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Rice false smut, caused by Ustilaginoidea virens, threatens rice production by reducing yields and contaminating grains with harmful ustiloxins. However, studies on resistance genes are scarce. In this study, the resistance level of IR28 (resistant cultivar) to U. virens was validated through artificial inoculation. Notably, a reactivation of resistance genes after transient down-regulation during the first 3 to 5 dpi was observed in IR28 compared to WX98 (susceptible cultivar). Cluster results of a principal component analysis and hierarchical cluster analysis of differentially expressed genes (DEGs) in the transcriptome exhibited longer expression patterns in the early infection phase of IR28, consistent with its sustained resistance response. Results of GO and KEGG enrichment analyses highlighted the suppression of immune pathways when the hyphae first invade stamen filaments at 5 dpi, but sustained up-regulated DEGs were linked to the 'Plant–pathogen interaction' (osa04626) pathway, notably disease-resistant protein RPM1 (K13457, CNLs, coil-coiled NLR). An analysis of CNLs identified 245 proteins containing Rx-CC and NB-ARC domains in the Oryza sativa Indica genome. Partial candidate CNLs were shown to exhibit up-regulation at both 1 and 5 dpi in IR28. This study provides insights into CNLs' responses to U. virens in IR28, potentially informing resistance mechanisms and genetic breeding targets. [ABSTRACT FROM AUTHOR]

The NLR (nucleotide-binding leucine-rich repeat) class immune receptor Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the TSWV movement protein NSm, we know very little about the downstream events that lead to resistance. Here, we investigated the Sw-5b–mediated early transcriptomic changes that occur in response to mechanical and thrips-mediated inoculation of TSWV, using near-isogenic tomato lines CNPH-LAM 147 (Sw5b+/+) and Santa Clara (Sw-5b−/−). We observed earlier Sw-5b–mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b responses specific to the method of TSWV inoculation. Although TSWV is transmitted in nature primarily by the thrips, Sw-5b responses to thrips inoculation have not been previously studied. Therefore, the DEGs we have identified in response to thrips-mediated inoculation provide a new foundation for understanding the mechanistic roles of these genes in the Sw-5b–mediated resistance. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rice false smut, caused by Ustilaginoidea virens, threatens rice production by reducing yields and contaminating grains with harmful ustiloxins. However, studies on resistance genes are scarce. In this study, the resistance level of IR28 (resistant cultivar) to U. virens was validated through artificial inoculation. Notably, a reactivation of resistance genes after transient down-regulation during the first 3 to 5 dpi was observed in IR28 compared to WX98 (susceptible cultivar). Cluster results of a principal component analysis and hierarchical cluster analysis of differentially expressed genes (DEGs) in the transcriptome exhibited longer expression patterns in the early infection phase of IR28, consistent with its sustained resistance response. Results of GO and KEGG enrichment analyses highlighted the suppression of immune pathways when the hyphae first invade stamen filaments at 5 dpi, but sustained up-regulated DEGs were linked to the ‘Plant–pathogen interaction’ (osa04626) pathway, notably disease-resistant protein RPM1 (K13457, CNLs, coil-coiled NLR). An analysis of CNLs identified 245 proteins containing Rx-CC and NB-ARC domains in the Oryza sativa Indica genome. Partial candidate CNLs were shown to exhibit up-regulation at both 1 and 5 dpi in IR28. This study provides insights into CNLs’ responses to U. virens in IR28, potentially informing resistance mechanisms and genetic breeding targets.

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation. [ABSTRACT FROM AUTHOR]

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.

Summary: Plant intracellular nucleotide‐binding leucine‐rich repeat immune receptors (NLRs) perceive the activity of pathogen‐secreted effector molecules that, when undetected, promote colonisation of hosts. Signalling from activated NLRs converges with and potentiates downstream responses from activated pattern recognition receptors (PRRs) that sense microbial signatures at the cell surface. Efficient signalling of both receptor branches relies on the host cell nucleus as an integration point for transcriptional reprogramming, and on the macromolecular transport processes that mediate the communication between cytoplasm and nucleoplasm. Studies on nuclear pore complexes (NPCs), the nucleoporin proteins (NUPs) that compose NPCs, and nuclear transport machinery constituents that control nucleocytoplasmic transport, have revealed that they play important roles in regulating plant immune responses. Here, we discuss the contributions of nucleoporins and nuclear transport receptor (NTR)‐mediated signal transduction in plant immunity with an emphasis on NLR immune signalling across the nuclear compartment boundary and within the nucleus. We also highlight and discuss cytoplasmic and nuclear functions of NLRs and their signalling partners and further consider the potential implications of NLR activation and resistosome formation in both cellular compartments for mediating plant pathogen resistance and programmed host cell death. [ABSTRACT FROM AUTHOR]

