Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity.

Transcriptional corepressors of the Topless (TPL) family regulate plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of the TPL family roles in transcriptional regulation. Chromatin immunoprecipitation with sequencing (ChIP-Seq) was performed on Arabidopsis thaliana lines expressing GFP-tagged Topless-related 1 (TPR1-GFP) with and without constitutive immunity via Enhanced Disease Susceptibility 1 (EDS1). RNA-Seq profiling of the TPR1-GFP lines and pathogen-infected tpl/tpr mutants, combined with measuring immunity, growth, and physiological parameters was employed to investigate TPL/TPR roles in immunity and defense homeostasis. TPR1 was enriched at promoter regions of c. 1400 genes and c. 10% of the detected binding required EDS1 immunity signaling. In a tpr1 tpl tpr4 (t3) mutant, resistance to bacteria was slightly compromised, and defense-related transcriptional reprogramming was weakly reduced or enhanced, respectively, at early (< 1 h) and late 24 h stages of bacterial infection. The t3 plants challenged with bacteria or pathogen-associated molecular pattern nlp24 displayed photosystem II dysfunctions. Also, t3 plants were hypersensitive to phytocytokine pep1 at the level of root growth inhibition. Transgenic expression of TPR1 rescued these t3 physiological defects. We propose that TPR1 and TPL family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity.

Cold Exposure Memory Reduces Pathogen Susceptibility in Arabidopsis Based on a Functional Plastid Peroxidase System.

Chloroplasts serve as cold priming hubs modulating the transcriptional response of Arabidopsis thaliana to a second cold stimulus for several days by postcold accumulation of thylakoid ascorbate peroxidases (tAPX). In an attempt to investigate cross-priming effects of cold on plant pathogen protection, we show here that such a single 24-h cold treatment at 4°C decreased the susceptibility of Arabidopsis to virulent Pseudomonas syringae pv. tomato DC3000 but did not alter resistance against the avirulent P. syringae pv. tomato avRPM1 and P. syringae pv. tomato avrRPS4 strains or the effector-deficient P. syringae pv. tomato strain hrcC-. The effect of cold priming against P. syringae pv. tomato was active immediately after cold exposure and memorized for at least 5 days. The priming benefit was established independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) or activation of the immune-related genes NHL10, FRK1, ICS1 and PR1 but required thylakoid-bound as well as stromal ascorbate peroxidase activities because the effect was absent or weak in corresponding knock-out-lines. Suppression of tAPX postcold regulation in a conditional-inducible tAPX-RNAi line led to increased bacterial growth numbers. This highlights that the plant immune system benefits from postcold regeneration of the protective chloroplast peroxidase system.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility.

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogen's advantage. One class of oomycete effectors share a conserved 'RxLR' motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens.

Arabidopsis thaliana DM2h (R8) within the Landsberg RPP1-like Resistance Locus Underlies Three Different Cases of EDS1-Conditioned Autoimmunity.

Plants have a large panel of nucleotide-binding/leucine rich repeat (NLR) immune receptors which monitor host interference by diverse pathogen molecules (effectors) and trigger disease resistance pathways. NLR receptor systems are necessarily under tight control to mitigate the trade-off between induced defenses and growth. Hence, mis-regulated NLRs often cause autoimmunity associated with stunting and, in severe cases, necrosis. Nucleocytoplasmic ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) is indispensable for effector-triggered and autoimmune responses governed by a family of Toll-Interleukin1-Receptor-related NLR receptors (TNLs). EDS1 operates coincidently or immediately downstream of TNL activation to transcriptionally reprogram cells for defense. We show here that low levels of nuclear-enforced EDS1 are sufficient for pathogen resistance in Arabidopsis thaliana, without causing negative effects. Plants expressing higher nuclear EDS1 amounts have the genetic, phenotypic and transcriptional hallmarks of TNL autoimmunity. In a screen for genetic suppressors of nuclear EDS1 autoimmunity, we map multiple, independent mutations to one gene, DM2h, lying within the polymorphic DANGEROUS MIX2 cluster of TNL RPP1-like genes from A. thaliana accession Landsberg erecta (Ler). The DM2 locus is a known hotspot for deleterious epistatic interactions leading to immune-related incompatibilities between A. thaliana natural accessions. We find that DM2hLer underlies two further genetic incompatibilities involving the RPP1-likeLer locus and EDS1. We conclude that the DM2hLer TNL protein and nuclear EDS1 cooperate, directly or indirectly, to drive cells into an immune response at the expense of growth. A further conclusion is that regulating the available EDS1 nuclear pool is fundamental for maintaining homeostatic control of TNL immune pathways.

