Phytocytokine signalling reopens stomata in plant immunity and water loss

Abstract

Stomata exert considerable effects on global carbon and water cycles by mediating gas exchange and water vapour^1,2. Stomatal closure prevents water loss in response to dehydration and limits pathogen entry^3,4. However, prolonged stomatal closure reduces photosynthesis and transpiration and creates aqueous apoplasts that promote colonization by pathogens. How plants dynamically regulate stomatal reopening in a changing climate is unclear. Here we show that the secreted peptides SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREWs) and the cognate receptor kinase PLANT SCREW UNRESPONSIVE RECEPTOR (NUT) counter-regulate phytohormone abscisic acid (ABA)- and microbe-associated molecular pattern (MAMP)-induced stomatal closure. SCREWS sensed by NUT function as immunomodulatory phytocytokines and recruit SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors to relay immune signalling. SCREWS trigger the NUT-dependent phosphorylation of ABA INSENSITIVE 1 (ABI1) and ABI2, which leads to an increase in the activity of ABI phosphatases towards OPEN STOMATA 1 (OST1)–a key kinase that mediates ABA- and MAMP-induced stomatal closure^5,6–and a reduction in the activity of S-type anion channels. After induction by dehydration and pathogen infection, SCREW–NUT signalling promotes apoplastic water loss and disrupts microorganism-rich aqueous habitats to limit pathogen colonization. The SCREW–NUT system is widely distributed across land plants, which suggests that it has an important role in preventing uncontrolled stomatal closure caused by abiotic and biotic stresses to optimize plant fitness.

Plant genomes encode thousands of small peptides, the functions of which remain mostly unknown⁷. Some secreted peptides regulate plant development, reproduction, immunity and environmental adaptations, and function as short- and long-distance signalling molecules^7,8. Plant peptides are usually perceived by cell-surface-resident receptor kinases^9,10. Some receptor kinases function as pattern recognition receptors (PRRs) that recognize MAMPs or plant-derived damage-associated molecular patterns (DAMPs) and immunomodulatory phytocytokines^11–14. The immune responses induced by MAMPs, DAMPs and phytocytokines collectively contribute to plant pattern-triggered immunity (PTI). The expression of phytocytokines is often induced after the perception of MAMPs by PRRs to boost plant immunity^13,14. Some PRRs can perceive both phytocytokines and MAMPs, such as MALE DISCOVERER1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2), which recognizes SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) from plants and microorganisms^15,16. How immunity is mechanistically coordinated by the concerted action of microbial patterns and host phytocytokines remains mostly unknown.

SCREWS function as phytocytokines

We analysed Arabidopsis transcriptome datasets for genes encoding small peptides (fewer than 120 amino acids (aa)) that are induced upon the perception of flg22, a 22-aa peptide that corresponds to the MAMP flagellin¹⁷. Three closely related genes, AT1G06135, AT2G31345 and AT1G06137, were significantly upregulated at 30 and 60 min after flg22 treatment and were named SCREW1, SCREW2 and SCREW3, respectively (Extended Data Fig. 1a). SCREWS contain an N-terminal signal peptide domain, a variable domain and a C-terminal conserved domain (Fig. 1a, Extended Data Fig. 1b). Phylogenetic analysis revealed an additional SCREW homologue, AT2G31335 (SCREW4), in Arabidopsis (Fig. 1a, Extended Data Fig. 1b). The SCREW orthologues were detected in a wide range of dicot and some monocot species (Extended Data Fig. 1c). Quantitative PCR with reverse transcription (RT–qPCR) analysis confirmed that SCREW genes were upregulated by treatment with flg22 (Fig. 1b) and treatment with elf18 (an 18-aa peptide from the bacterial elongation factor Tu (EF-Tu)), phytocytokine PLANT ELICITOR PEPTIDE 1 (Pep1) and bacterial Pseudomonas syringae pv. tomato (Pst) DC3000 (Extended Data Fig. 1d–f), with SCREW1 and SCREW2 being the most highly induced. Histochemical analysis of GUS expression under the control of the SCREW1 or SCREW2 promoter in transgenic Arabidopsis plants confirmed that the SCREW promoters were substantially induced after treatment with flg22 and primarily active in leaf veins (Fig. 1c).

Fig. 1 |. SCREWs are phytocytokines.

a, Multiple sequence alignment and WebLogo analysis of SCREWs. Different domains are boxed, and conserved cysteine residues are highlighted with stars. b, SCREW genes are upregulated after flg22 treatment. Seedlings were treated with 200 nM flg22 for RT–qPCR analysis. Means (n = 4) of fold induction compared to non-treatment shown as log₂ were used for the heat map using TBtools⁴⁵. c, Flg22 upregulates the activity of SCREW promoters. pSCREW1/2::GUS seedlings were treated with 100 nM flg22 or ddH₂O (Ctrl) for 3 h before GUS staining. Scale bars, 2 mm. d, SCREWs induce MAPK activation. Seedlings were treated with protein elution buffer (Ctrl), GST or GST–SCREWs (1 μM). IB, immunoblot; RBC, Rubisco. e, SCREW1 inhibits seedling growth. Seedlings treated without (Ctrl) or with 1 μM SCREW1 were imaged. Fresh weights are shown as mean ± s.d. (n = 8). Scale bars, 1 cm. f, SCREW1 protects Arabidopsis from Pst DC3000 infection. Leaves were pre-infiltrated with ddH₂O (Ctrl) or 500 nM SCREW1, followed by inoculation with Pst DC3000. Bacterial growth was measured at 0 and 3 dpi. Data are mean ± s.d. (n = 8). Cfu, colony-forming units. g, Inducible expression of SCREW1 enhances resistance to Pst DC3000. Plants carrying pEst::SCREW1-HA were spray-treated with 0.05% DMSO (Ctrl) or 50 μM β-oestradiol (Oest) for 24 h, followed by inoculation with Pst DC3000. Bacterial growth was measured at 3 dpi and is shown as mean ± s.d. (n = 6). WT, wild type. h, Flg22-induced resistance is partially compromised in screw1/2 mutants. Leaves were pre-infiltrated with ddH₂O or 500 nM flg22, followed by inoculation with Pst DC3000. Data are mean ± s.d. (n = 8). Experiments were repeated at least three times with similar results. Data were analysed by two-sided Student’s t-test (e, f) or one-way (h) or two-way (g) ANOVA followed by Tukey’s test; n = biologically independent samples.

To determine whether SCREWS have a role in immunity, we examined their effects on various hallmarks of PTI responses using recombinant SCREW proteins purified from Escherichia coli or synthetic peptides. SCREW1, SCREW2 and SCREW3 without the signal peptide, and fused with either glutathione S-transferase (GST) or 6×His, induced the activation of mitogen-activated protein kinases (MAPKs) (Fig. 1d, Extended Data Fig. 2a) and the expression of PTI marker genes, including WRKY30, WRKY33, WRKY53 and FRK1 (Extended Data Fig. 2b, c). To define the immunogenic region, we generated a series of SCREW1 truncations and synthetic peptides (Extended Data Fig. 2d). The conserved C terminus of SCREW1 without the signal peptide and variable region (SCREW1^39–69, which was used as the SCREW1 peptide hereafter) was sufficient to activate MAPKs at a subnanomolar scale (Extended Data Fig. 2d–g). Although SCREW1 induced a comparable activation of MAPKs to flg22, SCREW1 triggered a weak burst of reactive oxygen species (ROS) and a moderate increase in cytosolic calcium ([Ca²⁺]_cyt) compared to flg22 (Extended Data Fig. 2h–j). MAMP-induced phosphorylation of the receptor-like cytoplasmic kinase (RLCK) BOTRYTIS-INDUCED KINASE 1 (BIK1) contributes to ROS bursts and [Ca²⁺]_cyt influx^18,19. Unlike flg22, SCREW1 did not induce detectable phosphorylation of BIK1 (Extended Data Fig. 2k). Similar to flg22 and Pep1, prolonged treatment with SCREW1 inhibited seedling growth (Fig. 1e). SCREWS contain two conserved cysteine residues, which are predicted to form a disulfide bond (Fig. 1a, Extended Data Fig. 3a). Mutation of the two cysteine residues to serine in SCREW2 (SCREW2(CC/SS)) reduced its effects on MAPK activation and seedling growth inhibition (Extended Data Fig. 3b, c), indicating the importance of the conserved cysteines for the activity of SCREWS. Synthetic SCREW2 peptides with an extra step of oxidation to facilitate the formation of a disulfide bond after synthesis (SCREW2(CC)) had a similar activity to that of SCREW2 in terms of the activation of MAPKs and inhibition of the growth of Arabidopsis seedlings (Extended Data Fig. 3b, c), suggesting that the two cysteine residues in SCREWS might be naturally oxidized to form a disulfide bond. SCREW1 peptides do not appear to be mobile (Extended Data Fig. 3d)–similar to previous reports on Pep peptides²⁰. Unlike Pep1, SCREW1 did not induce the expression of PATHOGENESIS-RELATED PROTEIN 1 (PR1) in the distal leaves with local infiltration (Extended Data Fig. 3e, f). Treatment with SCREW1 did not trigger systemic resistance to Pst DC3000 (Extended Data Fig. 3g). These data suggest that SCREWS might act in an autocrine or paracrine manner.

Pretreatment with SCREW1 protected Arabidopsis against infection with Pst DC3000 (Fig. 1f). In addition, oestrogen-inducible expression of C-terminal HA-tagged SCREW1 in the wild-type (WT) Col-0 background primed plant resistance against Pst DC3000 infection (Fig. 1g). The C-terminal HA tag did not affect the activity of SCREW1 (Extended Data Fig. 3h, i). Notably, these transgenic plants showed yellowish leaves and chlorosis, accompanied with increased PR1 expression (Extended Data Fig. 4a, b), after prolonged treatment with β-oestradiol at the late growth stage. Similarly, several lines of transgenic plants expressing SCREW1 or SCREW2 under the constitutive 35S promoter in the wild type showed retarded growth with curled leaves, chlorosis on the old leaves and increased expression of PR1 (Extended Data Fig. 4c, d).

As SCREW1 and SCREW2 were the most highly upregulated of the four SCREW genes after MAMP treatments (Fig. 1b), we generated screw1/screw2 (screw1/2) double mutants using CRISPR–Cas9 (Extended Data Fig. 4e, f). Two homozygous mutants, screw1/2-1 and screw1/2-2, showed enhanced susceptibility to Pst DC3000 and its type III secretion mutant hrcC⁻ (Extended Data Fig. 4g, h). In addition, flg22-primed resistance to Pst DC3000 was partially compromised in screw1/2 mutants compared to the wild type (Fig. 1h). Our data collectively indicate that SCREWs are pathogen-inducible phytocytokines and protect plants from infection.

NUT mediates SCREW-induced immunity

Receptor kinases, especially leucine-rich repeat (LRR) receptor kinases (LRR-RKs), can function as receptors of endogenous peptides^7,8,10. To identify the potential SCREW receptor, we screened receptor kinases with more than 18 LRRs that are induced by flg22 and for which the ligand remains unknown (Extended Data Fig. 5a). A mutant containing a T-DNA insertion in AT5G25930, but not others, blocked the SCREW1-triggered activation of MAPKs (Fig. 2a). AT5G25930–which we here term NUT–is an LRR-RK in the subfamily XI (Extended Data Fig. 5b). It is phylogenetically close to several LRR-RKs that are known to perceive endogenous peptides, including HAESA (HAE) and HAESA-LIKE2 (HSL2), which sense INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptides in floral organ abscission²¹, and PEP RECEPTOR 1 (PEPR1)/PEPR2 and RECEPTOR-LIKE KINASE 7 (RLK7), which sense Pep1 and PAMP-INDUCED SECRETED PEPTIDE 1 (PIP1)/PIP2, respectively^22,23 (Extended Data Fig. 5b). Similar to SCREWs, NUT is broadly conserved in dicots and monocots²⁴ (Extended Data Fig. 5c).

Fig. 2 |. SCREWs induce NUT-dependent immune responses.

a, NUT is required for SCREWl-induced MAPK activation. Plants were treated with 1 μM GST-SCREW1 for 15 min for MAPK detection. NILR1 regulates immunity to nematodes⁴⁶. MIK2L is the homologue of MIK2. IKU2 is the homologue ofRLK7. LET1 and LET2 are malectin-like receptor kinases that regulate autoimmunity^47,48. b, NUT is required for MAPK activation triggered by multiple SCREWs. Plants were treated with 1 μM GST–SCREWs. c, NUT is required for SCREW1-induced growth inhibition. Experiments were performed as in Fig. 1e. Scale bars, 1 cm. Data are mean ± s.d. (n = 8). d, NUT-HA restores SCREW1-induced FRK1 promoter activation in nut mutants. Protoplasts were co-transfected with pFRK1::LUC, pUBQ10::GUS, with or without p35S::NUT-HA, followed by treatment with 1 μM SCREW1. Data are shown as mean ± s.d. (n = 8). e, NUT-GFP restores SCREW1-induced MAPK activation in nut-2. Leaf discs were treated with 100 nM SCREW1 for 15 min. CBB, Coomassie brilliant blue; LP, left genomic primer; RP, right genomic primer; LB, T-DNA border primer. f, SCREW1-triggered leaf chlorosis is blocked in nut-2. Four-week-old plants were sprayed with 50 μM β-oestradiol and imaged five days later. Scale bars, 1 cm. g, NUT is required for SCREW1-induced resistance. Experiments were performed as in Fig. 1f. Bacterial numbers were detected at 3 dpi. Data are mean ± s.d. (n = 6). h, The nut mutants are more susceptible to Pst DC3000 hrcC⁻. Leaves were inoculated with Pst DC3000 hrcC⁻, and bacterial numbers were measured at 3 dpi and shown as mean ± s.d. (n = 8). i, Flg22-induced resistance is compromised in nut mutants. Experiments were performed as in Fig. 1h. Data are mean ± s.d. (n = 6). Experiments were repeated at least three times with similar results. Data were analysed by one-way (h, i) or two-way (c, d, g) ANOVA followed by Tukey’s test; n = biologically independent samples.

