Key message Plant U-box E3 ligases PUB20 and PUB21 are flg22-triggered signaling components and negatively regulate immune responses. Abstract Plant U-box proteins (PUBs) constitute a class of E3 ligases that are associated with various stress responses. Among the class IV PUBs featuring C-terminal Armadillo (ARM) repeats, PUB20 and PUB21 are closely related homologs. Here, we show that both PUB20 and PUB21 negatively regulate innate immunity in plants. Loss of PUB20 and PUB21 function leads to enhanced resistance to surface inoculation with the virulent bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the resistance levels remain unaffected after infiltration inoculation, suggesting that PUB20 and PUB21 primarily function during the early defense stages. The enhanced resistance to Pst DC3000 in PUB mutant plants (pub20-1, pub21-1, and pub20-1/pub21-1) correlates with extensive flg22-triggered reactive oxygen production, strong MPK3 activation, and enhanced transcriptional activation of early immune response genes. Additionally, PUB mutant plants (except pub21-1) exhibit constitutive stomatal closure after Pst DC3000 inoculation, implying the significant role of PUB20 in stomatal immunity. Comparative analyses of flg22 responses between PUB mutants and wild-type plants reveals that the robust activation of the pattern-induced immune responses may enhance resistance against Pst DC3000. Notably, the hypersensitivity responses triggered by RPM1/avrRpm1 and RPS2/avrRpt2 are independent of PUB20 and PUB21. These results suggest that PUB20 and PUB21 knockout mutations affect bacterial invasion, likely during the early stages, acting as negative regulators of plant immunity.

Carbohydrate-based cell wall signaling impacts plant growth, development, and stress responses; however, how cell wall signals are perceived and transduced remains poorly understood. Several cell wall breakdown products have been described as typical damage-associated molecular patterns that activate plant immunity, including pectin-derived oligogalacturonides (OGs). Receptor kinases of the WALL-ASSOCIATED KINASE (WAK) family bind pectin and OGs and were previously proposed as OG receptors. However, unambiguous genetic evidence for the role of WAKs in OG responses is lacking. Here, we investigated the role of Arabidopsis (Arabidopsis thaliana) WAKs in OG perception using a clustered regularly interspaced short palindromic repeats mutant in which all 5 WAK genes were deleted. Using a combination of immune assays for early and late pattern-triggered immunity, we show that WAKs are dispensable for OG-induced signaling and immunity, indicating that they are not bona fide OG receptors.

Plants have evolved an elaborate cell wall integrity (CWI) sensing system to monitor and modify cell wall formation. LRR-extensins (LRXs) are cell wall-anchored proteins that bind RAPID ALKALINIZATION FACTOR (RALF) peptide hormones and induce compaction of cell wall structures. At the same time, LRXs form a signaling platform with RALFs and the transmembrane receptor kinase FERONIA (FER) as a means to relay changes in CWI to the protoplast. LRX1 of Arabidopsis thaliana is predominantly expressed in root hairs and lrx1mutants develop defective root hairs. Here, we identify a regulator of LRX1-RALF-FER signaling as a suppressor of the lrx1 root hair phenotype. The repressor of lrx1_23 (rol23) gene encodes PP2C12, a type 2C phosphatase of clade H that interacts with FER and dephosphorylates Thr696 in the FER activation loop in vitro. The LRX1-related function of PP2Cs appears clade H-specific and was not observed for other PP2Cs investigated. Collectively, our data suggest that LRX1 acts upstream of the RALF1-FER signaling module and PP2C12 has an inhibitory activity via modulating FER activity to fine-tune CWI signaling.

Nucleotide-binding leucine-rich repeat (NLR) disease resistance genes typically confer resistance against races of a single pathogen. Here, we report that Yr87/Lr85, an NLR gene from Aegilops sharonensis and Aegilops longissima, confers resistance against both P. striiformis tritici (Pst) and Puccinia triticina (Pt) that cause stripe and leaf rust, respectively. Yr87/Lr85 confers resistance against Pst and Pt in wheat introgression as well as transgenic lines. Comparative analysis of Yr87/Lr85 and the cloned Triticeae NLR disease resistance genes shows that Yr87/Lr85 contains two distinct LRR domains and that the gene is only found in Ae. sharonensis and Ae. longissima. Allele mining and phylogenetic analysis indicate multiple events of Yr87/Lr85 gene flow between the two species and presence/absence variation explaining the majority of resistance to wheat leaf rust in both species. The confinement of Yr87/Lr85 to Ae. sharonensis and Ae. longissima and the resistance in wheat against Pst and Pt highlight the potential of these species as valuable sources of disease resistance genes for wheat improvement. Leaf rust and stripe rust of wheat are two important fungal diseases of cultivated wheat and they are caused by infection of different pathogens. Here, the authors report the nucleotide-binding leucine-rich repeat (NLR) protein encoding gene Yr87/Lr85 confers resistance to both diseases.

