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Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9's role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing. [ABSTRACT FROM AUTHOR]

The relationship among microbiome, immunity and cervical cancer has been targeted by several studies, yet many questions remain unanswered. We characterized herein the virome and bacteriome from cervical samples and correlated these findings with innate immunity gene expression in a Brazilian convenience sample of HPV-infected (HPV+) and uninfected (HPV-) women. For this purpose, innate immune gene expression data were correlated to metagenomic information. Correlation analysis showed that interferon (IFN) is able to differentially modulate pattern recognition receptors (PRRs) expression based on HPV status. Virome analysis indicated that HPV infection correlates to the presence of Anellovirus (AV) and seven complete HPV genomes were assembled. Bacteriome results unveiled that vaginal community state types (CST) distribution was independent of HPV or AV status, although bacterial phyla distribution differed between groups. Furthermore, TLR3 and IFNαR2 levels were higher in the Lactobacillus no iners-dominated mucosa and we detected correlations among RIG-like receptors (RLR) associated genes and abundance of specific anaerobic bacteria. Collectively, our data show an intriguing connection between HPV and AV infections that could foster cervical cancer development. Besides that, TLR3 and IFNαR2 seem to create a protective milieu in healthy cervical mucosa (L. no iners-dominated), and RLRs, known to recognize viral RNA, were correlated to anaerobic bacteria suggesting that they might be related to dysbiosis. [ABSTRACT FROM AUTHOR]

The relationship among microbiome, immunity and cervical cancer has been targeted by several studies, yet many questions remain unanswered. We characterized herein the virome and bacteriome from cervical samples and correlated these findings with innate immunity gene expression in a Brazilian convenience sample of HPV-infected (HPV+) and uninfected (HPV-) women. For this purpose, innate immune gene expression data were correlated to metagenomic information. Correlation analysis showed that interferon (IFN) is able to differentially modulate pattern recognition receptors (PRRs) expression based on HPV status. Virome analysis indicated that HPV infection correlates to the presence of Anellovirus (AV) and seven complete HPV genomes were assembled. Bacteriome results unveiled that vaginal community state types (CST) distribution was independent of HPV or AV status, although bacterial phyla distribution differed between groups. Furthermore, TLR3 and IFNαR2 levels were higher in the Lactobacillus no iners-dominated mucosa and we detected correlations among RIG-like receptors (RLR) associated genes and abundance of specific anaerobic bacteria. Collectively, our data show an intriguing connection between HPV and AV infections that could foster cervical cancer development. Besides that, TLR3 and IFNαR2 seem to create a protective milieu in healthy cervical mucosa (L. no iners-dominated), and RLRs, known to recognize viral RNA, were correlated to anaerobic bacteria suggesting that they might be related to dysbiosis.

Arabidopsis LIM proteins are named after the initials of three proteins Lin-11, Isl-1, and MEC-3, which belong to a class of transcription factors that play an important role in the developmental regulation of eukaryotes and are also involved in a variety of life processes, including gene transcription, the construction of the cytoskeleton, signal transduction, and metabolic regulation. Plant LIM proteins have been shown to regulate actin bundling in different cells, but their role in immunity remains elusive. Mitogen-activated protein kinases (MAPKs) are a family of conserved serine/threonine protein kinases that link upstream receptors to their downstream targets. Pathogens produce pathogen-associated molecular patterns (PAMPs) that trigger the activation of MAPK cascades in plants. Recently, we conducted a large-scale phosphoproteomic analysis of PAMP-induced Arabidopsis plants to identify putative MAPK targets. One of the identified phospho-proteins was WLIM2A, an Arabidopsis LIM protein. In this study, we investigated the role of WLIM2A in plant immunity. We employed a reverse-genetics approach and generated wlim2a knockout lines using CRISPR-Cas9 technology. We also generated complementation and phosphosite-mutated WLIM2A expression lines in the wlim2a background. The wlim2a lines were compromised in their response to Pseudomonas syringae Pst DC3000 but showed enhanced resistance to the necrotrophic fungus Botrytis cinereae . Transcriptome analyses of wlim2a mutants revealed the deregulation of immune hormone biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways. The wlim2a mutants also exhibited altered stomatal phenotypes. Analysis of plants expressing WLIM2A variants of the phospho-dead or phospho-mimicking MAPK phosphorylation site showed opposing stomatal behavior and resistance phenotypes in response to Pst DC3000 infection, proving that phosphorylation of WLIM2A plays a crucial role in plant immunity. Overall, these data demonstrate that phosphorylation of WLIM2A by MAPKs regulates Arabidopsis responses to plant pathogens.