Two independent T-DNA insertion alleles of NUT (nut-1 and nut-2) were insensitive to SCREW-triggered MAPK activation (Fig. 2b, Extended Data Fig. 5d–f), seedling growth inhibition (Fig. 2c) and FRK1 promoter induction (Fig. 2d). Expressing NUT in nut mutant protoplasts or transgenic plants restored SCREW1-induced FRK1 promoter induction and MAPK activation (Fig. 2d, e, Extended Data Fig. 6a). Furthermore, the inducible leaf yellowing and cell death triggered by SCREW1 expression in wild-type plants were blocked in nut-2 mutants (Fig. 2f, Extended Data Fig. 6b). SCREW1-induced disease resistance against Pst DC3000 was abolished in nut mutants (Fig. 2g). Similar to screw1/2, nut mutants were more susceptible to Pst DC3000 by either infiltration or spray inoculation (Extended Data Fig. 6c, d) and Pst DC3000 hrcC⁻ (Fig. 2h), and showed compromised flg22-induced disease resistance against Pst DC3000 (Fig. 2i) compared to wild-type plants. NUT expression was induced by treatment with different MAMPs and various pathogens, including Pst DC3000, Pst DC3000 avrRpm1 and the fungal pathogen Botrytis cinerea (Extended Data Fig. 6e), suggesting that NUT has a broad role in plant immunity. Histochemical analysis of pNUT::GUS transgenic plants implied that the NUT promoter was strongly induced by flg22 treatment (Extended Data Fig. 6f), similar to pSCREW1/2::GUS (Fig. 1c).

The induction of SCREW and NUT expression by different pathogens prompted us to examine whether SCREW–NUT signalling has a role in plant resistance against other pathogens and insects. The screw and nut mutants did not affect plant resistance or the hypersensitive response to Pst carrying the effector avrRpm1 or avrRpt2 (Extended Data Fig. 6g, h). However, both screw and nut mutants showed increased susceptibility to B. cinerea (Extended Data Fig. 6i). Moreover, the p35S::SCREW1 and p35S::SCREW2 transgenic plants were more resistant to green peach aphids (Myzus persicae)—a phloem-feeding insect—than wild-type plants (Extended Data Fig. 6j). Conversely, aphid reproduction was increased in the nut plants (Extended Data Fig. 6k), indicating that SCREW–NUT has a role in the resistance of plants to aphids. Moreover, aphid feeding induced the expression of SCREW and NUT genes, as well as the activity of pNUT::GUS (Extended Data Fig. 6l, m). This insect infests plants through its specialized mouthpart that penetrates plant tissues and sucks saps from the phloem²⁵. This is consistent with the observation of increased expression of pSCREW::GUS and pNUT::GUS in vascular tissues (Fig. 1c, Extended Data Fig. 6f). Together, the data show that SCREW–NUT has a vital role in plant resistance against various pests, including bacterial P. syringe, fungal B. cinerea, and sap-feeding green peach aphids.

NUT is the receptor of SCREWs

NUT consists of an extracellular domain of 22 LRRs, a transmembrane domain and a cytoplasmic kinase domain (Fig. 3a). NUT–GFP is primarily localized at the plasma membrane in Arabidopsis transgenic plants and Nicotiana benthamiana (Extended Data Fig. 7a, b). Treatment with SCREW1 induced NUT endocytosis (Fig. 3b). The NUT kinase domain is annotated as an arginine–aspartate (RD) kinase (Extended Data Fig. 7c). The GST-tagged NUT cytosolic domain (NUT_CD)—but not its kinase-inactive mutant NUT_CD(K714E), which contains a Lys-to-Glu mutation in the ATP-binding site—possessed an autophosphorylation activity (Fig. 3c, Extended Data Fig. 7c), implying that NUT is a functional kinase. Unlike NUT, NUT (K714E) did not restore the SCREW1-triggered activation of MAPKs in nut-1 (Fig. 3d), supporting the functional importance of the NUT kinase activity.

Fig. 3 |. NUT is the SCREW receptor.

a, Diagram of NUT protein domains with amino acid positions labelled. b, SCREW1 induces NUT–GFP endocytosis. Seedlings treated with H₂O or 100 nM SCREW1 were imaged using confocal microscopy. Insert is a 3× magnification. Scale bars, 20 μm. c, NUT exhibits autophosphorylation activity. Phosphorylation is shown by autoradiogram. d, NUT(K714E) does not restore SCREW1-induced MAPK activation in nut-1. Protoplasts expressing NUT–HA or NUT(K714E)–HA were treated with 100 nM SCREW1. e, SCREW1 binds to plant-expressed NUT. NUT–Flag or FLS2–Flag expressed in protoplasts were incubated with 100 nM biotin–SCREW1 for an immunoprecipitation (IP) assay, and non-labelled SCREW1 or flg22 (10 μM) was used as a competitor. f, SCREW1 binds to NUT_ECD in SPR assays. Top, SPR sensorgram profile of SCREW1 peptides at gradient concentrations on a sensor chip immobilized with NUT_ECD. Bottom, steady-state affinity (binding at equilibrium). g, SCREW2 binds to NUT_ECD in SPR assays. SPR assays (f, g) were performed at pH 7.5. h, SCREWs induce NUT and BAK1 association in plants. Seedlings were treated with 1 μM SCREW for 30 min. NUT–BAK1 association was detected with co-immunoprecipitation using anti-BAK1 or anti-GFP antibodies. i, bak1-5/serk4-1 is insensitive to SCREW1-induced growth inhibition. Experiments were performed as in Fig. 1e. Data are mean ± s.d. (n = 8). j, bak1-5/serk4-1 is compromised in SCREW1-induced resistance to Pst DC3000. Experiments were performed as in Fig. 1f. Disease symptoms were observed at 3 dpi. Scale bars, 1cm. Data are mean ± s.d. (n = 8). Experiments were repeated three (b–e, h–j) or two (f, g) times with similar results. Data were analysed by two-sided Student’s t-test (i, j) and two-way ANOVA followed by Tukey’s test (i); n = biologically independent samples.

When immunoprecipitated from plant cells, NUT—but not FLS2, the LRR-RK receptor of flg22—pulled down biotinylated SCREW1 (Fig. 3e). Excessive non-labelled SCREW1, and not flg22, competed for biotin–SCREW1 binding to NUT (Fig. 3e), indicating a specific interaction between SCREW1 and NUT. Surface plasmon resonance (SPR) assays with the NUT extracellular domain (NUT_ECD) purified from insect cells showed that NUT_ECD bound to SCREW1 with a dissociation constant (K_d) of 12.97 μM and SCREW2 with a K_d of 6.23 μM at pH 7.5 (Fig. 3f, g). NUT_ECD also bound to SCREW1, SCREW2 and SCREW1–HA at pH 5.7, similar to the apoplastic pH under the normal growth condition (Extended Data Fig. 7d–f). The two conserved cysteine residues are essential for the binding of SCREW2 to NUT_ECD (Extended Data Fig. 7g). Moreover, SCREW2(ΔC8), a deletion variant without the C-terminal eight amino acids, showed a substantial reduction in binding affinity to NUT and could no longer activate MAPKs and inhibit seedling growth compared to SCREW2 (Extended Data Figs. 3b, c, 7h). Together, the data indicate that NUT binds to SCREW1 and SCREW2 in vivo and in vitro, and that NUT is the receptor of SCREW1 and SCREW2.

SCREW–NUT functions through SERKs

BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and its related SERKs are co-receptors of multiple LRR-RKs²⁶. SCREW1 and SCREW2 stimulated the formation of complexes of NUT and BAK1 (Fig. 3h). SPR assays indicated that BAK1 markedly increased the binding between NUT_ECD and SCREW2, with a K_d of 0.38 μM (Extended Data Fig. 7i). SCREW1-induced seedling growth inhibition was abolished in the bak1-5/serk4-1 mutant (Fig. 3i). Moreover, SCREW1-mediated resistance against Pst DC3000 was compromised in bak1-5/serk4-1 (Fig. 3j).

RLCK BIK1 associates with multiple PRRs and relays downstream signalling events^18,27. Notably, we did not detect an interaction of BIK1 with NUT, although BIK1 formed a complex with PEPR1 (Extended Data Fig. 7j). BIK1 was also not required for the SCREW1-induced inhibition of seedling growth (Extended Data Fig. 7k). In line with the observations that SCREW1 did not induce detectable BIK1 phosphorylation and weakly activated ROS burst (Extended Data Fig. 2i, k), SCREW–NUT might activate a BIK1-independent signalling pathway. Alternatively, other RLCKs might function redundantly with BIK1 in relaying the SCREW–NUT signalling.

SCREW–NUT reopens stomata in immunity

Phyllosphere microorganisms exploit stomata as entry points to invade the leaf apoplasts, which are an aqueous habitat for microbial colonization⁴. As a defence mechanism, MAMPs and DAMPs induce stomatal closure to prevent pathogen entry²⁸. In contrast to flg22, treatment with SCREW1 did not induce stomatal closure in wild-type plants (Extended Data Fig. 8a). Of note, SCREW1 suppressed flg22-induced stomatal closure (Fig. 4a, Extended Data Fig. 8a). SCREW–NUT did not affect the flg22-induced formation of the FLS2–BAK1 complex or MAPK activation, suggesting that SCREW does not affect the activity of flg22 per se (Extended Data Fig. 8b–d). SCREW1-mediated suppression of flg22-induced stomatal closure was abolished in nut mutants (Fig. 4a). Thus, the SCREW1–NUT signalling counteracts flg22-induced stomatal closure. Furthermore, we observed a gradual reopening of stomata at three and four hours after flg22 treatment in wild-type plants, which was compromised in nut and screw1/2 mutants (Fig. 4b). These data suggest that SCREW–NUT might mediate the subsequent reopening of stomata, which are initially closed upon the perception of MAMPs during pathogen invasions.

Fig. 4 |. SCREW–NUT counter-regulates ABA- and MAMP-induced stomatal closure.

a, SCREW1 suppresses NUT-dependent flg22- and ABA-induced stomatal closure. Stomatal apertures were measured after treatment with flg22, ABA or in combination with SCREW1 for 2h. b, screw1/2 and nut are compromised in reopening stomata. c, Reduced stomatal conductance in nut and screw1/2 mutants after infection. Leaves were inoculated with MgCl₂ (buffer) or Pst DC3000 (n = 12). d, Increased water potential in nut after infection. Leaves were infiltrated with Pst DC3000 carrying pProU::GFP or pNptII::GFP. e, Quantification of GFP expression in d (n = 4). f, Enhanced sensitivity to mannitol treatment of pEst::SCREW1-HA plants. Scale bar, 2 mm. g, SCREW1 reduces ABA activation of S-type anion channels. h, SCREW1 induces ABI1 phosphorylation. Protoplasts expressing ABI1–HA were treated with 1 μM SCREW1. Proteins were separated with Mn²⁺-Phos-tag or regular SDS–PAGE (n = 4). i, SCREW1 suppresses ABA-induced OST1 phosphorylation in an ABI-dependent manner. Protoplasts expressing OST1–Flag were treated with SCREW1 for 5min and then ABA for 5min (n = 5). j, SCREW1 increases ABI1 phosphatase activity towards OST1. ABI1–HA proteins immunoprecipitated from protoplasts treated with and without SCREW1 were used for a kinase assay. Averages of three independent repeats are labelled. k, ABI1 and ABI2 are required for SCREW1 suppression on ABA-induced stomatal closure (n = 202). Experiments were repeated at least three times with similar results. Data are shown as box plots as defined in the Methods (a, b, k), mean ± s.d. (c, e, h, i), or mean ± s.e.m. (g). Data were analysed by one-way (a, b, e, h) or two-way (k) ANOVA followed by Tukey’s test, or two-sided Student’s t-test (g, i, k). Different letters (a, b, k) denote a statistically significant difference (P < 0.05). Statistical analysis was performed within the same genotype (a, b). n = biologically independent samples.

Water availability in apoplasts is a crucial determinant for pathogen colonization^29,30. After sensing MAMPs, plants close their stomata to restrict pathogen entry. Inevitably, closed stomata prevent water loss in apoplasts and create living niches for bacterial colonization. How plants counteract the detrimental effects that are caused by the prolonged stomatal closure remains unclear. We monitored the stomatal conductance—a quantitative measurement of stomatal opening—during infections. As reported, after infection with Pst DC3000, the stomatal conductance was initially decreased and then gradually increased in wild-type plants³¹ (Fig. 4c). The increased stomatal conductance was compromised in screw1/2 and nut mutants (Fig. 4c), reinforcing that SCREW–NUT mediates stomatal reopening during pathogen invasions. Thus, SCREW–NUT might function as a defence mechanism to subsequently reopen the stomata, which were closed upon MAMP perception, and promote water loss, thereby disrupting an aqueous habitat for pathogen colonization. The vesicle-trafficking-related HOPM iNTERACTOR 7 (MIN7) regulates the aqueous apoplasts³². Notably, the min7 mutant was partially compromised in SCREW1-induced resistance to Pst DC3000, but did not affect SCREW1-induced MAPK activation and growth inhibition (Extended Data Fig. 8e–g), suggesting that SCREW–NUT might function partially overlapping with MIN7 downstream or independently of MAPK activation and growth inhibition.