The plant immune system relies on germline-encoded pattern recognition receptors (PRRs) that sense foreign and plant-derived molecular patterns, and signal health threats. Genomic and pangenomic data sets provide valuable insights into the evolution of PRRs and their molecular triggers, which is furthering our understanding of plant–pathogen co-evolution and convergent evolution. Moreover, in silico and in vivo methods of PRR identification have accelerated the characterization of receptor–ligand complexes, and advances in protein structure prediction algorithms are revealing novel PRR sensor functions. Harnessing these recent advances to engineer PRRs presents an opportunity to enhance plant disease resistance against a broad spectrum of pathogens, enabling more sustainable agricultural practices. This Review summarizes both established and innovative approaches to leverage genomic data and translate resulting evolutionary insights into engineering PRR recognition specificities. Genomic and pangenomic data are yielding insights into the evolution of plant pattern recognition receptors (PRRs) and their molecular triggers. Recent advances in in silico and in vivo methods, alongside protein structure prediction, are helping to harness these insights for PRR engineering, offering sustainable solutions for broad-spectrum plant disease resistance.

Ethylene regulates plant growth, development, and stress adaptation. However, the early signaling events following ethylene perception, particularly in the regulation of ethylene receptor/CTRs (CONSTITUTIVE TRIPLE RESPONSE) complex, remains less understood. Here, utilizing the rapid phospho-shift of rice OsCTR2 in response to ethylene as a sensitive readout for signal activation, we revealed that MHZ3, previously identified as a stabilizer of ETHYLENE INSENSITIVE 2 (OsEIN2), is crucial for maintaining OsCTR2 phosphorylation. Genetically, both functional MHZ3 and ethylene receptors prove essential for OsCTR2 phosphorylation. MHZ3 physically interacts with both subfamily I and II ethylene receptors, e.g., OsERS2 and OsETR2 respectively, stabilizing their association with OsCTR2 and thereby maintaining OsCTR2 activity. Ethylene treatment disrupts the interactions within the protein complex MHZ3/receptors/OsCTR2, reducing OsCTR2 phosphorylation and initiating downstream signaling. Our study unveils the dual role of MHZ3 in fine-tuning ethylene signaling activation, providing insights into the initial stages of the ethylene signaling cascade. The early signalling events following ethylene perception by plants remain incompletely understood. Here the authors show that in the absence of ethylene, rice MHZ3, a known stabilizer of OsEIN2, promotes phosphorylation of OsCTR2 to suppress ethylene signalling.

Plants lack specialized and mobile immune cells. Consequently, any cell type that encounters pathogens must mount immune responses and communicate with surrounding cells for successful defence. However, the diversity, spatial organization and function of cellular immune states in pathogen-infected plants are poorly understood1. Here we infect Arabidopsis thaliana leaves with bacterial pathogens that trigger or supress immune responses and integrate time-resolved single-cell transcriptomic, epigenomic and spatial transcriptomic data to identify cell states. We describe cell-state-specific gene-regulatory logic that involves transcription factors, putative cis-regulatory elements and target genes associated with disease and immunity. We show that a rare cell population emerges at the nexus of immune-active hotspots, which we designate as primary immune responder (PRIMER) cells. PRIMER cells have non-canonical immune signatures, exemplified by the expression and genome accessibility of a previously uncharacterized transcription factor, GT-3A, which contributes to plant immunity against bacterial pathogens. PRIMER cells are surrounded by another cell state (bystander) that activates genes for long-distance cell-to-cell immune signalling. Together, our findings suggest that interactions between these cell states propagate immune responses across the leaf. Our molecularly defined single-cell spatiotemporal atlas provides functional and regulatory insights into immune cell states in plants. The development of a molecularly defined spatiotemporal atlas of pathogen-infected Arabidopsis thaliana leaves reveals specific cell states that have distinct roles in plant immunity.

Fungi are the most important group of plant pathogens, responsible for many of the world’s most devastating crop diseases. One of the reasons they are such successful pathogens is because several fungi have evolved the capacity to breach the tough outer cuticle of plants using specialized infection structures called appressoria. This is exemplified by the filamentous ascomycete fungus Magnaporthe oryzae, causal agent of rice blast, one of the most serious diseases affecting rice cultivation globally. M. oryzae develops a pressurized dome-shaped appressorium that uses mechanical force to rupture the rice leaf cuticle. Appressoria form in response to the hydrophobic leaf surface, which requires the Pmk1 MAP kinase signalling pathway, coupled to a series of cell-cycle checkpoints that are necessary for regulated cell death of the fungal conidium and development of a functionally competent appressorium. Conidial cell death requires autophagy, which occurs within each cell of the spore, and is regulated by components of the cargo-independent autophagy pathway. This results in trafficking of the contents of all three cells to the incipient appressorium, which develops enormous turgor of up to 8.0 MPa, due to glycerol accumulation, and differentiates a thickened, melanin-lined cell wall. The appressorium then re-polarizes, re-orienting the actin and microtubule cytoskeleton to enable development of a penetration peg in a perpendicular orientation, that ruptures the leaf surface using mechanical force. Re-polarization requires septin GTPases which form a ring structure at the base of the appressorium, which delineates the point of plant infection, and acts as a scaffold for actin re-localization, enhances cortical rigidity, and forms a lateral diffusion barrier to focus polarity determinants that regulate penetration peg formation. Here we review the mechanism of regulated cell death in M. oryzae, which requires autophagy but may also involve ferroptosis. We critically evaluate the role of regulated cell death in appressorium morphogenesis and examine how it is initiated and regulated, both temporally and spatially, during plant infection. We then use this synopsis to present a testable model for control of regulated cell death during appressorium-dependent plant infection by the blast fungus.