Summary: Calcium serves as a second messenger in a variety of developmental and physiological processes and has long been identified as important for plant immune responses. We discuss recent discoveries regarding plant immune‐related calcium‐permeable channels and how the two intertwined branches of the plant immune system are intricately linked to one another through calcium signalling. Cell surface immune receptors carefully tap the immense calcium gradient that exists between apoplast and cytoplasm in a short burst via tightly regulated plasma membrane (PM)‐resident cation channels. Intracellular immune receptors form atypical calcium‐permeable cation channels at the PM and mediate a prolonged calcium influx, overcoming the deleterious influence of pathogen effectors and enhancing plant immune responses. See also the Commentary on this article by Lee & Romeis, 234: 769–772. [ABSTRACT FROM AUTHOR]

Plant nucleotide-binding and leucine-rich repeat (NLR) receptors recognize avirulence effectors directly through their integrated domains (IDs) or indirectly via the effector-targeted proteins. Previous studies have succeeded in generating designer NLR receptors with new recognition profiles by engineering IDs or targeted proteins based on prior knowledge of their interactions with the effectors. However, it is yet a challenge to design a new plant receptor capable of recognizing effectors that function by unknown mechanisms. Several rice NLR immune receptors, including RGA5, possess an integrated heavy metal–associated (HMA) domain that recognizes corresponding Magnaporthe oryzae Avrs and ToxB-like (MAX) effectors in the rice blast fungus. Here, we report a designer rice NLR receptor RGA5HMA2 carrying an engineered, integrated HMA domain (RGA5-HMA2) that can recognize the noncorresponding MAX effector AvrPib and confers the RGA4-dependent resistance to the M. oryzae isolates expressing AvrPib, which originally triggers the Pib-mediated blast resistance via unknown mechanisms. The RGA5-HMA2 domain is contrived based on the high structural similarity of AvrPib with two MAX effectors, AVR-Pia and AVR1-CO39, recognized by cognate RGA5-HMA, the binding interface between AVR1-CO39 and RGA5-HMA, and the distinct surface charge of AvrPib and RAG5-HMA. This work demonstrates that rice NLR receptors with the HMA domain can be engineered to confer resistance to the M. oryzae isolates noncorresponding but structurally similar MAX effectors, which manifest cognate NLR receptor–mediated resistance with unknown mechanisms. Our study also provides a practical approach for developing rice multilines and broad race spectrum–resistant cultivars by introducing a series of engineered NLR receptors. [ABSTRACT FROM AUTHOR]

NLRs constitute intracellular immune receptors in both plants and animals. Direct or indirect ligand recognition results in formation of oligomeric NLR complexes to mediate immune signaling. Over the past 20 years, rapid progress has been made in our understanding of NLR signaling. Structural and biochemical studies provide insight into molecular basis of autoinhibition, ligand recognition, and resistosome/inflammasome formation of several NLRs. In this review, we summarize these studies focusing on the structural aspect of NLRs. We also discuss the analogies and differences between plant and animal NLRs in their mechanisms of action and how the available knowledge may shed light on the signaling mechanisms of other NLRs. [ABSTRACT FROM AUTHOR]

Proteins form complex networks through interaction to drive biological processes. Thus, dissecting protein-protein interactions (PPIs) is essential for interpreting cellular processes. To overcome the drawbacks of traditional approaches for analyzing PPIs, enzyme-catalyzed proximity labeling (PL) techniques based on peroxidases or biotin ligases have been developed and successfully utilized in mammalian systems. However, the use of toxic H 2 O 2 in peroxidase-based PL, the requirement of long incubation time (16-24 h), and higher incubation temperature (37 °C) with biotin in BioID-based PL significantly restricted their applications in plants. TurboID-based PL, a recently developed approach, circumvents the limitations of these methods by providing rapid PL of proteins under room temperature. We recently optimized the use of TurboID-based PL in plants and demonstrated that it performs better than BioID in labeling endogenous proteins. Here, we describe a step-by-step protocol for TurboID-based PL in studying PPIs in planta, including Agrobacterium-based transient expression of proteins, biotin treatment, protein extraction, removal of free biotin, quantification, and enrichment of the biotinylated proteins by affinity purification. We describe the PL using plant viral immune receptor N, which belongs to the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors, as a model. The method described could be easily adapted to study PPI networks of other proteins in Nicotiana benthamiana and provides valuable information for future application of TurboID-based PL in other plant species., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The NLR (nucleotide-binding leucine-rich repeat) class immune receptor Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the TSWV movement protein NSm, we know very little about the downstream events that lead to resistance. Here, we investigated the Sw-5b -mediated early transcriptomic changes that occur in response to mechanical and thrips-mediated inoculation of TSWV, using near-isogenic tomato lines CNPH-LAM 147 ( Sw5b +/+ ) and Santa Clara ( Sw-5b - / - -mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b -mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b responses specific to the method of TSWV inoculation. Although TSWV is transmitted in nature primarily by the thrips, Sw-5b responses to thrips inoculation have not been previously studied. Therefore, the DEGs we have identified in response to thrips-mediated inoculation provide a new foundation for understanding the mechanistic roles of these genes in the Sw-5b -mediated resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Summary: Intracellular nucleotide binding (NB) and leucine‐rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings have revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition.Here, by using the CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR‐NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1.Overaccumulation of SAUL1 is monitored by the SOC3−TN2 pair, while SAUL1's disappearance is guarded by the SOC3−CHS1 pair. SOC3 forms a head‐to‐head genomic arrangement with CHS1 and TN2, indicative of transcriptional co‐regulation.Such intricate cooperative interactions can probably enlarge the recognition spectrum and increase the functional flexibility of NLRs, which can partly explain the overwhelming occurrence of NLR gene clustering in higher plants. [ABSTRACT FROM AUTHOR]