To test whether SCREW–NUT-mediated stomatal reopening leads to a water potential change in apoplasts, we used a water-potential-responding reporter, pProU::GFP, in which GFP is under the promoter of the E. coli ProU operon, rapidly activated by the reduced water potential and thus used as a whole-cell biosensor for water availability³³. Pst DC3000 containing pNptII::GFP, the expression of which is driven by the constitutive neomycin phosphotransferase gene promoter and not affected by water potential, was included as a control for bacterial growth. The expression of pNptII::GFP was gradually increased at 6 and 9 hours post-infection (hpi) (Fig. 4d), indicating in planta bacterial multiplication. The expression of pNptII::GFP in nut was higher than that in the wild type at 9 hpi (Fig. 4d), corroborating the increased susceptibility of nut to Pst DC3000 (Extended Data Fig. 6c, d). Notably, the expression of pProU::GFP in nut was lower than that in the wild type at 9 hpi (Fig. 4d). Quantification of the expression of pProU::GFP relative to pNptII::GFP from three independent repeats indicated statistically significant differences between wild-type and nut mutant plants at 9 hpi (Fig. 4e). The data indicate that water potential in nut mutants is higher than in wild-type plants after infection with Pst DC3000, implying that SCREW–NUT negatively regulates the water potential during infection. High humidity promotes bacterial multiplication and the water-soaking disease phenotype by maintaining aqueous apoplasts during infection³². Under increased humidity (85–98%) with transient apoplast water supplementation³², the increased susceptibility and bacterial multiplication in screw1/2 or nut mutants became less apparent (Extended Data Fig. 8h). In addition, SCREW1 no longer induced plant resistance to Pst DC3000 under high humidity with transient apoplast water supplementation (Extended Data Fig. 8i). Together, our studies reveal the critical role of SCREW–NUT in plant immunity through modulating stomatal reopening and the apoplastic water level.

SCREW–NUT modulates plant water loss

Plants expressing p35S::SCREW1 or p35S::SCREW2 showed leaf wilting and curling, partially resembling plants under dehydration (Extended Data Fig. 9a). These transgenic plants also exhibited increased water loss in response to dehydration compared to wild-type plants (Extended Data Fig. 9b). In addition, inducible expression of SCREW1 rendered plants more sensitive to mannitol—a causative agent of the drought osmotic stress–than wild-type plants (Fig. 4f).

Conversely, screw1/2 and nut mutants exhibited reduced water loss and sensitivity to mannitol treatment compared to wild-type plants (Extended Data Fig. 9c, d). Notably, screw1/2 and nut mutants had the normal permeability and thickness of plant cuticle—one of the barriers protecting plants from water loss—as indicated by toluidine blue staining³⁴ or transmission electron microscopy analysis (Extended Data Fig. 9e–g). The plant hormone ABA mediates plant water usage and drought tolerance^35,36. Consistent with the reduced water loss, screw1/2 and nut mutants were more sensitive to ABA treatment than wild-type plants (Extended Data Fig. 9h). Under water-deficit stress, the increased levels of ABA promote stomatal closure to prevent water loss³. ABA-induced stomatal closure was suppressed by treatment with SCREW1 (Extended Data Fig. 8a). In addition, SCREW1 and SCREW2 suppressed the ABA-induced expression of the desiccation-responsive genes RESPONSIVE TO ABA 18 (RAB18) and RESPONSIVE TO DESICCATION 29A (RD29A) (Extended Data Fig. 9i). The SCREW1-mediated suppression of ABA-induced stomatal closure depended on NUT (Fig. 4a). Like flg22 treatment, the gradual reopening of ABA-induced stomatal closure depended on SCREW–NUT signalling (Fig. 4b). Moreover, SCREW and NUT transcripts were upregulated by ABA treatment, drought and mannitol treatment (Extended Data Fig. 9j, k). Thus, the data suggest that SCREW–NUT has a critical role in modulating plant water loss by antagonizing ABA-induced stomatal closure.

SCREW–NUT regulates the ABI–OST1 module

Stomatal aperture is mainly controlled by a core signalling module that consists of type 2C protein phosphatases (PP2Cs), including ABI1 and ABI2, and SNF1-related protein kinases (SnRK2s), most importantly OST1 (SnRK2.6), which regulates S-type anion channels such as SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) to drive plasma membrane depolarization and subsequent K⁺ efflux^2,37. The activation of S-type anion channels is a critical step in stomatal closure^38,39. Whole-cell recordings of anion channel currents in wild-type guard cells indicated that SCREW1 suppressed the ABA-induced activation of S-type anion channels (Fig. 4g). Of note, treatment with SCREW1 induced the phosphorylation of ABI1 and ABI2, but not OST1, as revealed by Phos-tag immunoblotting (Fig. 4h, Extended Data Fig. 10a, b). Furthermore, the SCREW1-induced ABI1 phosphorylation was no longer observed in nut-2, and expression of NUT–Flag restored ABI1 phosphorylation (Extended Data Fig. 10c).

ABA treatment induces the phosphorylation of OST1, a critical kinase in regulating stomatal closure^6,40 (Extended Data Fig. 11a, b). Expression of ABI1 or ABI2 blocked ABA-induced OST1 phosphorylation (Extended Data Fig. 11a, b), consistent with ABI phosphatase-mediated dephosphorylation and inhibition of OST1^2,37. Treatment with SCREW1 suppressed ABA-induced OST1 phosphorylation in a dosage-dependent manner (Fig. 4i, Extended Data Fig. 11c). We further tested whether ABI1 phosphorylation by SCREW–NUT could enhance its phosphatase activity. ABI1–HA immunoprecipitated from plant cells dephosphorylated purified MBP-tagged OST1 proteins (Fig. 4j). In addition, ABI1 stimulated with SCREW1 treatment exhibited an enhanced phosphatase activity towards OST1 (Fig. 4j). ABI1–HA co-immunoprecipitated with NUT–Flag in plant cells (Extended Data Fig. 11d). His-tagged ABI1 directly interacted with GST-tagged NUT_CD (Extended Data Fig. 11e). Together, the data indicate that SCREW–NUT regulates ABI phosphorylation and enhances its phosphatase activity, thereby reducing OST1 phosphorylation and the activity of S-type anion channels.

As SCREW–NUT targets ABIs, which dephosphorylate OST1, we tested the requirement of ABI1 and ABI2 for SCREW-regulated OST1 phosphorylation and stomatal opening. The SCREW1-triggered suppression of ABA-induced OST1 phosphorylation was compromised in the loss-of-function abi1-2/abi2-2 mutant (Fig. 4i). The SCREW1-mediated suppression of ABA-induced stomatal closure was also reduced in abi1-2/abi2-2 (Fig. 4k). In addition, ABI1 and ABI2 are partially required for the SCREW1-mediated suppression of flg22-induced stomatal closure (Extended Data Fig. 12a). Thus, our biochemical and genetic data indicate that SCREW–NUT regulates the stomatal aperture through the ABI–OST1 phosphorylation module.

Discussion

Terrestrial plants rely on stomata to facilitate gas exchange and water vapour between plants and the environment^1,2. Under water-deficit stress, leaf cells sense changes in water potential and accumulate ABA, which leads to stomatal closure and decreased transpiration to prevent water loss^3,36. As a defence strategy, plants close their stomata after sensing infections to limit the entry of pathogens, including bacteria and fungi^28,41. However, stomatal closure prevents the uptake of CO₂ and reduces transpiration and photosynthesis. Meanwhile, stomatal closure restricts water loss and leads to a rapid increase in apoplastic water content, favouring bacterial colonization^29,30. Thus, long-term stomatal closure is detrimental to plants, and plants must have evolved a mechanism to enable this process to be transient at the whole-plant level. Stomata also emerge as important mediators of plant–herbivore interactions⁴². We report here that a peptide–receptor pair, which is induced after pathogen exposure and dehydration, reopens the stomata and acts in concert with MAMP- and ABA-induced stomatal closure to enable stomatal movement to be a dynamic process in the plant response to pathogen and insect attacks and water deficiency.

Without infections, SCREWs and NUT are weakly expressed. The SCREW–NUT signalling may not be active at the initial pathogen invasion stage. After a MAMP is perceived, the expression of SCREWs and NUT is markedly increased. MAMP-PRR signalling induces SCREW–NUT to reopen the stomata, consequently disrupting the microorganism-rich aqueous habitat and suppressing the proliferation of pathogens at the post-invasion stage (Extended Data Fig. 12b). SCREW–NUT also counter-regulates ABA-induced stomatal closure and promotes water loss in response to dehydration, consistent with a previous report about the role of NUT (also named HAESA-LIKE3, HSL3) in stomatal closure and drought stress⁴³. A recent preprint also reports that HSL3 is the receptor for SCREWs (named CTNIPs there)⁴⁴. Cross-talk between ABA- and MAMP-induced stomatal closure has been observed⁵. OST1 is a crucial kinase that functions downstream of ABIs in ABA- and flg22-induced stomatal closure^5,6. We show here that SCREW–NUT induces ABI phosphorylation and enhances ABI phosphatase activity, thereby reducing OST1 phosphorylation and the activation of S-type anion channels. Thus, SCREW–NUT regulates the stomatal aperture by targeting the ABI-OST1 signalling module.

In summary, the SCREW–NUT peptide–receptor pair modulates two different facets of plant physiology in response to attacks by pathogens and dehydration by controlling stomatal reopening. SCREW–NUT homologues are broadly conserved in dicots and monocots. The SCREW–NUT-regulated stomatal movement dynamics are likely to be a widespread mechanism to ensure a balanced physiological response at the whole-plant level in response to biotic and abiotic stresses.

Methods

Plant materials and growth conditions

The Arabidopsis thaliana accession Columbia-0 (Col-0) was used as the wild type. T-DNA insertion mutants nut-1 (WiscDsLox450B04), mik2-1 (SALK_061769), mik2-like (SALK_112341) and nilr1 (SAIL_859-H01) were obtained from the Nottingham Arabidopsis Stock Centre (NASC), and nut-2 (SALK_207895) and at1g34420 (SALK_033924C) were from the Arabidopsis Biological Resource Center (ABRC). The fls2, bak1-3, bak1-4, bak1-5, sobir1-12, bik1, bak1-5/serk4-1, hae/hsl2, pepr1-2/pepr2-2, rlk7-2/iku2-1, mik2-1/mik2-l, let1/let2, min7 and abi1-2/abi2-2 mutants were reported in previous studies^{15,23,32,47,49–52}. The double mutants were generated by genetic crossing. The pNUT::GUS seeds were from J. Li⁵³. All Arabidopsis plants, unless otherwise stated, were grown on soil (Metro Mix 366, Sunshine LP5 or Sunshine LC1, Jolly Gardener C/20 or C/GP) in a growth room at 20–23 °C, 50% humidity and 100 μE m⁻² s⁻² light with a 12-h light–12-h dark photoperiod. Four- to five-week-old plants were used for protoplast isolation, disease assays and detection of ROS production, callose deposition and stomatal movement. Seedlings used for analyses of growth inhibition, MAPK activation, gene transcription and β-glucuronidase (GUS) staining were grown on half-strength Murashige and Skoog (½ MS) plates containing 0.5% (w/v) sucrose and 0.75% (w/v) agar, pH 5.7, under the same growth conditions as plants grown on soil. Tobacco (Nicotiana benthamiana) was grown on Sunshine LP5 soil under a 14-h light/10-h dark photoperiod at 23 °C.

Plasmid construction, protoplast transfection and Arabidopsis transformation

pFRK1::LUC, pUBQ10::GUS, pHBT-35S::SERK-HA, pHBT-35S::FLS2-Flag, and pHBT-35S::FLS2-GFP and pHBT-35S::PEPR1-Flag were described previously^49,54. SCREW1 and SCREW2 were PCR-amplified from Col-0 cDNA using gene-specific primers with BamHI at the 5′ end and StuI or SmaI at the 3′ end, followed by digestion with BamHI and StuI or SmaI and ligation into the pHBT vector with the HA sequence at the 3′ end to generate pHBT-35S::SCREW1, 2-HA. NUT was amplified from Col-0 genomic DNAs using gene-specific primers with NcoI at the 5′ end and StuI at the 3′ end and ligated into the pHBT vector with HA, Flag, or GFP sequences at the 3′ end to generate pHBT-NUT-Flag/HA/GFP. The pHBT-35S::NUT^K714E-HA vector was generated by site-directed mutagenesis using pHBT-35S::NUT-HA as the template.