NOD-like receptor cooperativity in effector-triggered immunity.

Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are basic elements of innate immunity in plants and animals. Whereas animal NLRs react to conserved microbe- or damage-associated molecular patterns, plant NLRs intercept the actions of diverse pathogen virulence factors (effectors). In this review, we discuss recent genetic and molecular evidence for functional NLR pairs, and discuss the significance of NLR self-association and heteromeric NLR assemblies in the triggering of downstream signaling pathways. We highlight the versatility and impact of cooperating NLR pairs that combine pathogen sensing with the initiation of defense signaling in both plant and animal immunity. We propose that different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and enhancing the host's pathogen recognition spectrum to keep pace with rapidly evolving microbial populations.

Elevated salicylic acid levels conferred by increased expression of ISOCHORISMATE SYNTHASE 1 contribute to hyperaccumulation of SUMO1 conjugates in the Arabidopsis mutant early in short days 4.

Post-translational modification of proteins by attachment of small ubiquitin-like modifier (SUMO) is essential for plant growth and development. Mutations in the SUMO protease early in short days 4 (ESD4) cause hyperaccumulation of conjugates formed between SUMO and its substrates, and phenotypically are associated with extreme early flowering and impaired growth. We performed a suppressor mutagenesis screen of esd4 and identified a series of mutants called suppressor of esd4 (sed), which delay flowering, enhance growth and reduce hyperaccumulation of SUMO conjugates. Genetic mapping and genome sequencing indicated that one of these mutations (sed111) is in the gene salicylic acid induction-deficient 2 (SID2), which encodes ISOCHORISMATE SYNTHASE I, an enzyme required for biosynthesis of salicylic acid (SA). Analyses showed that compared with wild-type plants, esd4 contains higher levels of SID2 mRNA and about threefold more SA, whereas sed111 contains lower SA levels. Other sed mutants also contain lower SA levels but are not mutant for SID2, although most reduce SID2 mRNA levels. Therefore, higher SA levels contribute to the small size, early flowering and elevated SUMO conjugate levels of esd4. Our results support previous data indicating that SUMO homeostasis influences SA biosynthesis in wild-type plants, and also demonstrate that elevated levels of SA strongly increase the abundance of SUMO conjugates.

A TIR-NBS protein encoded by Arabidopsis Chilling Sensitive 1 (CHS1) limits chloroplast damage and cell death at low temperature.

Survival of plants at low temperature depends on mechanisms for limiting physiological damage and maintaining growth. We mapped the chs1-1 (chilling sensitive1-1) mutation in Arabidopsis accession Columbia to the TIR-NBS gene At1g17610. In chs1-1, a single amino acid exchange at the CHS1 N-terminus close to the conserved TIR domain creates a stable mutant protein that fails to protect leaves against chilling stress. The sequence of another TIR-NBS gene (At5g40090) named CHL1 (CHS1-like 1) is related to that of CHS1. Over-expression of CHS1 or CHL1 alleviates chilling damage and enhances plant growth at moderate (24°C) and chilling (13°C) temperatures, suggesting a role for both proteins in growth homeostasis. chs1-1 mutants show induced salicylic acid production and defense gene expression at 13°C, indicative of autoimmunity. Genetic analysis of chs1-1 in combination with defense pathway mutants shows that chs1-1 chilling sensitivity requires the TIR-NBS-LRR and basal resistance regulators encoded by EDS1 and PAD4 but not salicylic acid. By following the timing of metabolic, physiological and chloroplast ultrastructural changes in chs1-1 leaves during chilling, we have established that alterations in photosynthetic complexes and thylakoid membrane integrity precede leaf cell death measured by ion leakage. At 24°C, the chs1-1 mutant appears normal but produces a massive necrotic response to virulent Pseudomonas syringae pv. tomato infection, although this does not affect bacterial proliferation. Our results suggest that CHS1 acts at an intersection between temperature sensing and biotic stress pathway activation to maintain plant performance over a range of conditions.

