Pathogen recognition by nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) plays roles in plant immunity. The
pv.
effector AvrAC uridylylates the
PBL2 kinase, and the latter (PBL2
) ...acts as a ligand to activate the NLR ZAR1 precomplexed with the RKS1 pseudokinase. Here we report the cryo-electron microscopy structures of ZAR1-RKS1 and ZAR1-RKS1-PBL2
in an inactive and intermediate state, respectively. The ZAR1
domain, compared with animal NLR
domains, is differently positioned to sequester ZAR1 in an inactive state. Recognition of PBL2
is exclusively through RKS1, which interacts with ZAR1
PBL2
binding stabilizes the RKS1 activation segment, which sterically blocks ZAR1 adenosine diphosphate (ADP) binding. This engenders a more flexible NB domain without conformational changes in the other ZAR1 domains. Our study provides a structural template for understanding plant NLRs.
Direct or indirect recognition of pathogen-derived effectors by plant nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) initiates innate immune responses. The
effector ATR1 activates the ...N-terminal Toll-interleukin-1 receptor (TIR) domain of
NLR RPP1. We report a cryo-electron microscopy structure of RPP1 bound by ATR1. The structure reveals a C-terminal jelly roll/Ig-like domain (C-JID) for specific ATR1 recognition. Biochemical and functional analyses show that ATR1 binds to the C-JID and the LRRs to induce an RPP1 tetrameric assembly required for nicotinamide adenine dinucleotide hydrolase (NADase) activity. RPP1 tetramerization creates two potential active sites, each formed by an asymmetric TIR homodimer. Our data define the mechanism of direct effector recognition by a plant NLR leading to formation of a signaling-active holoenzyme.
Nucleotide-binding domain, leucine-rich repeat receptors (NLRs) mediate innate immunity by forming inflammasomes. Activation of the NLR protein NLRP1 requires autocleavage within its function-to-find ...domain (FIIND)
. In resting cells, the dipeptidyl peptidases DPP8 and DPP9 interact with the FIIND of NLRP1 and suppress spontaneous NLRP1 activation
; however, the mechanisms through which this occurs remain unknown. Here we present structural and biochemical evidence that full-length rat NLRP1 (rNLRP1) and rat DPP9 (rDPP9) form a 2:1 complex that contains an autoinhibited rNLRP1 molecule and an active UPA-CARD fragment of rNLRP1. The ZU5 domain is required not only for autoinhibition of rNLRP1 but also for assembly of the 2:1 complex. Formation of the complex prevents UPA-mediated higher-order oligomerization of UPA-CARD fragments and strengthens ZU5-mediated NLRP1 autoinhibition. Structure-guided biochemical and functional assays show that both NLRP1 binding and enzymatic activity are required for DPP9 to suppress NLRP1 in human cells. Together, our data reveal the mechanism of DPP9-mediated inhibition of NLRP1 and shed light on the activation of the NLRP1 inflammasome.
Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation ...of meristem cells is large- ly regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION （CLE） peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF （tracheary elements differentiation inhibitory factor）, functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase （LRR-RK）. Our structure reveals that TDIF mainly adopts an ＂t＇l＂-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an op- portunity for the identification of receptors of other uncharacterized CLE peptides.
The endogenous peptides AtPepl-8 in Arabidopsis mature from the conserved C-terminal portions of their precursor proteins PROPEP1-8, respectively. The two homologous leucine-rich repeat-receptor ...kinases （LRR-RKs） PEPR1 and PEPR2 act as receptors of AtPeps. AtPep binding leads to stable association of PEPR1,2 with the shared receptor LRR-RK BAK1, eliciting immune responses similar to those induced by pathogens. Here we report a crystal structure of the extraceUular LRR domain of PEPRI （PEPR1LRR） in complex with AtPepl. The structure reveals that AtPepl adopts a fully extended conformation and binds to the inner surface of the superhelical PEPRILRR. Biochemical assays showed that AtPepl is capable of inducing PEPR1LRR-BAK1LRR heterodimerization. The conserved C-terminal portion of AtPepl dominates AtPepl binding to PEPRILRR, with the last amino acid of AtPepl Asn23 forming extensive interactions with PEPR1LRR. Deletion of the last residue of AtPepl significantly compromised AtPep1 interaction with PEPRILRR. Together, our data reveal a conserved structural mechanism of AtPep1 recognition by PEPR1, providing significant insight into prediction of recognition of other peptides by their cognate LRR-RKs.