To construct pSCREW::GUS vectors, 1,879-bp and 1,927-bp sequences upstream of the SCREW1 or SCREW2 start codon were amplified from genomic DNAs using the primer pair containing HindIII or BglII and ligated into the pGFPGUSplus vector²³. SCREW1 or SCREW2 were PCR-amplified using gene-specific primers containing KpnI or SalI, followed by KpnI and SalI digestion and ligation into the pCAMBIA1300 vector to generate pCAMBIA1300-35S::SCREW1, 2. SCREW1 was amplified using gene-specific primers containing XhoI or StuI and inserted into pTA7002 with an HA-tag at the 3′ end to generate pTA7002-Dex::SCREW1-HA. SCREW1-HA-NOS was then shuttled to the pTK103 vector by XhoI and SpeI digestion to create pTK103-Est::SCREW1-HA. Coding sequences of SCREW1, SCREW2, NUT, and ABI2 were PCR-amplified from Arabidopsis cDNA using gene-specific primers with overlapping sequences from the pMDC32-GFP or pMDC32-HA vector and then were recombined into the BamHI- and StuI-digested pMDC32-35S::GFP or pMDC32-35S::HA using the ClonExpress II One Step Cloning Kit (Vazyme) to generate pMDC32-35S::SCREW1/2, NUT-GFP, and pMDC32-35S::ABI2. ABI1 and OST1 were PCR-amplified from Col-0 cDNA using gene-specific primers with BamHI at the 5′ end and StuI at the 3′ end, followed by digestion with BamHI and StuI and ligation into the pMDC32 vector with the HA or Flag sequence at the 3′ end to generate pMDC32-35S::ABI1-HA and pMDC32-OST1-Flag.

To create the E. coli recombinant protein constructs, the coding sequences of SCREWs without the N-terminal signal peptide were PCR-amplified from Arabidopsis cDNA using gene-specific primers with BamHI or XhoI and inserted into pGEX-4T-1 to generate pGST-SCREW1, 2, 3. The SCREW1 coding sequence lacking the N-terminal signal peptide was PCR-amplified from cDNAs using gene-specific primers containing BamHI or StuI and inserted into pET28a-His-SUMO-BIK1 to create pET28a-His-SUMO-SCREW1. ABI1-HA was PCR-amplified from pMDC32-35S::ABI1-HA using gene-specific primers containing BamHI or StuI and inserted into pET28a-His-SUMO-BIK1 to create pET28a-His-SUMO-ABI1-HA. The cytosolic domain of NUT (NUT_CD) was PCR-amplified from Arabidopsis cDNA using gene-specific primers with BamHI or XhoI and inserted into pGEX-4T-1 to create pGST-NUT_CD. pGST-NUT_CD(K714E) was generated by site-directed mutagenesis using pGST-NUT_CD as a template. To build the construct used for recombinant NUT_ECD expression in insect cells, the nucleotide sequence encoding NUT_ECD (aa residues 23–626) was biosynthesized and cloned into the pFastBac-1 vector with a signal peptide (MKLCILLAVVAFVGLSLG) at the N terminus and a 6×His tag at the C terminus (Gene Universal).

To construct pHEE401E-gSCREW1-gSCREW2 for the CRISPR–Cas9-mediated gene editing, two guide RNAs (gRNAs) were designed using CHOPCHOP (http://chopchop.cbu.uib.no/). gRNA1 targeting SCREW1 and gRNA2 targeting SCREW2 were incorporated into a gRNA expression cassette through PCR amplification with primers carrying gRNA1, gRNA2, and pCBC-DT1T2 as a template. The PCR fragments were further amplified using primers containing BsaI and inserted into pHEE401E⁵⁵.

All primers are listed in Supplementary Table 1, and PCR-amplified DNA fragments were confirmed using Sanger sequencing.

Protoplast isolation and transient expression assays were described previously⁵⁶. Agrobacterium tumefaciens strain GV3101 carrying binary vectors was used for Arabidopsis transformation by floral dipping. Transformants were selected with 25 μg ml⁻¹ hygromycin, 50 μg ml⁻¹ kanamycin or 10 μg ml⁻¹ Basta. Multiple transgenic lines were analysed by immunoblotting for protein expression. Two lines with the 3:1 segregation ratio for antibiotic resistance in the T₃ generation were selected to obtain homozygous seeds for further studies.

Peptide synthesis

Peptide sequences were listed in Supplementary Table 2. Flg22 and Pep1 were synthesized from BIOMATIK (Delaware), and SCREWs and SCREW derivatives from ChinaPeptides. For the synthesis of N-terminally biotinylated SCREW1 (biotin–SCREW1), a lysine residue was added to the N terminus of the SCREW1 peptide, and biotin was bound to the lysine side chain through an amide bond. For the synthesis of SCREW2(CC), SCREW2 peptide was oxidized with the addition of 10% DMSO (v/v) to facilitate the formation of a disulfide bond at pH 8.0 adjusted by meglumine.

Identification of screw1/2 mutants

Genomic DNAs were extracted from wild-type plants and T₁ generation pHEE401E-gSCREW1-gSCREW2 transformants. Fragments of 672 bp for SCREW1 and 679 bp for SCREW2 spanning the targeted loci of the gRNAs were amplified and subjected to BslI digestion. Gene editing in the fragments was confirmed by Sanger sequencing. Transgenic plants containing mutations in both SCREW1 and SCREW2 were used for further analyses. T₂ and T₃ plants were further confirmed by BslI digestion and Sanger sequencing.

RNA isolation, reverse transcription and RT–qPCR analysis

Total RNAs were extracted from Arabidopsis seedlings or rosette leaves using TRIzol reagent (Invitrogen) and quantified with a spectrophotometer (NanoDrop2000, Thermo Fisher Scientific). One microgram of total RNAs were reverse-transcribed to synthesize the first-strand cDNA with M-MuLV Reverse Transcriptase and oligo(dT) primers after treatment with RNase-free DNase I (New England Biolabs). RT–qPCR analyses were performed in the CFX384 Touch Real-Time PCR Detection System (Bio-Rad) with gene-specific primers (Supplementary Table 1) and iTaq SYBR green Supermix (Bio-Rad) following a manufactural protocol. The expression of each gene was normalized to the expression of UBQ10 or ACTIN2.

MAPK activation assay

Ten-day-old seedlings grown on ½MS plates were transferred into ddH₂O, kept overnight, and then treated with flg22, Pep1 or SCREWs with the indicated concentrations for the indicated time. Each sample containing three seedlings was grounded in 40 μl extraction buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% [v/v] Triton X-100, 1 mM Na₃VO₄, 1 mM NaF, 1 mM DTT and 1:200 complete protease inhibitor cocktail from Sigma). The supernatant was collected after 13,000g centrifugation for 5 min at 4 °C, and protein samples with 1× SDS buffer were loaded on 10% (v/v) SDS–PAGE gel to detect phosphorylated MPK3 and MPK6 by immunoblotting with anti-pERK1/2 antibodies (1:2,000; Cell Signaling) followed by anti-rabbit (1:10,000; Cell Signaling).

Analysis of ROS production

The third or fourth pair of true leaves from four- to five-week-old soil-grown Arabidopsis plants were excised into leaf discs (5 mm diameter). Leaf discs were incubated in 100 μl ddH₂O in a 96-well plate with gentle shaking overnight to eliminate the wounding effect. Water was replaced by 100 μl of a reaction solution containing 50 μM luminol, and 10 μg ml⁻¹ horseradish peroxidase (HRP, Sigma-Aldrich) supplemented with or without 100 nM flg22, 100 nM or 1 μM SCREW1. Luminescence was measured with a luminometer (Glomax Multi-Detection System, Promega) for 40 min. ROS production was indicated as means of relative light units (RLU).

Quantitative Ca²⁺ measurements

Protoplasts were transfected with p35S::mCherry-AEQ⁵⁷ and incubated in WI solution (0.5 M mannitol, 20 mM KCl and 4 mM MES pH 5.7) supplemented with 2 mM CaCl₂ at 23 °C for 7 h, followed by a 2-h treatment with 10 μM coelenterazine-h (Thermo Fisher Scientific). Approximately 2 × 10⁴ protoplasts (100 μl with a density of 2 × 10⁵ cells per ml) were loaded into individual wells of a black 96-well microplate and incubated for 30 min in the dark followed by treatment with flg22 or SCREW1 and Ca²⁺ measurements. Luminescence was measured with a luminometer (Glomax Multi-Detection System, Promega) with a 1-s interval for 15 min. The values for cytosolic Ca²⁺ concentration were indicated as means of RLU.

Seedling growth inhibition assay

Three-day-old seedlings grown on vertical ½MS plates were transferred into a 24-well plate containing 500 μl liquid ½MS medium supplemented with or without 1 μM SCREWs. Two seedlings were placed in a single well, and a total of at least six seedlings was used for each treatment. Seedlings were photographed seven days after treatments, and the fresh weight of each seedling was measured.

Trypan blue staining

Trypan blue staining was performed as previously described with modifications⁴⁸. In brief, detached leaves were immersed in the trypan blue staining solution (2.5 mg ml⁻¹ trypan blue in lactophenol (lactic acid:glycerol:liquid phenol:H₂O = 1:1:1:1]) under vacuum infiltration for 5 min, followed by incubation at 23 °C for 8 h with shaking at 70 r.p.m. on a rocker. Leaves were destained in a solution (ethanol: lactophenol = 2:1) at 65 °C for 30 min and then incubated in fresh distaining solution 23 °C until completely destained.

Toluidine blue staining

Toluidine blue staining was performed as previously described³⁴. Shoots of three-week-old plants grown in soil were submerged in 0.05% toluidine blue solution at room temperature for 15 min. Abaxial surfaces of plant leaves from four-week-old plants were dipped with 5 μl of 0.05% toluidine blue solution and incubated for 5–60 min at room temperature. The samples were rinsed with water before being photographed.

Histochemical detection of GUS activity

Two-week-old Arabidopsis seedlings or rosette leaves of four-week-old plants were immersed and vacuumed in the GUS staining solution (10 mM EDTA, 0.01% Silwet L-77, 2 mM K₃Fe(CN)₆, 2 mM K₄Fe(CN)₆ and 2 mM X-Gluc in 50 mM PBS, pH 7.0) for 5 min, followed by incubation at 37 °C for 12–24 h. Samples were cleared in 75% ethanol for 6 h, followed by photographing using the Olympus SZX10 stereomicroscope.

FRK1 reporter assay

For detection of flg22- or SCREW-induced FRK1 promoter activity, protoplasts (100 μl at the concentration of 10⁵ cells per ml) of wild-type Col-0 were co-transfected with 8 μg of pFRK1::LUC and 2 μg of pUBQ10::GUS, aliquoted into individual wells to incubate for 4 h at 23 °C followed by treatment with 100 nM flg22, 1 μM GST or 1 μM GST–SCREW for another 4 h. For NUT complementation assays, protoplasts of wild-type Col-0 and nut mutants were co-transfected with pFRK1::LUC, pUBQ10::GUS, and an empty vector or p35S::NUT-HA as described above. Protoplasts were collected and lysed with 30 μl cell lysis buffer (25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100 and 2 mM DTT). Cell lysates (20 μl) were used for the detection of luciferase activity with the Glomax Multi-Detection System (Promega) and the luciferase assay substrate (Promega). For GUS activities, 4-MUG (4-methylumbelliferyl-β-d-glucuronide) was mixed with 10 μl cell lysates, and fluorescence signals were analysed with a Multilabel Plate Reader (Victor X3, PerkinElmer). The relative luciferase activity was indicated by the ratio of the activity of luciferase to GUS.

Laser confocal microscopy

For protoplast transient expression assay, protoplasts were transfected with pHBT-35S::GFP, pHBT-35S::NUT-GFP or pHBT-35S::FLS2-GFP and incubated for 12 h. For N. benthamiana transient expression assays, A. tumefaciens strain GV3101 containing pMDC32-35S::GFP, pMDC32-35S::SCREW2-GFP, or pMDC32-35S::NUT-GFP was cultured overnight in LB medium (10 g l⁻¹ tryptone, 5 g l⁻¹ yeast extract, 5 g l⁻¹ NaCl) at 28 °C. The bacterial suspension containing 10 mM MES, pH 5.7, 10 mM MgCl₂ and 200 μM acetosyringone at an optical density at 600 nm (OD_{600 nm}) of 1.0 was infiltrated into leaves of four-week-old soil-grown N. benthamiana using a needleless syringe and incubated for 72 h. To detect SCREW1-induced NUT endocytosis, true leaves of seven-day-old Arabidopsis transgenic seedlings carrying pMDC32-35S::NUT-GFP were treated with water or 100 nM SCREW1 for 5 and 45 min and imaged using the Leica SP8 confocal laser microscope. The cotyledons of ten-day-old transgenic seedlings carrying p35S::GFP or p35S::NUT-GFP grown on ½MS plates were used to observe GFP and NUT–GFP subcellular localization. Fluorescence images were taken with the Leica SP8 confocal laser microscope. The excitation laser of 488 nm and 561 nm was used for imaging GFP and chloroplast fluorescence, respectively.

Transmission electron microscopy

Leaves of four-week-old plants were fixed in 3.7% formaldehyde and 1% glutaraldehyde in PBS buffer. After primary fixation, samples were rinsed and postfixed in buffered 1% osmium tetroxide. Samples were then dehydrated in an acetone series and embedded in Quetol-ERL 421 resin. The sectioned samples were imaged on a JEOL1200Ex electron microscope.