Plant-specific receptor-like protein kinases (RLKs) are essential for pathogen recognition during pattern-triggered immunity. Together with coreceptors and associated proteins, they act as bona fide immune receptors, perceiving a variety of microbe-associated molecular patterns or damage-associated molecular patterns. The cysteine-rich receptor-like kinases (CRKs) form one of the biggest subgroups of RLKs, but so far, their ligands have not been identified. It has been shown that CRKs play important roles in plant immunity and defense responses as well as in response to abiotic stimuli and in control of plant development. However, molecular information on how CRKs integrate with the known framework of signaling components controlling early defense responses remains enigmatic., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael Wrzaczek reports financial support was provided by Biology Centre Czech Academy of Sciences. Michael Wrzaczek reports financial support was provided by Czech Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Antiviral responses induced by double-stranded RNA (dsRNA) include RNA interference (RNAi) and pattern-triggered immunity (PTI), but their relative contributions to antiviral defence are not well understood. We aimed at testing the impact of exogenous applied dsRNA on both layers of defence against potato virus X expressing GFP (PVX-GFP) in Nicotiana benthamiana. Co-inoculation of PVX-GFP with either sequence-specific (RNAi) or nonspecific dsRNA (PTI) showed that nonspecific dsRNA reduced virus accumulation in both inoculated and systemic leaves. However, nonspecific dsRNA was a poor inducer of antiviral immunity compared to a sequence-specific dsRNA capable of triggering the RNAi response, and plants became susceptible to systemic infection. Studies with a PVX mutant unable to move from cell to cell indicated that the interference with PVX-GFP triggered by nonspecific dsRNA operated at the single-cell level. Next, we performed RNA-seq analysis to examine similarities and differences in the transcriptome triggered by dsRNA alone or in combination with viruses harbouring sequences targeted or not by dsRNA. Enrichment analysis showed an over-representation of plant-pathogen signalling pathways, such as calcium, ethylene and MAPK signalling, which are typical of antimicrobial PTI. Moreover, the transcriptomic response to the virus targeted by dsRNA had a greater impact on defence than the non-targeted virus, highlighting qualitative differences between sequence-specific RNAi and nonspecific PTI responses. Together, these results further our understanding of plant antiviral defence, particularly the contribution of nonspecific dsRNA-mediated PTI. We envisage that both sequence-specific RNAi and nonspecific PTI pathways may be triggered via topical application of dsRNA, contributing cumulatively to plant protection against viruses., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Plants employ distinct mechanisms to respond to environmental changes. Modification of mRNA by N  6 -methyladenosine (m 6 A), known to affect the fate of mRNA, may be one such mechanism to reprogram mRNA processing and translatability upon stress. However, it is difficult to distinguish a direct role from a pleiotropic effect for this modification due to its prevalence in RNA. Through characterization of the transient knockdown-mutants of m 6 A writer components and mutants of specific m 6 A readers, we demonstrate the essential role that m 6 A plays in basal resistance and pattern-triggered immunity (PTI). A global m 6 A profiling of mock and PTI-induced Arabidopsis plants as well as formaldehyde fixation and cross-linking immunoprecipitation-sequencing of the m 6 A reader, EVOLUTIONARILY CONSERVED C-TERMINAL REGION2 (ECT2) showed that while dynamic changes in m 6 A modification and binding by ECT2 were detected upon PTI induction, most of the m 6 A sites and their association with ECT2 remained static. Interestingly, RNA degradation assay identified a dual role of m 6 A in stabilizing the overall transcriptome while facilitating rapid turnover of immune-induced mRNAs during PTI. Moreover, polysome profiling showed that m 6 A enhances immune-associated translation by binding to the ECT2/3/4 readers. We propose that m 6 A plays a positive role in plant immunity by destabilizing defense mRNAs while enhancing their translation efficiency to create a transient surge in the production of defense proteins., Competing Interests: Competing interests statement:X.D. is a founder of Upstream Biotechnology Inc. and a member of its scientific advisory board, as well as a scientific advisory board member of Inari Agriculture Inc. and Aferna Bio, Inc. G.H.G. is a founder of Upstream Biotechnology Inc. C.H. is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, Inc. and Ellis Bio Inc., a scientific cofounder and equity holder of Accent Therapeutics, Inc., and a member of the scientific advisory board of Rona Therapeutics and Element Biosciences.