Abstract
Sessile plants encode a large number of small peptides and cell surface-resident receptor kinases, most of which have unknown functions. Here, we report that the
Arabidopsis
receptor kinase ...MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) recognizes the conserved signature motif of SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) from
Brassicaceae
plants as well as proteins present in fungal
Fusarium
spp. and bacterial
Comamonadaceae
, and elicits various immune responses. SCOOP signature peptides trigger immune responses and altered root development in a MIK2-dependent manner with a sub-nanomolar sensitivity. SCOOP12 directly binds to the extracellular leucine-rich repeat domain of MIK2 in vivo and in vitro, indicating that MIK2 is the receptor of SCOOP peptides. Perception of SCOOP peptides induces the association of MIK2 and the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) and SERK4 and relays the signaling through the cytosolic receptor-like kinases
BOTRYTIS
-INDUCED KINASE 1 (BIK1) and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1). Our study identifies a plant receptor that bears a dual role in sensing the conserved peptide motif from phytocytokines and microbial proteins via a convergent signaling relay to ensure a robust immune response.
Peptide-mediated cell-to-cell signaling has crucial roles in coordination and definition of cellular functions in plants. Peptide-receptor matching is important for understanding the mechanisms ...underlying peptide-mediated sig- naling. Here we report the structure-guided identification of root meristem growth factor （RGF） receptors important for plant development. An assay based on a signature ligand recognition motif （Arg-x-Arg） conserved in a subfamily of leucine-rich repeat receptor kinases CLRR-RKs） identified the functionally uncharacterized LRR-RK At4926540 as a receptor of RGF1 （RGFR1）. We further solved the crystal structure of RGF1 in complex with the LRR domain of RGFR1 at a resolution of 2.6 A, which reveals that the Arg-x-Gly-Gly （RxGG） motif is responsible for specific rec- ognition of the sulfate group of RGF1 by RGFR1. Based on the RxGG motif, we identified additional four RGFRs. Participation of the five RGFRs in RGF-induced signaling is supported by biochemical and genetic data. We also of- fer evidence showing that SERKs function as co-receptors for RGFs. Taken together, our study identifies RGF receptors and co-receptors that can link RGF signals with their downstream components and provides a proof of principle for structure-based matching of LRR-RKs with their peptide ligands.
Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment
. Endogenous RAPID ...ALKALINIZATION FACTOR (RALF) peptides
have previously been proposed as ligands for several members of the CrRLK1L family
. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.
Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained ...unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.
Sexual reproduction in angiosperms relies on precise communications between the pollen and pistil. The molecular mechanisms underlying these communications remain elusive. We established that in
, a ...stigmatic gatekeeper, the ANJEA-FERONIA (ANJ-FER) receptor kinase complex, perceives the RAPID ALKALINIZATION FACTOR peptides RALF23 and RALF33 to induce reactive oxygen species (ROS) production in the stigma papillae, whereas pollination reduces stigmatic ROS, allowing pollen hydration. Upon pollination, the POLLEN COAT PROTEIN B-class peptides (PCP-Bs) compete with RALF23/33 for binding to the ANJ-FER complex, leading to a decline of stigmatic ROS that facilitates pollen hydration. Our results elucidate a molecular gating mechanism in which distinct peptide classes from pollen compete with stigma peptides for interaction with a stigmatic receptor kinase complex, allowing the pollen to hydrate and germinate.
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