In vivo co-immunoprecipitation assays

For co-IP in protoplasts, protoplasts were co-transfected with the indicated vectors and incubated for 12 h. After treatment with 1 μM SCREWs or flg22 for 15 min, protoplasts were collected by centrifugation at 100g for 2 min and lysed in 300 μl of IP buffer (20 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100 and protease inhibitor cocktail from Sigma) by vortexing. After centrifugation at 10,000g for 10 min at 4 °C, 30 μl of supernatant was collected as input control. The remaining supernatant was pre-incubated with protein G–agarose beads at 4 °C for 1 h with gentle shaking at 70 r.p.m. on a rocker. Immunoprecipitation was performed with anti-Flag agarose (Sigma-Aldrich) for 3 h at 4 °C. Beads were collected by centrifugation at 500g for 2 min and washed three times with washing buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100) and once with 50 mM Tris-HCl, pH 7.5. Immunoprecipitated proteins and input proteins were analysed by immunoblotting with anti-HA (1:2,000; Roche) or anti-Flag antibodies (1:2,000; Sigma-Aldrich).

For biotin-labelled SCREW1 (biotin–SCREW1) immunoprecipitation with NUT-Flag, protoplasts were transfected with p35S:: NUT-Flag, p35S::FLS2-Flag or an empty vector (control) and incubated for 12 h. Samples were lysed with lysis buffer as indicated above and incubated with 100 nM biotin–SCREW1 at 4 °C with shaking at 70 r.p.m. on a rocker for 3 h. Ten micromoles of non-labelled SCREW1 or flg22 was added together with biotin–SCREW1 as competitors. Five microlitres of anti-Flag agarose beads was added into the samples and they were incubated for another 1 h at 4 °C. Agarose beads were collected by centrifugation at 500g for 2 min and washed three times with the aforementioned washing buffer and one time with 50 mM Tris-HCl, pH 7.5. Immunoprecipitated proteins and input proteins were analysed by immunoblotting with anti-Flag antibodies (1:2,000; Sigma-Aldrich) for NUT–Flag and FLS2–Flag and HRP-labelled streptavidin (1:5,000; Thermo Fisher Scientific) for biotin–SCREW1.

For co-immunoprecipitation in plants, ten-day-old transgenic seedlings carrying p35S::GFP or p35S::NUT-GFP grown on the liquid ½MS were treated with or without SCREW1 or SCREW2 for 15 min. Seedlings of 500 mg were ground into powders with liquid nitrogen and lysed in 2 ml IP buffer by vortexing. After centrifugation at 10,000g for 10 min at 4 °C, the supernatant was collected for incubation with GFP-trap beads (Chromotek, Germany) at 4 °C for 3 h with gentle shaking at 70 r.p.m. on a rocker. Beads were collected by centrifugation at 500g for 2 min and washed three times with washing buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100) and once with 50 mM Tris-HCl, pH 7.5. Immunoprecipitated proteins and input proteins were analysed by immunoblotting with anti-GFP (1:2,000, Roche) followed by anti-mouse (1:10,000, Cell Signaling) or anti-BAK1 antibodies (1:2,000)⁵⁸ followed by anti-rabbit (1:10,000, Cell Signaling).

Disease assays

For bacteria disease assays, Pst DC3000, Pst DC3000 hrcC⁻, PstDC3000 avrRpm1 and Pst DC3000 avrRpt2 were cultured in King’s B (KB) liquid medium (Bacto proteose peptone 20 g l⁻¹, K₂HPO₄ 1.5 g l⁻¹, MgSO₄·7H₂O 1.5 g l⁻¹, glycerol 20 ml l⁻¹ and agar 15 g l⁻¹, pH 7.2) containing 50 μg ml⁻¹ rifampicin at 28 °C for 12 h. The bacteria were collected by centrifugation at 2,000g for 2 min at 23 °C and diluted into the indicated titres with 10 mM MgCl₂. Fully expanded leaves of four-week-old plants were spray inoculated or infiltrated with the bacterial suspension using a 1-ml needleless syringe. For flg22 or SCREW1-mediated protection assays, leaves were infiltrated with 500 nM flg22 or SCREW1 24 or 36 h before inoculation with PstDC3000. Leaf disks were collected for the bacterial counting at 0 or 3 days post-inoculation (dpi). Bacteria in two leaf disks as one sample were released into 200 μl H₂O by grinding, diluted into gradient titres and spread on tryptone soya agar (TSA) plates (1% (w/v) Bacto tryptone, 1% (w/v) sucrose, 0.1% (w/v) glutamic acid and 1.5% (w/v) agar) containing 25 μg ml⁻¹ rifampicin. Bacterial colonies were counted to determine colony-forming units (cfu) after incubation at 28 °C for two days.

For Botrytis cinerea disease assays, the B. cinerea strain BO5-10 was inoculated on potato dextrose agar and cultivated at 23 °C for two days. Conidia were collected and diluted to 10⁷ spores per ml with potato dextrose broth. Leaves detached from four-week-old plants were dropped with 5 μl spore suspension and kept in 98% humidity. Diameters of disease spots were measured at 2 dpi.

Aphid bioassays

Aphid no-choice tests were performed as described previously⁵⁹. Green peach aphids (Myzus persicae) used in this study are a tobacco (Nicotiana tabacum)-adapted red lineage that were maintained on cabbage (Brassica oleracea). Both adults and nymphs used in our experiments were female. Six age-synchronized second-instar nymphs (within 24 h) were placed on four-week-old plants. The total number of neonates on each plant was counted 10 days after infestation. Each genotype had at least 10 replicates.

Measurement of stomatal aperture

Stomatal apertures were measured on epidermal peels excised from the abaxial side of leaves of three- to four-week-old plants as described previously^60,61. For detection of flg22, ABA or SCREW1 induction of stomatal closure, two epidermal peels from two independent plants were incubated for 30 min in the dark in a bathing solution containing 30 mM KCl and 10 mM MES/Tris, pH 6.0. Epidermal peels were first maintained under darkness to keep them closed or exposed to white light in a growth chamber (120 μE m⁻² s⁻¹) for 3 h to induce maximal stomatal opening. Then, flg22 (1 μM), ABA (10 μM), SCREW1 (1 μM) or a combination of SCREW1 and flg22 or ABA was added to the bathing solution, and stomatal apertures were monitored for the indicated time in over 60 stomata for each independent repetition. The width and the length of the stomatal aperture were measured using ImageJ, and the stomatal aperture index was calculated by division of the aperture width through the length as described⁶².

Measurement of stomatal conductance

Stomatal conductance in leaves of six-week-old Arabidopsis plants was measured using a portable photosynthesis system (CIRAS-3, PP Systems). For detection of stomatal conductance under pathogen infections, the sixth or seventh pairs of true leaves were hand-inoculated with Pst DC3000 (OD_{600 nm} = 0.01) after exposure under light (120 μE m⁻² s⁻¹) for 2 h in the growth chamber, and stomatal conductance was measured at the indicated time point. Measurement parameters were CO₂ 400 μmol mol⁻¹ and photosynthetic photon flux density of 200 μmol m⁻² s⁻¹.

Measurement of water potential

Measurement of water potential was achieved with the water-responding pProU::GFP reporter described previously with modifications³³. The plasmid carrying pProU::GFP or pNptII::GFP was transformed into Pst DC3000. To measure the water potential, leaves from four-week-old soil-grown Arabidopsis plants were hand-inoculated with Pst DC3000 pProU::GFP or Pst DC3000 pNptII::GFP (OD_{600 nm} = 0.1), respectively. Two leaf discs were then collected at 0, 3, 6, and 9 h post-inoculation, and proteins were extracted with 1× SDS loading buffer. The expression of GFP in leaf discs was detected by immunoblotting with anti-GFP anti-body (1:2,000; Roche) followed by anti-mouse (1:10,000; Cell Signaling) and quantified by ImageJ. Quantification data are shown as the ratio of signal intensities of pProU::GFP to pNptII::GFP.

Patch-clamp experiments

S-type anion channel recordings were performed as previously described^63,64. Arabidopsis guard cell protoplasts were isolated from the epidermis of rosette leaves by a 12-h incubation at 21 °C with shaking in an enzyme solution containing 1.0% (w/v) Cellulase R10 (Yakult Pharmaceutical Industry), 0.5% (w/v) Macerozyme R10 (Yakult Pharmaceutical Industry), 0.5% (w/v) bovine serum albumin, 0.1% (w/v) kanamycin, 0.1 mM KCl, 0.1 mM CaCl₂, 10 mM ascorbic acid and 500 mM d-mannitol (pH 5.6 with KOH). Isolated guard cell protoplasts were collected through a nylon mesh with 10 μm size openings and then washed twice by centrifugation at 200g for 5 min with wash solution containing 0.1 mM KCl, 0.1 mM CaCl₂ and 500 mM d-sorbitol (pH 5.6 with KOH). Guard cell protoplasts were treated with or without 5 μM SCREW1 for 1 h, followed by treatment with or without 10 μM ABA for 0.5 h. The patch-clamp pipette solution was composed of 150 mM CsCl₂, 2 mM MgCl₂, 6.7 mM EGTA, 5.58 mM CaCl₂, 5 mM Mg-ATP and 10 mM HEPES-Tris (pH 7.1). The patch-clamp bath solution was composed of 30 mM CsCl₂, 2 mM MgCl₂, 1 mM CaCl₂ and 10 mM MES-Tris (pH 5.6). Osmolarities of the pipette and bath solutions were adjusted with d-sorbitol to 500 mosmol kg⁻¹ and 485 mosmol kg⁻¹, respectively. Whole-cell S-type anion current recordings were performed using a CEZ-2200 patch-clamp amplifier (Nihon Kohden) and pCLAMP 8.1 software (Molecular Devices).

ABA and mannitol treatment and measurement of water loss

For measurement of water loss, rosette leaves were detached from four-week-old plants and laid on dry filter paper under the light with the abaxial side of the leaves facing up. The weights of leaves were measured every hour over a period of six hours. The water-loss rate was presented as the leaf weight ratio at each time point relative to the initial weight. For analyses of the effects of ABA treatment on cotyledon greening rate, seeds after stratification at 4 °C for two days were germinated on ½MS plates with or without 1 μM ABA for seven days; this was followed by photographing, and the percentages of seedlings with radical and expanded green cotyledons were calculated. Approximately 80 seeds were counted for each genotype in each biologically independent repeat. For analyses of mannitol-treated phenotypes, seeds were germinated on ½MS plates containing 3 μM β-oestradiol supplemented with or without 100 or 200 mM mannitol after surface sterilization and stratification. Seedlings were photographed after 15 days of growth.

Purification of recombinant proteins, GST pull-down and in vitro kinase and phosphatase assays

Recombinant proteins were expressed in E. coli BL21 strain with an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible system. GST and GST-tagged proteins were purified with Pierce glutathione agaroses (Thermo Fisher Scientific), and His-tagged proteins were purified with Ni Sepharose beads (Qiagen) according to manufacturer’s protocol. For the GST pull-down assay, 10 mg of His–ABI1–HA proteins were incubated with prewashed GST or GST–NUT_CD glutathione beads in 0.5 ml pull-down buffer (10 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol and 1% Triton X-100) for 2 h at 4 °C with gentle shaking. The pulled-down proteins were analysed by immunoblotting with anti-HA (1:2,000) antibodies. For in vitro kinase assays, GST, GST–NUT_CD or GST–NUT_CD (K714E) were incubated in a kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 5 mM EDTA, 1 mM DTT and 100 mM ATP) containing 5 μCi[γ-³²P]ATP for 2 h at 23 °C with gentle shaking. The reaction was stopped by adding 4× SDS loading buffer (250 mM Tris-HCl, pH 6.8, 40% (v/v) glycerol, 4% (w/v) SDS, 0.1% (w/v) bromophenol and 4% (v/v) β-mercaptoethanol). Protein phosphorylation was visualized by autoradiography after running with SDS–PAGE. For in vitro phosphatase assay, protoplasts were transfected with pMDC32-35S::ABI1-HA and incubated for 12 h. After treatment with 1 μM SCREW1 for 5 min, protoplasts were collected by centrifugation at 100g for 2 min and lysed in 300 μl of IP buffer by vortexing. ABI1–HA was immunoprecipitated from the supernatant with anti-HA magnetic beads (Thermo Fisher Scientific) and resuspended in IP buffer. ABI1–HA and MBP–OST1 were incubated in a kinase reaction buffer containing 5 μCi[γ-³²P]ATP for 2 h at 23 °C with gentle shaking. The reaction was stopped by adding 4× SDS loading buffer. MBP–OST1 phosphorylation was visualized by autoradiography after 10% SDS–PAGE. Band intensities of phosphorylated MBP–OST1 (pOST1) normalized to input MBP–OST1 were quantified by ImageJ, and averages of three independent repeats were labelled below the gel (n = 3, biologically independent repeats).

Phos-tag immunoblotting

For detecting the phosphorylation of ABI1, ABI2 or OST1 with Phos-tag gel, protoplasts were transfected with pMDC32-35S::ABI1/2-HA or pMDC32-35S::OST1-Flag and incubated for 12 h. After treatment with SCREW or ABA, protoplasts were collected by centrifugation at 100g for 2 min and then lysed with 1× SDS loading buffer. The total proteins were separated in the 8% SDS–PAGE containing 25 μM Phos-tag (Fujifilm Wako Chemicals) and 100 mM MnCl₂, and immunoblotted with anti-HA (1:2,000) or anti-Flag antibodies (1:2,000, Sigma-Aldrich). For certain Phos-tag SDS–PAGE, molecular weight cannot be exactly indicated. For relative pABI1, band intensities of phosphorylated ABI1 (pABI1) normalized to input ABI1 in regular immunoblotting were quantified. For relative pOST1, band intensities of phosphorylated OST1 (pOST1) and unphosphorylated OST1 were quantified by ImageJ and the relative pOST1 represents the ratio of phosphorylated to unphosphorylated OST1.