In this research, a high-throughput RNA sequencing-based transcriptome analysis technique (RNA-Seq) was used to evaluate differentially expressed genes (DEGs) in the wild type Arabidopsis seedlings in response to AtPep1, a well-known peptide representing an endogenous damage-associated molecular pattern (DAMP), and flg22, a well-known microbe-associated molecular pattern (MAMP). We compared and dissected the global transcriptional landscape of Arabidopsis thaliana in response to AtPep1 and flg22 and could identify shared and unique DEGs in response to these elicitors. We found that while a remarkable number of flg22 up-regulated genes were also induced by AtPep1, 256 genes were exclusively up-regulated in response to flg22, and 328 were exclusively up-regulated in response to AtPep1. Furthermore, among down-regulated DEGs upon flg22 treatment, 107 genes were exclusively down-regulated by flg22 treatment, while 411 genes were exclusively down-regulated by AtPep1. We found a number of hitherto overlooked genes to be induced upon treatment with either flg22 or with AtPep1, indicating their possible involvement general pathways in innate immunity. Here, we characterized two of them, namely PP2-B13 and ACLP1. pp2-b13 and aclp1 mutants showed increased susceptibility to infection by the virulent pathogen Pseudomonas syringae DC3000 and its mutant Pst DC3000 hrcC (lacking the type III secretion system), as evidenced by increased proliferation of the two pathogens in planta. Further, we present evidence that the aclp1 mutant is deficient in ethylene production upon flg22 treatment, while the pp2-b13 mutant is deficient in the production of reactive oxygen species (ROS). The results from this research provide new information for a better understanding of the immune system in Arabidopsis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Safaeizadeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest class of membrane-localized receptor-like kinases in plants. Leucine-rich repeat receptor-like kinases are key immune sectors contributing to pattern-triggered immunity (PTI), but whether LRR-RLK mediates effector-triggered immunity (ETI) in plants remains unclear. In this study, we evaluated the function of LRR-RLKs in regulating ETI by using a virus-induced gene silencing (VIGS)-based reverse genetic screening assay, and identified a LRR-RLK named ETI-dependent receptor-like kinase 1 (EDK1) required for ETI triggered by the avirulence effector AVRblb2 secreted by Phytophthora infestans and its cognate receptor Rpi-blb2. Silencing or knockout of EDK1 compromised immunity mediated by Rpi-blb2 and the cell death triggered by recognition of AVRblb2. NLR-required for cell death 4 (NRC4), a signaling component acts downstream of Rpi-blb2, was identified that interacts with EDK1 using the LC-MS analysis and the interaction was further evaluated by co-immunoprecipitation. EDK1 promotes protein accumulation of NRC4 in a kinase-dependent manner and positively regulates resistance to P. infestans in Nicotiana benthamiana. Our study revealed that EDK1 positively regulates plant ETI through modulating accumulation of the NLR signaling component NRC4, representing a new regulatory role of the membrane-localized LRR-RLKs in plant immunity., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

Rice is a staple crop continually threatened by bacterial and fungal pathogens. OsWRKY transcription factors are involved in various disease responses. However, the functions of many OsWRKYs are still elusive. In this study, we demonstrated that OsWRKY7 enhances rice immunity against Xanthomonas oryzae pv. oryzae (Xoo). OsWRKY7 localized in the nucleus, and gene expression of OsWRKY7 was induced by Xoo inoculation. The OsWRKY7-overexpressing lines showed enhanced resistant phenotype against Xoo, and gene expressions of OsPR1a, OsPR1b, and OsPR10a were significantly increased in the transgenic lines after Xoo inoculation. Moreover, OsWRKY7 activated the OsPR promoters, and the promoter activities were synergistically upregulated by flg22. Genetic- and cell-based analysis showed OsWRKY7 is involved in pattern-triggered immunity against Xoo. These results suggest that OsWRKY7 plays a role as a positive regulator of disease resistance to Xoo through pattern-triggered immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP) 1-7 . The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA 8-15 . However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation., (© 2024. The Author(s).)

