effects in plants remain unknown. In this study, we investigated the effects of soil amendment with crab chitin flakes (hereafter chitin) on pattern-triggered immunity (PTI) and systemic disease resistance in various plant species. We found that soil amendment with chitin potentiates PTI and disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in lettuce, tomato and Arabidopsis as well as against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), which causes powdery mildew in wheat. Using micrografting in Arabidopsis, we demonstrated that this systemic effect is dependent on active chitin perception in the roots. We also showed that induced systemic resistance (ISR) and pattern-recognition receptors (PRRs)/coreceptors, but not systemic acquired resistance (SAR), are involved in the systemic effects triggered by chitin soil amendment. These systemic effects correlated with the transcriptional upregulation of key PTI regulators in distal leaves upon chitin soil amendment. Notably, chitin-triggered systemic immunity was independent of microbes present in soil or chitin flakes. Together, these findings contribute to a better understanding of chitin-triggered systemic immunity, from active chitin perception in roots to the potentiation of PTI in the leaves, ultimately priming plants to mount enhanced defence responses against pathogen attacks. Our study provides valuable insights into the molecular mechanisms of chitin soil amendment and resulting induced immunity and highlights its potential use for sustainable crop protection strategies.

Leucine-rich repeat (LRR) receptor kinases (RKs) and receptor proteins (RPs) are important classes of plant pattern recognition receptors (PRRs) activating pattern-triggered immunity. While both classical and AI-based structural approaches have recently provided crucial insights into ligand-LRR-RK binding mechanisms, our understanding of ligand perception by LRR-RPs remains limited. In contrast to LRR-RKs, many LRR-RPs typically embed one or more loopout regions in their extracellular domains that are crucial for functionality. Here, we employed an AI-based approach to reveal a novel ligand-binding mechanism shared by the Arabidopsis LRR-RPs RLP23 and RLP42 – the PRRs for the short peptide ligands nlp20 and pg13, derived from NECROSIS- AND ETHYLENE-INDUCING PEPTIDE 1-like proteins (NLPs) and fungal endopolygalacturonases (PGs), respectively. This mechanism relies on a β-strand interaction with the N-terminal part of the island domain (ID) loopout, which adopts an antiparallel β-sheet conformation. Additionally, we investigated the larger and more complex binding interface of RLP32 – the PRR for proteobacterial TRANSLATION INITIATION FACTOR 1 (IF1), a folded protein ligand that requires its tertiary structure for recognition. Finally, we describe a mechanistic role of the ID for co-receptor recruitment conserved across LRR-RPs. Together, our results shed light on the ligand-binding mechanisms and receptor complex formation of this important class of PRRs. This opens avenues for a molecular understanding of the plant-pathogen co-evolution, as well as the engineering of plant immune receptors for crop disease resistance.

Effector-triggered immunity (ETI) is a central component of host defense, but whether all cell types execute ETI similarly remains unknown. We combined chemically imposed immune activation with single-cell transcriptomics to profile ETI responses across all leaf cell types in Arabidopsis. Despite uniform ETI perception, we find striking divergence between transcriptional outputs: a core set of defense genes is broadly induced, while distinct cell types activate specialized immune modules. We infer that downstream immune execution is shaped not only by immune receptor activation, but also by cell identity and its associated transcriptional regulatory context, including local transcription factor availability and chromatin accessibility. We further demonstrate that transcriptional regulators preferentially induced in epidermal cells are required to restrict invasion by non-adapted pathogens. Their absence permits pathogen entry into deeper tissues despite intact recognition, revealing a spatial division of immune functions. Our findings uncover a layered immune architecture in plants, challenges the assumption of uniform immune execution, and provides a framework for exploring cell-type-specific resistance logic in multicellular hosts.

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) can function in networks of sensors and helpers to induce hypersensitive cell death and immunity against pathogens. The tomato sensor NLR Prf guards the Pto kinase from AvrPto and AvrPtoB effector perturbation and activates the downstream helpers NRC2 and NRC3. Prf is conserved across the Solanaceae and its ortholog in the model species Nicotiana benthamiana is also required for detection of AvrPto/AvrPtoB function on Pto. A recent study reported that cell death induction after transient expression of an autoactive mutant of tomato NRC3 is abolished upon RNAi silencing of Prf in N. benthamiana. Here we generated loss-of-function prf mutants in N. benthamiana and demonstrate that autoactive mutants of eight canonical tomato NRCs (NRC0, NRC1, NRC2, NRC3, NRC4a, NRC4b, NRC6, and NRC7) still induce hypersensitive cell death when expressed transiently in the prf mutant background. Autoactive tomato NRCs also triggered cell death when expressed in lettuce (Lactuca sativa), an Asteraceae plant that does not have a Prf ortholog. These results confirm a unidirectional dependency of sensors and helpers in the NRC network and underscore the value of the N. benthamiana and lettuce model systems for studying functional relationships between paired and networked NLRs.