NUT_ECD expression in insect cells and SPR analyses

NUT_ECD fused with a signal peptide (MKLCILLAVVAFVGLSLG) at the N terminus and a 6× His at the C terminus was expressed using the Bac-to-Bac baculovirus expression system (Invitrogen) in SF9 cells (Thermo Fisher Scientific, cat. no. 11496015) at 27 °C. No additional authentication or mycoplasma contamination testing was done by authors in this study. Five hundred millilitres of SF9 cells (2 × 10⁶ per ml) cultured in the Sf-900 II SFM medium (Invitrogen) were infected with 25 ml recombinant baculovirus and were cultured for another four days with gentle shaking at 27 °C. Secreted NUT_ECD in the supernatant was purified with Ni-NTA beads (Novagan) and then was dialysed into 10 mM PBS solution (2 mM KH₂PO₄, 8 mM Na₂HPO₄, 136 mM NaCl, 2.6 mM KCl, pH 7.4). Expression and purification of BAK1_ECD protein (residues 1–220) were described previously⁶⁵.

The binding kinetics and affinities of NUT_ECD with SCREW or SCREW derivative peptides were performed on a Biacore T200 instrument (GE Healthcare) with CM5 chips (GE Healthcare) at 25 °C. NUT_ECD proteins were exchanged to 10 mM NaAc (pH 5.0) and the peptides were dissolved in HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% (v/v) Surfactant P20, pH 7.5 or pH 5.7) (GE Healthcare). About 4,700 response units of NUT_ECD proteins were immobilized on the CM5 chip, and a blank channel was used as a negative control. The peptides were diluted into indicated concentrations and injected at a flow rate of 30 μl min⁻¹ in the absence or presence of 0.1 μM BAK1_ECD for 120 s, followed by dissociation for 5 min. After dissociation, 5 mM NaOH was injected for 30 s to remove any non-covalently bound proteins from the chip surface. The binding kinetics were analysed with the software Biaevaluation v.4.1 using the 1:1 Langmuir binding mode.

Multiple sequence alignment and phylogenetic tree analysis

Protein sequences were accessed from the NCBI database. Amino acid sequences were aligned using ClustalW and visualized in ESPript3 (https://espript.ibcp.fr/ESPript/ESPript/). WebLogo was conducted with WebLogo 3 (http://weblogo.threeplusone.com/). Phylogenetic trees were constructed using MEGAX with the neighbour-joining method. The trees in Extended Data Figs. 1b, c, 5b, 5c were visualized in an interactive tree of life (iTOL, https://itol.embl.de/).

Quantification and statistical analysis

No statistical methods were used to predetermine sample size. Blinding and randomization were not used. Data for quantification analyses are presented as mean ± s.e.m. or standard deviation (s.d.), or as box plots with the interquartile range as the upper and lower confines, minima and maxima as whiskers, and the median as a solid line. Statistical analyses were performed by two-sided Student’s t-test, or one-way or two-way ANOVA followed by Tukey’s or Dunnett’s test. The number of biologically independent replicates is indicated in graphs or figure legends. Exact P values are provided in the graphs and Supplementary Table 3.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Extended Data

Extended Data Fig. 1 |. Identification of SCREWs in Arabidopsis.

a, Upregulation of Arabidopsis peptide genes by flg22 in an FLS2-dependent manner. The gene expression data were extracted from an RNA-Seq analysis¹⁷ and subjected to data adjustment by log₂ transformation using TBtools for the heat map. b, Phylogenetic analysis of Arabidopsis SCREW homologs and schematic diagrams of different domains. The phylogenetic tree was constructed with MEGAX using neighbour-joining methods. The bootstrap values from 1,000 replications are indicated on the branches. c, SCREW orthologs are present in dicots and monocots. Protein sequences were blast-searched against the NCBI database, and wheat, rice, and maize genomes using Arabidopsis SCREW1 as a query. The phylogenetic tree was constructed as indicated in (b) and displayed with iTOL v5 online software (https://itol.embl.de/). The bootstrap values from 1,000 replications are shown on the branches. d, SCREWs are upregulated after elf18 treatments. Ten-day-old plate-grown seedlings were treated with 200 nM elf18. The expression of SCREWs normalized to UBQ10 was analysed by RT–qPCR. Means (n = 4, biologically independent samples) of fold induction shown as log₂ values were used to construct heat map using TBtools. e, SCREWs are upregulated after Pep1 treatments. Ten-day-old plate-grown seedlings were treated with 200 nM Pep1. The expression of SCREWs was detected and shown as in (d) (n = 4, biologically independent samples). f, SCREWs are upregulated after Pst DC3000 infections. Ten-day-old plate-grown seedlings were treated with Pst DC3000 at OD_{600 nm} = 0.01, and gene expression was analysed as in (d) (n = 3, biologically independent samples).

Extended Data Fig. 2 |. SCREWs activate PTI responses.

a, Recombinant His-SCREW1 induces MAPK activation. Ten-day-old plate-grown seedlings were treated with protein elution buffer (Ctrl) or 1 μM His-SCREW1. MAPK activation was analysed by immunoblotting with anti-pERK1/2 antibodies (top), and protein loading is shown by Ponceau S staining (Ponc.) for RBC (bottom). b, SCREW1 upregulates the expression of WRKY30, WRKY33, and WRKY53. Ten-day-old plate-grown seedlings were treated with 1 μM GST or GST-SCREW1 for 1 h, and gene expression was analysed by RT–qPCR. Means (n = 3, biologically independent samples) of fold induction shown as log₂ values were used to construct heat map using TBtools. c, SCREWs induce FRK1 promoter activities. Protoplasts were co-transfected with pFRK1::LUC and pUBQ10::GUS followed by treatment with ddH₂O (Ctrl), 100 nM flg22, 1 μM GST, GST-SCREW1, GST-SCREW2, or GST-SCREW3 for 4 h. The FRK1 promoter activity was presented as the ratio of luciferase to GUS values. Data were analysed by one-way ANOVA followed by Tukey’s test, and are shown as mean ± s.d. (n = 12, biologically independent samples). P values are provided in the graph and Supplementary Table 3. d, Schematic diagrams of full-length (FL) and truncated SCREW1 variants. Domains of the signal peptide, variable region, and C-terminal conserved region are shown with aa positions labelled. e, SCREW1-induced MAPK activation requires its conserved C-terminus. Ten-day-old seedlings were treated with 1 μM GST or different GST-SCREW1 truncation proteins for 15 min, and the MAPK activation was detected as in (a). f, Synthesized peptides corresponding to the conserved C-terminal domain in SCREW1 induce MAPK activation. Ten-day-old plate-grown seedlings were incubated with 100 nM flg22, 100 nM Pep1, or 100 nM different SCREW1 variants, and the MAPK activation was detected as in (a). g, SCREW1 induces MAPK activation at a subnanomolar scale. Ten-day-old plate-grown seedlings were treated with synthesized peptides corresponding to SCREW1³⁹⁻⁶⁹ with indicated concentrations for 15 min, and MAPK activation was detected as in (a). h, SCREW1 induces a comparable MAPK activation with flg22. Ten-day-old plate-grown WT seedlings were treated with 500 nM SCREW1 or 100 nM flg22 for the indicated time. i, SCREW1 induces a weak ROS burst. Leaf discs from four-week-old soil-grown WT plants were treated with or without 100 nM flg22, 100 nM or 1 μM SCREW1, and ROS production was measured as relative light units (RLU) by a luminometer over 60 min. Data are shown as mean ± s.e.m. (n = 12, biologically independent samples). j, SCREW1 induces a moderate cytoplasmic Ca²⁺ increase relative to flg22. Protoplasts were transfected with p35S::mCherry-AEQ and incubated for 6 h, followed by treatment with 1 μM SCREW1 or 200 nM flg22. Cytoplasmic Ca²⁺ concentration was detected over 15 min. Data are shown as mean ± s.d. (n = 3, biologically independent samples). k, SCREW1 does not induce BIK1 phosphorylation. Protoplasts expressing BIK1-HA were treated with 100 nM flg22, 100 nM, or 1 μM SCREW1 for 10 min. BIK1-HA proteins were detected by immunoblotting using anti-HA antibodies (top) with protein loading shown by Coomassie brilliant blue (CBB) staining for RBC (bottom). Experiments were repeated at least three times with similar results.

Extended Data Fig. 3 |. Two conserved cysteine residues are required for SCREW activities, and SCREW1 is not mobile.

a, Predicted structures of SCREW1 and SCREW2 C-terminal 23 aa. Structures were predicted using AlphaFold Protein Structure Database (https://www.alphafold.ebi.ac.uk/). Disulfide bonds are shown by yellow sticks. b, Two cysteine residues are required for SCREW2 activation of MAPKs. Ten-day-old plate-grown WT seedlings were treated with or without 100 nM SCREW2, SCREW2(CC), SCREW2(CC/SS) and SCREW2 (ΔC8). MAPK activation was analysed by immunoblots with anti-pERK1/2 antibodies (top), and the protein loading is shown by CBB staining for RBC (bottom). c, Two cysteine residues are required for SCREW2-induced seedling growth inhibition. Three-day-old plate-grown WT seedlings were transferred into liquid ½MS medium without (Ctrl) or with 1 μM peptides. Images were taken (left), and fresh weights of seedlings (right) were measured seven days later. Data are shown as box plots with the interquartile range as the upper and lower confines, minima and maxima as whiskers, and the median as a solid line (n = 12, biologically independent samples). d, The biotin-SCREW1 peptide is not mobile. The third pair of true leaves of four-week-old plants were infiltrated with biotin-SCREW1, and both third (local) and fourth (systemic) pairs of true leaves were collected for the detection of biotin-SCREW1 by immunoblotting with HRP-labelled Streptavidin (top). The protein loading control is shown by CBB staining for RBC (bottom). e, Local application of SCREW1 does not induce PR1 expression in distal leaves. The third pair of true leaves from four-week-old plants were infiltrated with H₂O, 500 nM Pep1, or 500 nM SCREW1, and both third (local) and fourth (systemic) pairs of true leaves were collected 24 h later for RT–qPCR using ACTIN2 as internal controls. Data of induction fold compared to H₂O treatment are shown as mean ± s.d. (n = 3, biologically independent samples). f, Local application of SCREW1 does not induce PR1 accumulation in distal leaves. The experiment was performed as in e, and PR1 proteins were detected by immunoblotting with anti-PR1 antibodies (top). The protein loading control is shown by CBB staining for RBC (bottom). g, Local application of SCREW1 does not induce disease resistance in distal leaves. The third pair of leaves were pre-infiltrated with 500 nM SCREW1 followed by Pst DC3000 inoculation 24 h later on both third and fourth pairs of leaves. Bacterial growth was detected at three days post-inoculation (dpi). Data are shown as the means ± s.d. (n = 8, biologically independent samples). h, The biotin-SCREW1 and SCREW1-HA peptides have similar activities with SCREW1 for MAPK activation. Ten-day-old plate-grown WT seedlings were treated with or without 100 nM SCREW1, biotin-SCREW1, and SCREW1-HA. MAPK activation was analysed by immunoblots using anti-pERK1/2 antibodies (top) with the protein loading shown by CBB staining for RBC (bottom). i, The biotin-SCREW1 and SCREW1-HA peptides have similar activities with SCREW1 for seedling growth inhibition. The experiment was performed as in (c). Data are shown as box plots with the interquartile range as the upper and lower confines, minima and maxima as whiskers, and the median as a solid line (n = 12, biologically independent samples). Experiments were repeated three times with similar results. Data were analysed by one-way (c, i), or two-way (e, g) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.

Extended Data Fig. 4 |. SCREW1 and SCREW2 are involved in plant immunity.

a, Inducible overexpression of SCREW1 leads to leaf chlorosis. Four-week-old transgenic plants carrying pEst::SCREW1-HA in WT (L12 & L19) were sprayed with 0.05% DMSO (Ctrl) or 50 μM β-oestradiol (Est), and then imaged five days later (Scale bar, 1 cm) with trypan blue staining (Scale bar, 0.5 cm). SCREW1-HA proteins were detected by immunoblots with anti-HA antibodies (bottom). b, SCREW1 overexpression elevates PR1 expression. Four-week-old soil-grown pEst::SCREW1-HA transgenic plants were sprayed with 50 μM β-oestradiol. The PR1 expression normalized to ACTIN2 was analysed by RT–qPCR. Data are shown as mean ± s.d. (n = 3, biologically independent samples). c, Overexpression of SCREW1 or SCREW2 leads to plant growth retardation and leaf curling. Transgenic lines carrying p35S::SCREW1 or p35S::SCREW2 were grown on soil for five weeks before photography. Scale bar, 1 cm. The expression of SCREWs normalized to ACTIN2 in ten-day-old plate-grown seedlings was analysed with RT–qPCR. Data are shown as mean ± s.d. (n = 3, biologically independent samples). d, Overexpression of SCREW1 or SCREW2 upregulates PR1 expression. Relative PR1 expression levels normalized to UBQ10 in four-week-old soil-grown plants were analysed with RT–qPCR. Data are shown as mean ± s.d. (n = 3, biologically independent samples). e, CRISPR/Cas9-mediated gene editing of SCREW1 and SCREW2. Mutations of SCREW1 and SCREW2 in screw1/2-1 and screw1/2-2 were detected by DNA sequencing and shown as chromatographs. Two homozygous lines, screw1/2-1 and screw1/2-2, carry the same nucleotide insertion in SCREW1 for both lines, and a nucleotide insertion and a sixteen-nucleotide deletion in SCREW2 for screw1/2-1 and screw1/2-2, respectively. f, The screw1/2 mutants are morphologically indistinguishable from WT plants. Plants were grown on soil for four weeks and photographed. Scale bar, 1 cm. g, The screw1/2 mutants are more susceptible to Pst DC3000. Leaves of four-week-old WT and screw1/2 mutant lines (1 & 2) were hand-inoculated with Pst DC3000 at OD_{600 nm} = 5 × 10⁻⁴. Bacterial numbers were measured at 0 and 3 dpi and shown as mean ± s.d. (n = 8, biologically independent samples). h, The screw1/2 mutants show enhanced susceptibility to Pst DC3000 hrcC⁻. Leaves of four-week-old soil-grown WT and screw1/2 mutant lines (1 & 2) were hand-inoculated with Pst DC3000 hrcC⁻ at OD_{600 nm} = 0.005, and the bacterial number was measured at 3 dpi. Data are shown as mean ± s.d. (n = 8, biologically independent samples). Experiments were repeated at least three times with similar results. Data were analysed by one-way (b-d, h) or two-way (g) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.