The pattern-triggered immunity (PTI) response is triggered at the plant cell surface by the recognition of microbe-derived molecules known as microbe- or pathogen-associated molecular patterns or molecules derived from compromised host cells called damage-associated molecular patterns. Membrane-localized receptor proteins, known as pattern recognition receptors, are responsible for this recognition. Although much of the machinery of PTI is conserved, natural variation for the PTI response exists within and across species with respect to the components responsible for pattern recognition, activation of the response, and the strength of the response induced. This review describes what is known about this variation. We discuss how variation in the PTI response can be measured and how this knowledge might be utilized in the control of plant disease and in developing plant varieties with enhanced disease resistance., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.

Genomic stability relies on a multifaceted and evolutionarily conserved DNA damage response (DDR). In multicellular organisms, an integral facet of the DDR involves the activation of the immune system to eliminate cells with persistent DNA damage. Recent research has shed light on a complex array of nucleic acid sensors crucial for innate immune activation in response to oncogenic stress-associated DNA damage, a process vital for suppressing tumor formation. Yet, these immune sensing pathways may also be co-opted to foster tolerance of chromosomal instability, thereby driving cancer progression. This review aims to provide an updated overview of how the innate immune system detects and responds to DNA damage. An improved understanding of the regulatory intricacies governing this immune response may uncover new avenues for cancer prevention and therapeutic intervention., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: G.P.G. is a co-inventor on a patent held by the University of North Carolina at Chapel Hill on methods for detecting tumor-derived viral DNA in blood (U.S. Patent 11,168,373), receives patent licensing fees from and has equity in Naveris, Inc, and is the recipient of research funding from Breakpoint Therapeutics and Merck.A.M.G, M.G.C, and L.M.L. have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Transient and rapid increase in cytosolic Ca 2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca 2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca 2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca 2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Key Message: Plant U-box E3 ligases PUB20 and PUB21 are flg22-triggered signaling components and negatively regulate immune responses. 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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacterial biofilm-like aggregates have been observed in plants, but their role in pathogenicity is underinvestigated. In the present study, we observed that extracellular DNA and polysaccharides colocalized with green fluorescent protein (GFP)-expressing Pseudomonas syringae pv. tomato (Pst) aggregates in Arabidopsis leaves, suggesting that Pst aggregates are biofilms. GFP-expressing Pst, Pst ΔalgU ΔmucAB (Pst algU mutant), and Pst ΔalgD ΔalgU ΔmucAB (Pst algU algD mutant) were examined to explore the roles of (1) alginate, a potential biofilm component; (2) Pst AlgU, thought to regulate alginate biosynthesis and some type III secretion system effector genes; and (3) intercellular salicylic acid (SA) accumulation during pathogen-associated molecular pattern-triggered immunity (PTI). Pst formed extensive aggregates in susceptible plants, whereas aggregate numbers and size were reduced in Pst algU and Pst algD algU mutants, and both multiplied poorly in planta, suggesting that aggregate formation contributes to Pst success in planta. However, in SA-deficient sid2-2 