Cold priming on pathogen susceptibility in the Arabidopsis eds1 mutant background requires a functional stromal Ascorbate Peroxidase.

24 h cold exposure (4°C) is sufficient to reduce pathogen susceptibility in Arabidopsis thaliana against the virulent Pseudomonas syringae pv. tomato (Pst) strain even when the infection occurs five days later. This priming effect is independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the immune-compromised eds1-2 null mutant. In contrast, cold priming-reduced Pst susceptibility is strongly impaired in knock-out lines of the stromal and thylakoid ascorbate peroxidases (sAPX/tAPX) highlighting their relevance for abiotic stress-related increased immune resilience. Here, we extended our analysis by generating an eds1 sapx double mutant. eds1 sapx showed eds1-like resistance and susceptibility phenotypes against Pst strains containing the effectors avrRPM1 and avrRPS4. In comparison to eds1-2, susceptibility against the wildtype Pst strain was constitutively enhanced in eds1 sapx. Although a prior cold priming exposure resulted in reduced Pst titers in eds1-2, it did not alter Pst resistance in eds1 sapx. This demonstrates that the genetic sAPX requirement for cold priming of basal plant immunity applies also to an eds1 null mutant background.

The role of Arabidopsis heterotrimeric G-protein subunits in MLO2 function and MAMP-triggered immunity.

Heterotrimeric G-proteins, composed of Gα, Gß, and Gγ subunits, regulate many fundamental processes in plants. In animals, ligand binding to seven transmembrane (7TM) cell surface receptors designated G-protein coupled receptors (GPCR) leads to heterotrimeric G-protein activation. Because the plant G-protein complex is constitutively active, the exact role of plant 7TM proteins in this process is unclear. Members of the mildew resistance locus O (MLO) family represent the best-characterized 7TM plant proteins. Although genetic ablation of either MLO2 or G-proteins alters susceptibility to pathogens in Arabidopsis thaliana, it is unknown whether G-proteins directly couple signaling through MLO2. Here, we exploited two well-documented phenotypes of mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition in leaf mesophyll cells, to assess the relationship of MLO2 proteins to the G-protein complex. Although our data reveal modulation of antifungal defense responses by Gß and Gγ subunits and a role for the Gγ1 subunit in mlo2-conditioned callose deposition, our findings overall are inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants lacking the Gß subunit show delayed accumulation of a subset of defense-associated genes following exposure to the microbe-associated molecular pattern flg22. Moreover, Gß mutants were found to be hypersusceptible to spray inoculation with the bacterial pathogen Pseudomonas syringae.

Cytokinins mediate resistance against Pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling.

Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated cytokinin synthesis in response to pathogen infection showed that cytokinins mediate enhanced resistance against the virulent hemibiotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous cytokinin production and by exogenous supply of adenine- and phenylurea-derived cytokinins. The cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of cytokinins in the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and cytokinin-producing pathogens and the practical application in agriculture are discussed.

A role for beta-sitosterol to stigmasterol conversion in plant-pathogen interactions.

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana-Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from beta-sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen-inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild-type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild-type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen-associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/beta-sitosterol ratio as whole-leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid-mediated defence signalling but attenuate pathogen-induced expression of the defence regulator flavin-dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to beta-sitosterol ratio in plant membranes.

Determining the ROS and the Antioxidant Status of Leaves During Cold Acclimation.