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector's host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops., Competing Interests: JM, MS, AB, SV, MF, AL, CS, JD, AB, RT, MB No competing interests declared, SK receives funding from industry on NLR biology, (© 2023, Maidment et al.)

Plant-parasitic nematodes (PPNs) secrete an array of molecules that can lead to their detection by or promote infection of their hosts. However, the function of these molecules in plant cells is often unknown or limited to phenotypic observations. Similarly, how plant cells detect and/or respond to these molecules is still poorly understood. Here, we highlight recent advances in mechanistic insights into the molecular dialogue between PPNs and plants at the cellular level. New discoveries reveal a) the essential roles of extra- and intracellular plant receptors in PPN perception and the manipulation of host immune- or developmental pathways during infection and b) how PPNs target such receptors to manipulate their hosts. Finally, the plant secretory pathway has emerged as a critical player in PPN peptide delivery, feeding site formation and non-canonical resistance. • Perception of PPNs at the cell surface induces immune and developmental pathways. • PPNs exploit the host cell post-translational trafficking machinery. • The host secretory pathway is critical in feeding site formation and PPN resistance. • Intracellular NLR immune receptors act in concert to trigger plant resistance to PPN. • PPN effectors, including peptide mimics, target plant immune receptors for virulence. [ABSTRACT FROM AUTHOR]

Calcium serves as a second messenger in a variety of developmental and physiological processes and has long been identified as important for plant immune responses. We discuss recent discoveries regarding plant immune-related calcium-permeable channels and how the two intertwined branches of the plant immune system are intricately linked to one another through calcium signalling. Cell surface immune receptors carefully tap the immense calcium gradient that exists between apoplast and cytoplasm in a short burst via tightly regulated plasma membrane (PM)-resident cation channels. Intracellular immune receptors form atypical calcium-permeable cation channels at the PM and mediate a prolonged calcium influx, overcoming the deleterious influence of pathogen effectors and enhancing plant immune responses., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Plant nucleotide-binding and leucine-rich repeat (NLR) receptors recognize avirulence effectors directly through their integrated domains (IDs) or indirectly via the effector-targeted proteins. Previous studies have succeeded in generating designer NLR receptors with new recognition profiles by engineering IDs or targeted proteins based on prior knowledge of their interactions with the effectors. However, it is yet a challenge to design a new plant receptor capable of recognizing effectors that function by unknown mechanisms. Several rice NLR immune receptors, including RGA5, possess an integrated heavy metal-associated (HMA) domain that recognizes corresponding Magnaporthe oryzae Avrs and ToxB-like (MAX) effectors in the rice blast fungus. Here, we report a designer rice NLR receptor RGA5 HMA2 carrying an engineered, integrated HMA domain (RGA5-HMA2) that can recognize the noncorresponding MAX effector AvrPib and confers the RGA4-dependent resistance to the M. oryzae isolates expressing AvrPib, which originally triggers the Pib-mediated blast resistance via unknown mechanisms. The RGA5-HMA2 domain is contrived based on the high structural similarity of AvrPib with two MAX effectors, AVR-Pia and AVR1-CO39, recognized by cognate RGA5-HMA, the binding interface between AVR1-CO39 and RGA5-HMA, and the distinct surface charge of AvrPib and RAG5-HMA. This work demonstrates that rice NLR receptors with the HMA domain can be engineered to confer resistance to the M. oryzae isolates noncorresponding but structurally similar MAX effectors, which manifest cognate NLR receptor-mediated resistance with unknown mechanisms. Our study also provides a practical approach for developing rice multilines and broad race spectrum-resistant cultivars by introducing a series of engineered NLR receptors., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)