Nutrient acquisition is crucial for sustaining life. Plants develop beneficial intracellular partnerships with arbuscular mycorrhiza (AM) and nitrogen-fixing bacteria to surmount the scarcity of soil nutrients and tap into atmospheric dinitrogen, respectively1,2. Initiation of these root endosymbioses requires symbiont-induced oscillations in nuclear calcium (Ca2+) concentrations in root cells3. How the nuclear-localized ion channels, cyclic nucleotide-gated channel (CNGC) 15 and DOESN’T MAKE INFECTIONS1 (DMI1)4 are coordinated to specify symbiotic-induced nuclear Ca2+ oscillations remains unknown. Here we discovered an autoactive CNGC15 mutant that generates spontaneous low-frequency Ca2+ oscillations. While CNGC15 produces nuclear Ca2+ oscillations via a gating mechanism involving its helix 1, DMI1 acts as a pacemaker to specify the frequency of the oscillations. We demonstrate that the specificity of symbiotic-induced nuclear Ca2+ oscillations is encoded in its frequency. A high frequency activates endosymbiosis programmes, whereas a low frequency modulates phenylpropanoid pathways. Consequently, the autoactive cngc15 mutant, which is capable of generating both frequencies, has increased flavonoids that enhance AM, root nodule symbiosis and nutrient acquisition. We transferred this trait to wheat, resulting in field-grown wheat with increased AM colonization and nutrient acquisition. Our findings reveal a new strategy to boost endosymbiosis in the field and reduce inorganic fertilizer use while sustaining plant growth. Nuclear calcium oscillations initiate plant–arbuscular mycorrhiza and nitrogen-fixing bacteria symbioses for nutrient acquisition, with a newly discovered autoactive CNGC15 mutant enhancing these partnerships, potentially improving crop nutrition and reducing inorganic fertilizer dependence.

As much as 10% of plant immune receptors from the nucleotide-binding domain leucine-rich repeat (NLR) family carry integrated domains (IDs) that can directly bind pathogen effectors. However, it remains unclear whether direct binding to effectors is a universal feature of ID-containing NLRs given that only a few NLR-IDs have been functionally characterized. Here we show that the rice (Oryza sativa) sensor NLR-ID Pii2 confers resistance to strains of the rice blast fungus Magnaporthe oryzae that carry the effector AVR-Pii without directly binding this protein. First, we show that AVR-Pii binds the exocyst subunit OsExo70F2 in rice (Oryza sativa) to dissociate preformed complexes of OsExo70F2 with host RPM1 INTERACTING PROTEIN4 (RIN4) at the conserved NOI motif, facilitating a possible virulence function. Second, we show that in its resting state, Pii2 binds OsExo70F2 and OsExo70F3, essential components of Pii-mediated resistance, through its integrated NOI domain. Remarkably, AVR-Pii binding to OsExo70F2/F3 leads to dissociation of the Pii2–OsExo70F2 and Pii2–OsExo70F3 complexes, destabilization of Pii2, and activation of immunity. These findings support a novel conceptual model in which an NLR-ID monitors alterations of tethered host proteins targeted by pathogen effectors, providing insight into pathogen recognition mechanisms.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. Therefore, understanding the mechanisms of blast fungus infection and resistance of rice against the disease is important for global food security. In this study, we show that the M. oryzae effector protein AVR-PikD binds rice sHMA proteins and stabilizes them, presumably to enhance pathogen infection. We show that loss-of-function mutants in one rice sHMA, OsHIPP20, reduced the level of susceptibility against a compatible isolate of M. oryzae, suggesting that M. oryzae requires host sHMA to facilitate invasion. Remarkably, OsHIPP20 knockout rice line showed no growth defect, suggesting editing sHMA genes may present a novel source of resistance against blast disease.

The emergence of fungal antimicrobial resistance—fAMR—is having a growing impact on human and animal health, and food security. This roadmap charts inter-related actions that will enhance our ability to mitigate the risk of fAMR. As humanity’s reliance on antifungal chemicals escalates, our understanding of their one-health consequences needs to scale accordingly if we are to protect our ability to manage the global spectrum of fungal disease sustainably.