Extended Data Fig. 5 |. Identification of the SCREW receptor NUT.

a, Scheme to identify SCREW receptor candidates. Among 269 LRR-RKs in Arabidopsis, 26 members are upregulated by flg22 treatment, among which 11 members contain at least 18 LRRs in the extracellular domain. The cognate ligands of five of them remain unknown at the time of the study. b, NUT belongs to the XI subfamily of LRR-RK and is phylogenetically close to HAE and HSLs. Phylogenetic analysis of 52 LRR-RKs from the subfamily VII (orange curved line), X (purple curved line), XI (green curved line), and XII (grey curved line) is shown. Purple and red bars indicate the induction folds after elf18 and flg22 treatments, respectively. Olive green bars with numbers indicate the number of LRRs. Blue squares indicate cognate ligands of LRR-RKs. AT5G25930 (NUT) is highlighted in bold red font. The protein sequences were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/) for MEGAX phylogenetic analysis using the neighbour-joining method with 1,000 bootstrap replicates. The phylogenetic tree was displayed by iTOL (https://itol.embl.de/). The expression data were from GENEVESTIGATOR V3. c, SCREWs and NUT are conserved in dicots and monocots. Protein sequences were blast-searched in NCBI using Arabidopsis SCREW1, NUT, or FLS2 as queries, and the phylogenetic analysis was performed as in (b). Red, purple, and olive curved lines indicate monocots, dicots, and other plant classes, respectively; Orange and teal bars indicate the percentage of homology of FLS2 and NUT in different plant species, respectively. Blue dots, olive stars, and red squares indicate FLS2, NUT, and SCREW homologs, respectively. Peach, lime green, grey, and brown fans denote different plant families. d, Diagram of AT5G25930 (NUT) with annotated T-DNA insertion sites in nut-1 (WiscDslox450B04) and nut-2 (SALK_207895). Solid bars indicate exons, lines for introns, and open boxes for UTRs. Arrows indicate primers used for genotyping. e, Genotyping of nut-1 and nut-2. The T-DNA insertions in the NUT coding region were PCR-amplified using genomic DNAs of WT, nut-1, or nut-2 as templates and primers shown in (d). f, RT–qPCR analysis of NUT transcripts. NUT expression levels in ten-day-old plate-grown seedlings were analysed using RT–qPCR with UBQ10 as an internal control. Data are shown as mean ± s.d. (n = 3, biologically independent samples) with one-way ANOVA followed by Tukey’s test. Exact P values are provided in the graph and Supplementary Table 3. Experiments were repeated three times with similar results (e, f).

Extended Data Fig. 6 |. NUT is required for SCREW-triggered responses and resistance to B. cinerea and green peach aphids.

a, NUT-Flag restores SCREW1-induced MAPK activation in nut protoplasts. Protoplasts were transfected with an empty vector (Ctrl) or NUT-Flag followed by treatment with 100 nM SCREW1 for 15 min. MAPK activation and NUT-Flag proteins were detected with anti-pERK1/2 (top) and anti-Flag antibodies (middle), respectively. Protein loading is shown by Ponceau S staining (Ponc.) for RBC (bottom). b, Inducible overexpressing SCREW1-triggered leaf chlorosis is blocked in nut-2. Five-week-old pEst::SCREW1-HA transgenic plants in WT and nut-2 were sprayed with 50 μM β-oestradiol and photographed five days later. Scale bar, 1cm. c, The nut mutants are more susceptible to Pst DC3000 than WT. Leaves of four-week-old plants were hand-inoculated with Pst DC3000 at OD_{600 nm} = 5 × 10⁻⁴. The bacterial numbers were measured at 0 and 3 dpi. Data are shown as mean ± s.d. (n = 6, biologically independent samples). d, The nut and screw1/2 mutants are more susceptible to spray-inoculated Pst DC3000. Four-week-old plants were sprayed with Pst DC3000 at OD_{600 nm} = 0.2. The disease symptoms and bacterial numbers were determined at 72 hpi. Data are shown as mean ± s.d. (n = 8, biologically independent samples). e, NUT is upregulated by MAMPs and pathogens. Data were retrieved from the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) and shown as histograms. Grey squares indicate no data available from eFP browser. Flg22, NLP, HrpZ, and lipopolysaccharide (LPS) are MAMPs. Pst DC3000, Pst DC3000 hrcC⁻, Pst DC3000 avrRpm1 and P. syringae pv. phaseolicola (Psp) are bacterial pathogens. Phytophthora infestans is an oomycete, and B. cinerea is a fungal pathogen. f, Flg22 upregulates NUT promoter activities. Two-week-old plate-grown pNUT::GUS/WT transgenic seedlings were treated without (Ctrl) or with 100 nM flg22 for 2 h, followed by GUS staining and microscopic imaging under a stereomicroscope. Scale bar, 2 mm. g, The nut and screw1/2 mutants do not affect plant resistance to Pst DC3000 avrRpm1 (left) and Pst DC3000 avrRpt2 (right). Bacteria were infiltrated into four-week-old plant leaves at OD_{600 nm} = 0.001, and bacterial populations were determined at 2 dpi. Data are shown as mean ± s.d. (n = 6, biologically independent samples). h, The nut and screw1/2 mutants do not affect plant HR to Pst DC3000 avrRpm1 (left) and Pst DC3000 avrRpt2 (right). Bacteria were infiltrated into four-week-old plant leaves at OD_{600 nm} = 0.08, and wilting leaves were counted at the indicated time points. At least 20 leaves were inoculated for each genotype and inoculum. The cell death rate was presented as the ratio of wilting leaves to total inoculated leaves. Data are shown as mean ± s.d. (n = 3, biologically independent samples). i, The nut and screw1/2 mutants are more susceptible to B. cinerea. Detached leaves of four-week-old plants were drop-inoculated with B. cinerea at 10⁷ spores/mL. Disease phenotype was recorded at 48 hpi. Data are shown as mean ± s.d. (left to right: n = 30, 29, 27, 25, 28, biologically independent samples). j, The p35S::SCREW1 and p35S::SCREW2 plants show enhanced resistance to aphids. Six-age-synchronized second instar nymphs of Myzus persicae were inoculated onto leaves of four-week-old plants. The number of neonates per plant (n) was counted at 10 dpi. Data are shown as mean ± s.d. (left to right: n = 19, 12, 12, 12, 8, biologically independent samples). k, The nut mutants are more susceptible to aphid infections than WT plants. The experiments and statistics were performed as in j (left to right: n = 10, 10, 10, 12, 12, biologically independent samples). l, The expression of SCREWs and NUT is induced by aphid infections. Leaves of two-week-old plants were inoculated with or without aphid nymphs for 24, 48, and 72 h. The expression of SCREWs and NUT normalized to UBQ10 was analysed by RT–qPCR. Means (n = 3, biologically independent samples) of fold induction compared to nontreatment are shown as log₂ transformation to construct heat map using TBtools. m, The NUT promoter activity is induced by aphid infections. Two-week-old soil-grown transgenic plants carrying pNUT::GUS were inoculated with aphid nymphs and subjected to GUS staining at 1 and 3 dpi. The pictures were taken under a stereomicroscope. Scale bar, 2 mm. Experiments except e were repeated three times with similar results. Data were analysed by one-way (d, i) or two-way (c, g) ANOVA followed by Tukey’s test, or one-way ANOVA followed by Dunnett’s test (j, k). Exact P values are provided in the graphs and Supplementary Table 3.

Extended Data Fig. 7 |. SPR assays of SCREW and SCREW derivatives binding to NUT_ECD, and bik1 does not affect SCREW1-induced inhibition of seedling growth.

a, NUT-GFP is localized on the plasma membrane. Transgenic seedlings carrying p35S::GFP or p35S::NUT-GFP were grown on ½MS plates for seven days, and true leaves were imaged using a confocal laser scanning microscopy. Scale bar, 25 μm. b, NUT-GFP and SCREW2-GFP are localized on the plasma membrane in N. benthamiana. N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying p35S::NUT-GFP, p35S::SCREW2-GFP or p35S::GFP and imaged under a confocal microscope at 3 dpi. Scale bar, 25 μm. c, Sequence alignment of parts of cytosolic kinase domains of different RKs, including NUT, FLS2, BAK1, EFR, and BRI1. The sequences were aligned by MEGAX and visualized with ESPript 3.0 (http://espript.ibcp.fr/). The lysine (K) required for ATP-binding (blue asterisk) and the RD/non-RD motif (blue rectangle) were marked. The positions of amino acids in NUT were labelled on the top. d-g, SPR assays of SCREW1 (d), SCREW2 (e), SCREW1-HA (f), SCREW2^CC/SS (g) binding to NUT_ECD under pH 5.7. NUT_ECD was immobilized on a sensor chip, and synthesized SCREW peptides were used as flow-through analytes. The top panel shows the SPR sensorgram profile of SCREW peptides at gradient concentrations flowing through the NUT_ECD-immobilized chip. The bottom panel shows the steady-state affinity (binding at equilibrium) with different K_d. h, The C-terminus of SCREW2 is essential for its binding to NUT_ECD. The SPR assays were performed as in Fig. 3f, g. i, BAK1 promotes the SCREW2-NUT binding affinity. (Left) SPR detection of BAK1_ECD binding to NUT_ECD in the absence of SCREW peptides. BAK1_ECD was used as flow-through analytes on a sensor chip immobilized with NUT_ECD. At the concentration tested, no binding between NUT_ECD and BAK1_ECD was detected. (Right) SPR detection of SCREW2 binding to NUT_ECD in the presence of BAK1_ECD. NUT_ECD was immobilized on a sensor chip, and SCREW2 peptides at gradient concentrations together with 0.1 μM BAK1_ECD proteins were used as flow-through analytes. The K_d of SCREW2 binding to NUT_ECD in the presence of BAK1 is 0.38 ± 0.074 μM. The SPR assays were performed at pH 5.7. j, NUT does not associate with BIK1. Protoplasts were co-transfected with BIK1-HA and NUT-Flag, PEPR1-Flag, or a control vector (Ctrl). Total proteins were immunoprecipitated with anti-Flag agarose beads and detected with anti-HA or anti-Flag antibodies (top two). Proteins before immunoprecipitation (IP) are shown as input controls (bottom two). k, SCREW1 inhibits seedling growth in the bik1 mutant. Three-day-old plate-grown seedlings were transferred into liquid ½MS medium with or without 1 μM SCREW1 and grown for seven days. Fresh weights of seedlings are shown as as mean ± s.d. (n = 8, biologically independent samples). Data were analysed by two-sided Student’s t-test and two-way ANOVA followed by Tukey’s test. P values are provided in the graph and Supplementary Table 3. Experiments were repeated twice (a, b, d–i) or three times (j, k) with similar results.