plants, Pst algD algU mutant multiplication and aggregate formation were partially restored, suggesting plant-produced SA contributes to suppression of Pst aggregate formation. Pst algD algU mutants formed fewer and smaller aggregates than Pst algU mutants, suggesting both AlgU and AlgD contribute to Pst aggregate formation. Col-0 plants accumulated low levels of SA in response to Pst and both mutants (Pst algU and Pst algD algU), suggesting the regulatory functions of AlgU are not involved in suppressing SA-mediated plant defence. Plant PTI was associated with highly reduced Pst aggregate formation and accumulation of intercellular SA in flg22-induced PTI-responding wild-type Col-0, but not in PTI-incompetent fls2, suggesting intercellular SA accumulation by Arabidopsis contributes to suppression of Pst biofilm-like aggregate formation during PTI., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The plant cell wall (CW) is one of the most important physical barriers that phytopathogens must conquer to invade their hosts. This barrier is a dynamic structure that responds to pathogen infection through a complex network of immune receptors, together with CW-synthesizing and CW-degrading enzymes. Callose deposition in the primary CW is a well-known physical response to pathogen infection. Notably, callose and cellulose biosynthesis share an initial substrate, UDP-glucose, which is the main load-bearing component of the CW. However, how these 2 critical biosynthetic processes are balanced during plant-pathogen interactions remains unclear. Here, using 2 different pathogen-derived molecules, bacterial flagellin (flg22) and the diffusible signal factor (DSF) produced by Xanthomonas campestris pv. campestris, we show a negative correlation between cellulose and callose biosynthesis in Arabidopsis (Arabidopsis thaliana). By quantifying the abundance of callose and cellulose under DSF or flg22 elicitation and characterizing the dynamics of the enzymes involved in the biosynthesis and degradation of these 2 polymers, we show that the balance of these 2 CW components is mediated by the activity of a β-1,3-glucanase (BG2). Our data demonstrate balanced cellulose and callose biosynthesis during plant immune responses., Competing Interests: Conflict of interest statement. The authors declare no conflict of interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Bacterial wilt, caused by Ralstonia solanacearum , one of the most destructive phytopathogens, leads to significant annual crop yield losses. Type III effectors (T3Es) mainly contribute to the virulence of R. solanacearum , usually by targeting immune-related proteins. Here, we clarified the effect of a novel E3 ubiquitin ligase (NEL) T3E, RipAW, from R. solanacearum on pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and further explored its action mechanism. In the susceptible host Arabidopsis thaliana , we monitored the expression of PTI marker genes, flg22-induced ROS burst, and callose deposition in RipAW - and RipAW C177A -transgenic plants. Our results demonstrated that RipAW suppressed host PTI in an NEL-dependent manner. By Split-Luciferase Complementation, Bimolecular Fluorescent Complimentary, and Co-Immunoprecipitation assays, we further showed that RipAW associated with three crucial components of the immune receptor complex, namely FLS2, XLG2, and BIK1. Furthermore, RipAW elevated the ubiquitination levels of FLS2, XLG2, and BIK1, accelerating their degradation via the 26S proteasome pathway. Additionally, co-expression of FLS2, XLG2, or BIK1 with RipAW partially but significantly restored the RipAW-suppressed ROS burst, confirming the involvement of the immune receptor complex in RipAW-regulated PTI. Overall, our results indicate that RipAW impairs host PTI by disrupting the immune receptor complex. Our findings provide new insights into the virulence mechanism of R. solanacearum .