Cold slows down Calvin cycle activity stronger than photosynthetic electron transport, which supports production of reactive oxygen species (ROS). Even under extreme temperature conditions, most ROS are detoxified by the combined action of low-molecular weight antioxidants and antioxidant enzymes. Subsequent regeneration of the low-molecular weight antioxidants by NAD(P)H and thioredoxin/thiol-dependent pathways relaxes the electron pressure in the photosynthetic electron transport chain. In general, the chloroplast antioxidant system protects plants from severe damage of enzymes, metabolites, and cellular structures by both ROS detoxification and antioxidant recycling. Various methods have been developed to quantify ROS and antioxidant levels in photosynthetic tissues. Here, we summarize a series of exceptionally fast and easily applicable methods that show local ROS accumulation and provide information on the overall availability of reducing sugars, mainly ascorbate, and of thiols.

Reprogramming of plants during systemic acquired resistance.

Genome-wide microarray analyses revealed that during biological activation of systemic acquired resistance (SAR) in Arabidopsis, the transcript levels of several hundred plant genes were consistently up- (SAR(+) genes) or down-regulated (SAR(-) genes) in systemic, non-inoculated leaf tissue. This transcriptional reprogramming fully depended on the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1). Functional gene categorization showed that genes associated with salicylic acid (SA)-associated defenses, signal transduction, transport, and the secretory machinery are overrepresented in the group of SAR(+) genes, and that the group of SAR(-) genes is enriched in genes activated via the jasmonate (JA)/ethylene (ET)-defense pathway, as well as in genes associated with cell wall remodeling and biosynthesis of constitutively produced secondary metabolites. This suggests that SAR-induced plants reallocate part of their physiological activity from vegetative growth towards SA-related defense activation. Alignment of the SAR expression data with other microarray information allowed us to define three clusters of SAR(+) genes. Cluster I consists of genes tightly regulated by SA. Cluster II genes can be expressed independently of SA, and this group is moderately enriched in H2O2- and abscisic acid (ABA)-responsive genes. The expression of the cluster III SAR(+) genes is partly SA-dependent. We propose that SA-independent signaling events in early stages of SAR activation enable the biosynthesis of SA and thus initiate SA-dependent SAR signaling. Both SA-independent and SA-dependent events tightly co-operate to realize SAR. SAR(+) genes function in the establishment of diverse resistance layers, in the direct execution of resistance against different (hemi-)biotrophic pathogen types, in suppression of the JA- and ABA-signaling pathways, in redox homeostasis, and in the containment of defense response activation. Our data further indicated that SAR-associated defense priming can be realized by partial pre-activation of particular defense pathways.

Methyl salicylate production and jasmonate signaling are not essential for systemic acquired resistance in Arabidopsis.

Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway.

Light regulation and daytime dependency of inducible plant defenses in Arabidopsis: phytochrome signaling controls systemic acquired resistance rather than local defense.

We have examined molecular and physiological principles underlying the light dependency of defense activation in Arabidopsis (Arabidopsis thaliana) plants challenged with the bacterial pathogen Pseudomonas syringae. Within a fixed light/dark cycle, plant defense responses and disease resistance significantly depend on the time of day when pathogen contact takes place. Morning and midday inoculations result in higher salicylic acid accumulation, faster expression of pathogenesis-related genes, and a more pronounced hypersensitive response than inoculations in the evening or at night. Rather than to the plants' circadian rhythm, this increased plant defense capability upon day inoculations is attributable to the availability of a prolonged light period during the early plant-pathogen interaction. Moreover, pathogen responses of Arabidopsis double mutants affected in light perception, i.e. cryptochrome1cryptochrome2 (cry1cry2), phototropin1phototropin2 (phot1phot2), and phytochromeAphytochromeB (phyAphyB) were assessed. Induction of defense responses by either avirulent or virulent P. syringae at inoculation sites is relatively robust in leaves of photoreceptor mutants, indicating little cross talk between local defense and light signaling. In addition, the blue-light receptor mutants cry1cry2 and phot1phot2 are both capable of establishing a full systemic acquired resistance (SAR) response. Induction of SAR and salicylic-acid-dependent systemic defense reactions, however, are compromised in phyAphyB mutants. Phytochrome regulation of SAR involves the essential SAR component FLAVIN-DEPENDENT MONOOXYGENASE1. Our findings highlight the importance of phytochrome photoperception during systemic rather than local resistance induction. The phytochrome system seems to accommodate the supply of light energy to the energetically costly increase in whole plant resistance.