Extended Data Fig. 8 |. SCREW–NUT does not affect flg22-triggered early signalling events but partially requires MIN7.

a, SCREW1 suppresses flg22- or ABA-induced stomatal closure. The stomatal apertures were measured after treatment without or with 1 μM SCREW1, 1 μM flg22, 10 μM ABA, or a combination of SCREW1 with flg22 or ABA for 2h under the light. Data are shown as box plots with the interquartile range as the upper and lower confines, minima and maxima as whiskers, and the median as a solid line. Different letters denote a statistically significant difference (P < 0.05). n = number of stomata in the graph. b, SCREW1 does not affect the flg22-induced FLS2-BAK1 association. Four-day-old plate-grown WT seedlings were transferred into liquid ½MS for six days, followed by 100 nM flg22, 100 nM SCREW, or a combination of 100 nM flg22 and 100 nM SCREW for 10 min. Proteins were subjected for IP assays using anti-FLS2 antibodies and followed by immunoblotting using anti-BAK1 or anti-FLS2 antibodies. Top two panels show the IPed products, and bottom two panels show protein inputs. c, The nut and screw mutants do not affect the flg22-induced FLS2-BAK1 association. Four-day-old plate-grown seedlings were transferred into liquid ½MS for six days, followed by 100 nM flg22 for 10 min. Co-IP analysis was similar to the above in b. d, SCREW1 and NUT do not affect flg22-induced MAPK activation. Ten-day-old seedlings were treated with 100 nM flg22, 100 nM SCREW1, or a combination of 100 nM flg22 and 100 nM SCREW1. MAPK activation was analysed by immunoblotting with anti-pERK1/2 antibodies (top), and protein loading is shown by Ponceau S staining (Ponc.) for RBC (bottom). e, SCREW1-triggered resistance to Pst DC3000 is partially abolished in min7. Four-week-old plants were pre-infiltrated with 0.5 μM SCREW1, or H₂O as a control. After 24 h, Pst DC3000 was infiltrated at a concentration of OD_{600 nm} = 0.02 or 0.002, and bacterial counting was performed at 1 dpi for OD_{600 nm} = 0.02, and 2 dpi for OD_{600 nm} = 0.002. Data are shown as means ± s.d. (n = 3, biologically independent samples) (left). The picture was taken at 3 dpi with OD_{600 nm} = 0.002 (right). f, MIN7 is not required for SCREW1 activation of MAPKs. Ten-day-old plate-grown seedlings were treated with 100 nM SCREW1. MAPK activation was analysed by immunoblots with anti-pERK1/2 antibodies (top), and the protein loading is shown by CBB staining for RBC (bottom). g, MIN7 is not required for SCREW1 suppression of seedling growth. Three-day-old plate-grown seedlings were transferred into liquid ½MS medium with or without 1 μM SCREW1. Seedlings were imaged (left) and weighed (right) seven days post-treatment. Scale bar, 1 cm. Fresh weights of seedlings are shown as box plots with the interquartile range as the upper and lower confines, minima and maxima as whiskers, and the median as a solid line (n = 12, biologically independent samples). h, The susceptibility of nut and screw1/2 mutants to Pst DC3000 is comparable to WT plants under high humidity with transient apoplast water supplementation. Leaves of four-week-old plants were inoculated with Pst DC3000 at OD_{600 nm} = 1 × 10⁻⁴ and kept under 85–98% humidity for three days before bacterial counting. Transient water supplementation (+H₂O) was performed by keeping plants under high humidity after syringe-infiltration without air-drying inoculated leaves as described previously³². Data are shown as means ± s.d. (n = 8, biologically independent samples). i, SCREW1-triggered plant resistance to Pst DC3000 is compromised under high humidity with transient apoplast water supplementation. Leaves of four-week-old WT plants were pre-infiltrated with H₂O (Ctrl) or 1 μM SCREW1 for 24 h followed by Pst DC3000 inoculation. Plants were kept under 50% or 85–98% humidity for three days before bacterial counting. Data are shown as means ± s.d. (n = 8, biologically independent samples). Experiments were repeated three times with similar results. Data were analysed by two-sided Student’s t-test (e, g), one-way (a) or two-way (h, i) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.

Extended Data Fig. 9 |. SCREW–NUT regulates leaf water loss and ABA responses.

a, Transgenic plants carrying p35S::SCREW1 or p35S::SCREW2 exhibit curled leaves and increased sensitivity to dehydration stress. Leaves of five-week-old soil-grown plants were detached and imaged at 0 and 6 h after detachment. Scale bar, 1 cm. b, Increased water-loss rate in transgenic plants carrying p35S::SCREW1 or p35S::SCREW2. The rates of cumulative water loss from rosette leaves of five-week-old plants were measured at 6 h post-detachment. Data are shown as means ± s.d. (n = 6, biologically independent samples). c, Reduced water-loss rate in nut and screw1/2. The rate of cumulative water loss from detached leaves of four-week-old plants was measured at the indicated time points after detachment. Data are shown as means ± s.d. (n = 6, biologically independent samples). d, Enhanced resistance to mannitol treatment in nut mutants. Seedlings were grown on ½MS plates with 0, 50, 100, 150, or 200 mM mannitol for 15 days. Scale bar, 2 mm. e, Cuticle permeability of nut and screw1/2 seedlings is similar to that of WT. Three-week-old plate-grown plants were soaked with 0.05% toluidine blue for 15 min and washed with ddH₂O before imaging. Scale bar, 1 cm. At least six seedlings for each genotype were analysed for the presence of the blue-coloured patches, which indicate an increased permeability of the stain into the leaf through the cuticle. No apparent differences were observed between WT and mutants. f, Leaf cuticle permeability of nut and screw1/2 is similar to that of WT. Leaves of four-week-old soil-grown plants were drop-stained with 0.05% toluidine blue on the adaxial surface for the indicated time. The red circles and rectangles indicate the sites of inoculation. Inserts show zoomed-in areas. No blue-coloured patches were observed, indicating the intact cuticle for each genotype. Scale bar, 20 μm. Scale bar, 1 cm. Ten leaves for each genotype were analysed. g, Leaf cuticle layers of nut and screw1/2 are similar to those of WT. Three-week-old plate-grown plant leaves were examined by transmission electron microscopy from the adaxial side. Red arrows indicate cuticles observed as a thin (~80–100 nm) electron-dense layer on the surface of the cell wall. Scale bar, 1 μM. Four leaves of each genotype were analysed. No apparent differences in thickness were detected among different genotypes. h, The nut and screw1/2 mutants are more sensitive to ABA treatment than WT plants. Seedlings were grown on ½MS plates without (Ctrl) or with 1 μM ABA for seven days (left). Cotyledon greening rates are shown as means ± s.d. (right, n = 4, biologically independent repeats). i, SCREW1 and SCREW2 suppress ABA-induced expression of RAB18 and RD29A in plants. Ten-day-old plate-grown WT seedlings were treated with H₂O, 10 μM ABA, or combinations of 10 μM ABA and 1 μM SCREW1 or SCREW2 for 3 h. Transcript levels of RAB18and RD29A normalized to UBQ10 were determined via RT–qPCR. Data are shown as mean ± s.d. (n = 4, biologically independent samples). j, NUT is upregulated by ABA, mannitol, and drought treatments. The expression data were extracted from Genevestigator V3 and shown as histograms. Grey squares indicate no data available. k, SCREWs and NUT are up-regulated after ABA treatments. Ten-day-old plate-grown WT seedlings were treated with 100 μM ABA for 0, 3, and 6 h. Transcript levels of SCREWs and NUT normalized to UBQ10 were determined via RT–qPCR. Data are shown as mean ± s.d. (n = 3, biologically independent samples). Experiments were repeated three times with similar results. Data were analysed by one-way (b) or two-way (c, h, i, k) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.

Extended Data Fig. 10 |. SCREW1 induces NUT-dependent ABI phosphorylation.

a, SCREW1 induces ABI2 phosphorylation. Protoplasts were transfected with ABI2-HA followed by treatment with 1 μM SCREW1 for the indicated time. Proteins were separated with Mn²⁺-Phos-tag (top two) or regular SDS-PAGE (middle two) and detected with anti-HA antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of the top two bands corresponding to the phosphorylated ABI2 (pABI2) normalized to the input ABI2 in the regular SDS-PAGE from six independent immunoblots were quantified by ImageJ (bottom). The phosphorylation of ABI2 without SCREW1 treatment was set as 1. Data are shown as mean ± s.d. (n = 6, biologically independent repeats). b, SCREW1 does not induce OST1 phosphorylation. Protoplasts were transfected with OSTl-Flag, followed by treatment with 1 μM SCREW1 for the indicated time. Proteins were separated with Mn²⁺-Phos-tag (top two) or regular SDS-PAGE (middle two) and detected with anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands from three independent immunoblots were analysed by ImageJ (bottom). The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. Data are shown as mean ± s.d. (n = 3, biologically independent repeats). c, SCREW1 induces NUT-dependent ABI1 phosphorylation. Protoplasts were co-transfected with ABI1-HA and NUT-Flag or a control vector followed by treatment with 1 μM SCREW1 for the indicated time. Proteins were separated by SDS-PAGE with (top two) or without Mn²⁺-Phos-tag (middle four) and detected with anti-HA or anti-pERK1/2 antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands corresponding to phosphorylated ABI1 (pABI1) normalized to the input ABI1 in the regular SDS-PAGE from four independent immunoblots were quantified by ImageJ (bottom). The phosphorylation of ABI1 without SCREW1 treatment was set as 1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). Experiments were repeated three times with similar results. Data were analysed by one-way (a, b) or two-way (c) ANOVA followed by Tukey’s test. Exact P values are provided in graphs and Supplementary Table 3.

Extended Data Fig. 11 |. SCREW1 and ABI suppress ABA-induced OST1 phosphorylation.

a, ABI1 suppresses ABA-induced OST1 phosphorylation. Protoplasts were co-transfected with OST1-Flag and ABI1-HA with the indicated ratio of DNAs, followed by treatment with 1 μM SCREW1 for 5 min before adding 1 μM ABA for an additional 5 min. Proteins were separated with Mn²⁺-Phos-tag (top three) or regular SDS-PAGE (middle two) and detected with anti-HA or anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands corresponding to phosphorylated ABI1 (pABI1) and OST1 (pOST1) from four independent immunoblots were quantified by ImageJ (bottom two). The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. The phosphorylation of ABI1 without treatment was set as 1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). b, ABI2 suppresses ABA-induced OST1 phosphorylation. Protoplasts were co-transfected with ABI2-HA and OST1-Flag followed by treatment with 1 μM SCREW1 before adding 1 μM ABA. Proteins were separated with Mn²⁺-Phos-tag (top two) or regular SDS-PAGE (bottom three) and detected with anti-HA or anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands from four independent immunoblots were analysed by ImageJ (bottom). The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). c, SCREW1 suppresses ABA-induced OST1 phosphorylation in a dosage-dependent manner. Protoplasts were transfected with OST1-Flag followed by treatments with 0, 10, 100 or 1,000 nM SCREW1 for 5 min before adding 1 μM ABA for another 5 min. Proteins were separated with Mn²⁺-Phos-tag (top two) and regular SDS-PAGE (middle two) and detected with anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands from four independent immunoblots were analysed by ImageJ. The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). d, NUT interacts with ABI1 in protoplasts. Protoplasts were co-transfected with ABI1-HA and NUT-Flag or a control vector. Proteins were immunoprecipitated with anti-Flag agarose beads and detected with anti-HA or anti-Flag antibodies (top two). Proteins before IP are shown as input controls (bottom two). e, GST-NUT_CD interacts with ABI1 in vitro. GST or GST-NUT_CD proteins were immobilized on glutathione sepharose beads and incubated with His-ABI1-HA followed by immunoblotting with anti-HA antibodies (top). The protein loading is shown by CBB (bottom). Experiments were repeated three times with similar results. Data were analysed by one-way (b, c) or two-way (a) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.

Extended Data Fig. 12 |. SCREW1 suppresses flg22-induced stomatal closure through the ABI–OST1 module.

a, ABI1 and ABI2 are required for SCREW1 suppression of flg22-induced stomatal closure. The stomatal apertures from epidermal peels of WT and abi1-2/abi2-2 were measured after treatment without or with 1 μM SCREW1, 1 μM flg22, or a combination of SCREW1 and flg22 for 2h. Data are shown as the box plots with the interquartile range as the upper and lower confines, minima and maxima as whiskers, and the median as a solid line (n = 202, the number of stomata). The different letters denote a statistically significant difference (P < 0.05, two-way ANOVA followed by the Tukey’s test). The effect of SCREW1 on flg22-induced stomatal closure in WT and abi1-2/abi2-2 was compared by a two-sided Student’s t-test. The experiment was repeated three times with similar results. Exact P values are provided in the graph and Supplementary Table 3. b, A model of SCREW–NUT in protecting plants against infections via promoting stomatal reopening and reducing apoplastic water levels. MAMP perception by PRRs induces stomatal closure to limit pathogen entry. Inevitably, stomatal closure increases the apoplastic water levels and creates aqueous habitats favourable for pathogen multiplication. To counteract, MAMP–PRR signalling induces the expression of SCREWs and NUT. Upon SCREW perception, NUT complexes with BAK1 and promotes stomatal reopening via regulating the ABI–OST1–SLAC1 phosphorylation module, thereby increasing water loss and reducing apoplastic water levels to prevent the pathogen colonization. To invade hosts, pathogens deliver effectors or toxins, some of which can open stomata. The blue lines indicate SCREW–NUT induction and function in plant immunity revealed in this study.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files (uncropped blots and gel images, primers, peptides, and exact P values). The sequences of proteins were obtained from TAIR (https://www.arabidopsis.org/), UniProt (https://www.uniprot.org/), NCBI (https://www.ncbi.nlm.nih.gov/) and Phytozome (https://phytozome-next.jgi.doe.gov/). The protein structures were obtained from AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/). Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank–EMBL databases under the following accession numbers: SCREW1 (AT1G06135), SCREW2 (AT2G31345), SCREW3 (AT1G06137), SCREW4 (AT2G31335), NUT (AT5G25930), WRKY30 (AT5G24110), WRKY33 (AT2G38470), WRKY53 (AT4G23810), PR1 (AT2G14610), FRK1 (AT2G19190), MPK3 (AT3G45640), MPK4 (AT4G01370), MPK6 (AT2G43790), UBQ10 (AT4G05320), ACTIN2 (AT3G18780), BAK1 (AT4G33430), SERK1 (AT1G71830), SERK2 (AT1G34210), SERK4 (AT2G13790), FLS2 (AT5G46330), BIK1 (AT2G39660), MIK2 (AT4G08850), MIK2L (AT1G35710), RLK7 (AT1G09970), IKU2 (AT3G19700), HAE (AT4G28490), HSL2 (AT5G65710), LET1 (AT2G23200), LET2 (AT5G38990), NILR1 (AT1G74360), MIN7 (AT3G43300), ABI1 (AT4G26080), ABI2 (AT5G57050) and OST1 (AT4G33950).