Bacterial leaf streak (BLS) of maize is an emerging foliar disease of maize in the Americas. It is caused by the gram-negative nonvascular bacterium Xanthomonas vasicola pv. vasculorum . There are no chemical controls available for BLS, and thus, host resistance is crucial for managing X. vasicola pv. vasculorum . The objective of this study was to examine the genetic determinants of resistance to X. vasicola pv. vasculorum in maize, as well as the relationship between other defense-related traits and BLS resistance. Specifically, we examined the correlations among BLS severity, severity for three fungal diseases, flg-22 response, hypersensitive response, and auricle color. We conducted quantitative trait locus (QTL) mapping for X. vasicola pv. vasculorum resistance using the maize recombinant inbred line population Z003 (B73 × CML228). We detected three QTLs for BLS resistance. In addition to the disease resistance QTL, we detected a single QTL for auricle color. We observed significant, yet weak, correlations among BLS severity, levels of pattern-triggered immunity response and leaf flecking. These results will be useful for understanding resistance to X. vasicola pv. vasculorum and mitigating the impact of BLS on maize yields., Competing Interests: The author(s) declare no conflict of interest.

Cyclic di-guanosine monophosphate (c-di-GMP) is a unique bacterial second messenger but is hijacked by host cells during bacterial infection as a pathogen-associated molecular pattern (PAMP) to trigger STING-dependent immune responses. Here, we show that upon infection, VopY, an effector of Vibrio parahaemolyticus, is injected into host cells by type III secretion system 2 (T3SS2), a secretion system unique to its pathogenic strains and indispensable for enterotoxicity. VopY is an EAL-domain-containing phosphodiesterase and is capable of hydrolyzing c-di-GMP. VopY expression in host cells prevents the activation of STING and STING-dependent downstream signaling triggered by c-di-GMP and, consequently, suppresses type I interferon immune responses. The presence of VopY in V. parahaemolyticus enables it to cause both T3SS2-dependent enterotoxicity and cytotoxicity. These findings uncover the destruction of self-derived PAMPs by injecting specific effectors to suppress PAMP-triggered immune responses as a unique strategy for bacterial pathogens to subvert immunity and cause disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this issue of JEM, companion articles from Pinilla et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230104) and Robinson et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230105) demonstrate that ribotoxic stress induced by Pseudomonas aeruginosa and Corynebacterium diphtheriae EEF2-targeting exotoxins leads to NLRP1 inflammasome activation, representing a new mechanism of effector-triggered immunity., Competing Interests: Disclosures: The authors declare no competing financial interests., (© 2023 Tibble et al.)

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M. polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although many studies have shown that microbes can ectopically stimulate or suppress plant immune responses, the fundamental question of whether the entire preexisting microbiota is indeed required for proper development of plant immune response remains unanswered. Using a recently developed peat-based gnotobiotic plant growth system, we found that Arabidopsis grown in the absence of a natural microbiota lacked age-dependent maturation of plant immune response and were defective in several aspects of pattern-triggered immunity. Axenic plants exhibited hypersusceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and the fungal pathogen Botrytis cinerea. Microbiota-mediated immunocompetence was suppressed by rich nutrient conditions, indicating a tripartite interaction between the host, microbiota and abiotic environment. A synthetic microbiota composed of 48 culturable bacterial strains from the leaf endosphere of healthy Arabidopsis plants was able to substantially restore immunocompetence similar to plants inoculated with a soil-derived community. In contrast, a 52-member dysbiotic synthetic leaf microbiota overstimulated the immune transcriptome. Together, these results provide evidence for a causal role of a eubiotic microbiota in gating proper immunocompetence and age-dependent immunity in plants., (© 2023. The Author(s).)

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites 1-4 . However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming., (© 2023. The Author(s).)

The detection of microbial infections by plants induces the rapid formation of immune receptor complexes at the plasma membrane. However, how this process is controlled to ensure proper immune signaling remains largely unknown. Here, we found that the Nicotiana benthamiana membrane-localized leucine-rich repeat receptor-like kinase BAK1-INTERACTING RLK 2 (NbBIR2) constitutively associates with BRI1-ASSOCIATED RECEPTOR KINASE 1 (NbBAK1) in vivo and in vitro and promotes complex formation with pattern recognition receptors. In addition, NbBIR2 is targeted by 2 RING-type ubiquitin E3 ligases, SNC1-INFLUENCING PLANT E3 LIGASE REVERSE 2a (NbSNIPER2a) and NbSNIPER2b, for ubiquitination and subsequent degradation in planta. NbSNIPER2a and NbSNIPER2b interact with NbBIR2 in vivo and in vitro and are released from NbBIR2 upon treatment with different microbial patterns. Furthermore, accumulation of NbBIR2 in response to microbial patterns is tightly associated with NbBAK1 abundance in N. benthamiana. NbBAK1 acts as a modular protein that stabilizes NbBIR2 by competing with NbSNIPER2a or NbSNIPER2b for association with NbBIR2. Similar to NbBAK1, NbBIR2 positively regulates pattern-triggered immunity and resistance to bacterial and oomycete pathogens in N. benthamiana, whereas NbSNIPER2a and NbSNIPER2b have the opposite effect. Together, these results reveal a feedback regulatory mechanism employed by plants to tailor pattern-triggered immune signaling., Competing Interests: Conflict of interest statement. The authors have no conflicts of interest to declare., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Receptor-like cytoplasmic kinases (RLCKs) mediate the intracellular signaling downstream of pattern-recognition receptors (PRRs). Several RLCKs from subfamily VII of rice (Oryza sativa) have important roles in plant immunity, but the role of RLCK VII-4 in pattern-triggered immune (PTI) signaling and resistance to pathogens has not yet been investigated. Here, we generated by multiplex clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing rice sextuple mutant lines where the entire RLCK VII-4 subfamily is inactivated and then analyzed the resulting lines for their response to chitin and flg22 and for their immunity to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae. Analysis of the rlckvii-4 mutants revealed that they have an impaired reactive oxygen system burst and reduced defense gene expression in response to flg22 and chitin. This indicates that members of the rice RLCK VII-4 subfamily are required for immune signaling downstream of multiple PRRs. Furthermore, we found that the rice RLCK VII-4 subfamily is important for chitin-induced callose deposition and mitogen-activated protein kinase activation and that it is crucial for basal resistance against Xoo and M. oryzae pathogens. This establishes that the RLCK VII-4 subfamily has critical functions in the regulation of multiple PTI pathways in rice and opens the way for deciphering the precise role of its members in the control of rice PTI., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is the first line of defense in plant disease resistance. However, the molecular mechanisms of plant PTI vary across species, making it challenging to identify a core set of trait-associated genes. This study aimed to investigate key factors that influence PTI and identify the core molecular network in Sorghum bicolor, a C4 plant. We performed comprehensive weighted gene co-expression network analysis and temporal expression analysis of large-scale transcriptome data from various sorghum cultivars under different PAMP treatments. Our results revealed that the type of PAMP had a stronger influence on the PTI network than did the sorghum cultivar. Following PAMP treatment, 30 genes with stable downregulated expression and 158 genes with stable upregulated expression were identified, including genes encoding potential pattern recognition receptors whose expression was upregulated within 1 h of treatment. PAMP treatment altered the expression of resistance-related, signaling, salt-sensitive, heavy metal-related, and transporter genes. These findings provide novel insights into the core genes involved in plant PTI and are expected to facilitate the identification and application of resistance genes in plant breeding studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus 1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer 3 . Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life 4-8 . FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Testing effector knockout strains of the Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) for reduced in planta growth in their native kiwifruit host revealed a number of nonredundant effectors that contribute to Psa3 virulence. Conversely, complementation in the weak kiwifruit pathogen P. syringae pv. actinidifoliorum (Pfm) for increased growth identified redundant Psa3 effectors. Psa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus (ΔEEL; 10 effectors) were significant contributors to bacterial colonisation of the host and were additive in their effects on virulence. Four of the EEL effectors (HopD1a, AvrB2b, HopAW1a and HopD2a) redundantly contribute to virulence through suppression of pattern-triggered immunity (PTI). Important Psa3 effectors include several redundantly required effectors early in the infection process (HopZ5a, HopH1a, AvrPto1b, AvrRpm1a and HopF1e). These largely target the plant immunity hub, RIN4. This comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Pattern recognition receptors (PRRs) are plasma membrane-localised proteins that sense molecular patterns to initiate pattern-triggered immunity (PTI). Receptor-like cytoplasmic kinases (RLCKs) function downstream of PRRs to propagate signal transduction via the phosphorylation of substrate proteins. The identification and characterisation of RLCK-regulated substrate proteins are critical for our understanding of plant immunity. We showed that SHOU4 and SHOU4L are rapidly phosphorylated upon various patterns elicitation and are indispensable for plant resistance to bacterial and fungal pathogens. Protein-protein interaction and phosphoproteomic analysis revealed that BOTRYTIS-INDUCED KINASE 1, a prominent protein kinase of RLCK subfamily VII (RLCK-VII), interacted with SHOU4/4L and phosphorylated multiple serine residues on SHOU4L N-terminus upon pattern flg22 treatment. Neither phospho-dead nor phospho-mimic SHOU4L variants complemented pathogen resistance and plant development defect of the loss-of-function mutant, suggesting that reversible phosphorylation of SHOU4L is critical to plant immunity and plant development. Co-immunoprecipitation data revealed that flg22 induced SHOU4L dissociation from cellulose synthase 1 (CESA1) and that a phospho-mimic SHOU4L variant inhibited the interaction between SHOU4L and CESA1, indicating the link between SHOU4L-mediated cellulose synthesis and plant immunity. This study thus identified SHOU4/4L as new components of PTI and preliminarily revealed the mechanism governing SHOU4L regulation by RLCKs., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

The innate immune system detects pathogens via germline-encoded receptors that bind to conserved pathogen ligands called pathogen-associated molecular patterns (PAMPs). Here we consider an additional strategy of pathogen sensing called effector-triggered immunity (ETI). ETI involves detection of pathogen-encoded virulence factors, also called effectors. Pathogens produce effectors to manipulate hosts to create a replicative niche and/or block host immunity. Unlike PAMPs, effectors are often diverse and rapidly evolving and can thus be unsuitable targets for direct detection by germline-encoded receptors. Effectors are instead often sensed indirectly via detection of their virulence activities. ETI is a viable strategy for pathogen sensing and is used across diverse phyla, including plants, but the molecular mechanisms of ETI are complex compared to simple receptor/ligand-based PAMP detection. Here we survey the mechanisms and functions of ETI, with a particular focus on emerging insights from animal studies. We suggest that many examples of ETI may remain to be discovered, hiding in plain sight throughout immunology.

Plants rely on innate immune systems to defend against a wide variety of biotic attackers. Key components of innate immunity include cell-surface pattern-recognition receptors (PRRs), which recognize pest- and pathogen-associated molecular patterns (PAMPs). Unlike other classes of receptors that often have visible cell-death immune outputs upon activation, PRRs generally lack rapid methods for assessing function. Here, we describe a genetically encoded bioluminescent reporter of immune activation by heterologously expressed PRRs in the model organism Nicotiana benthamiana . We characterized N. benthamiana transcriptome changes in response to Agrobacterium tumefaciens and subsequent PAMP treatment to identify pattern-triggered immunity (PTI)-associated marker genes, which were then used to generate promoter-luciferase fusion fungal bioluminescence pathway (FBP) constructs. A reporter construct termed pFBP_2xNbLYS1::LUZ allows for robust detection of PTI activation by heterologously expressed PRRs. Consistent with known PTI signaling pathways, reporter activation by receptor-like protein (RLP) PRRs is dependent on the known adaptor of RLP PRRs, i.e., SOBIR1. The FBP reporter minimizes the amount of labor, reagents, and time needed to assay function of PRRs and displays robust sensitivity at biologically relevant PAMP concentrations, making it ideal for high throughput screens. The tools described in this paper will be powerful for investigations of PRR function and characterization of the structure-function of plant cell-surface receptors. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability 1 , which has been linked to the action of endonucleases such as XPG 2-4 . However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7 ), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref.  8 ), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref.  9 ) and in BRCA1-mutated cancer cells 10 . These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors 1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence 3 . However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor-TCP21-which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Polyamines are small polycationic amines whose levels increase during defense. Previous studies support the contribution of the polyamine spermine to defense responses. However, the potential contribution of spermine to pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) has not been completely established. Here, we compared the contribution of spermine and putrescine to early and late PTI responses in Arabidopsis. We found that putrescine and spermine have opposite effects on PAMP-elicited reactive oxygen species (ROS) production, with putrescine increasing and spermine lowering the flg22-stimulated ROS burst. Through genetic and pharmacological approaches, we found that the inhibitory effect of spermine on flg22-elicited ROS production is independent of polyamine oxidation, nitric oxide, and salicylic acid signaling but resembles chemical inhibition of RBOHD (RESPIRATORY BURST OXIDASE HOMOLOG D). Spermine can also suppress ROS elicited by FLS2-independent but RBOHD-dependent pathways, thus pointing to compromised RBOHD activity. Consistent with this, we found that spermine but not putrescine dampens flg22-stimulated cytosolic Ca2+ influx. Finally, we found that both polyamines differentially reshape transcriptional responses during PTI and disease resistance to Pseudomonas syringae. Overall, we provide evidence for the differential contributions of putrescine and spermine to PTI, with an impact on plant defense., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae .