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SUMMARYPlant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. We reveal a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid (ABA) pathways via a fungal secondary metabolism gene cluster related to sesquiterpene ABA and botrydial (BOT) biosynthesis. ABA-BOT cluster activation during root infection suppresses host nutrient uptake-related genes and changes the mineral contents, suggesting its role in manipulating host nutrition states. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic–mutualistic continuum in fluctuating environments.

In plants, a first layer of inducible immunity is conferred by pattern recognition receptors (PRRs) that bind microbe- and damage-associated molecular patterns to activate pattern-triggered immunity (PTI). PTI is strengthened or followed by another potent form of immunity when intracellular receptors recognize pathogen effectors, termed effector-triggered immunity. Immunity signaling regulators have been reported to influence abiotic stress responses as well, yet the governing principles and mechanisms remain ambiguous. Here, we report that PRRs of a leucine-rich repeat ectodomain also confer salt tolerance in Arabidopsis thaliana, following recognition of cognate ligands such as bacterial flagellin (flg22 epitope) and elongation factor Tu (elf18 epitope), and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires authentic PTI signaling components; namely, the PRR-associated kinases BAK1 and BIK1 and the NADPH oxidase RBOHD. Exposure to salt stress induces the release of Pep precursors, pointing to the involvement of the endogenous immunogenic peptides in developing plant tolerance to high salinity. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following a preexposure to immunogenic patterns. In good accordance, plants challenged with nonpathogenic bacteria also acquired salt tolerance in a manner dependent on PRRs. Our findings provide insight into signaling plasticity underlying biotic or abiotic stress cross-tolerance in plants conferred by PRRs.[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 .

・In Arabidopsis thaliana, PROPEPs and their derived elicitor‐active Pep epitopes provide damage‐associated molecular patterns (DAMPs), which trigger defence responses through cell‐surface receptors PEPR1 and PEPR2. In addition, Pep peptides induce root growth inhibition and root hair formation, however their relationships and coordinating mechanisms are poorly understood. ・Here, we reveal that Pep1‐mediated root hair formation requires PEPR‐associated kinases BAK1/BKK1 and BIK1/PBL1, ethylene, auxin and root hair differentiation regulators, in addition to PEPR2. ・Our analysis on 69 accessions unravels intraspecies variations in Pep1‐induced root hair formation and growth inhibition. The absence of a positive correlation between the two traits suggests their separate regulation and diversification in natural populations of A. thaliana. ・Restricted PEPR2 expression to certain root tissues is sufficient to induce root hair formation and growth inhibition in response to Pep1, indicating the capacity of non‐cell‐autonomous receptor signalling in different root tissues. Of particular note, root hair cell‐specific PEPR2 expression uncouples defence activation from root growth inhibition and root hair formation, suggesting a unique property of root hairs in root defence activation following Pep1 recognition.

Initial exposure of plants to osmotic stress caused by drought, cold, or salinity leads to acclimation, termed acquired tolerance, to subsequent severe stresses. Acquired osmotolerance induced by salt stress is widespread across Arabidopsis (Arabidopsis thaliana) accessions and is conferred by disruption of a nucleotide-binding leucine-rich repeat gene, designated ACQUIRED OSMOTOLERANCE. De-repression of this gene under osmotic stress causes detrimental autoimmunity via ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4). However, the mechanism underlying acquired osmotolerance remains poorly understood. Here, we isolated an acquired osmotolerance-defective mutant (aod13) by screening 30,000 seedlings of an ion beam-mutagenized M2 population of Bu-5, an accession with acquired osmotolerance. We found that AOD13 encodes the dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1), which negatively regulates MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6). Consistently, MPK3/6 activation was greater in aod13 than in the Bu-5 wild-type (WT). The aod13 mutant was sensitive to osmotic stress but tolerant to salt stress. Under osmotic stress, pathogenesis-related genes were strongly induced in aod13 but not in the Bu-5 WT. Loss of PAD4 in pad4 aod13 plants did not restore acquired osmotolerance, implying that activation of immunity independent of PAD4 renders aod13 sensitive to osmotic stress. These findings suggest that AOD13 (i.e. MKP1) promotes osmotolerance by suppressing the PAD4-independent immune response activated by MPK3/6.

Plants constantly monitor and cope with the fluctuating environment while hosting a diversity of plant-inhabiting microbes. The mode and outcome of plant-microbe interactions, including plant disease epidemics, are dynamically and profoundly influenced by abiotic factors, such as light, temperature, water and nutrients. Plants also utilize associations with beneficial microbes during adaptation to adverse conditions. Elucidation of the molecular bases for the plant-microbe-environment interactions is therefore of fundamental importance in the plant sciences. Following advances into individual stress signaling pathways, recent studies are beginning to reveal molecular intersections between biotic and abiotic stress responses and regulatory principles in combined stress responses. We outline mechanisms underlying environmental modulation of plant immunity and emerging roles for immune regulators in abiotic stress tolerance. Furthermore, we discuss how plants coordinate conflicting demands when exposed to combinations of different stresses, with attention to a possible determinant that links initial stress response to broad-spectrum stress tolerance or prioritization of specific stress tolerance.

Like animals, plants accommodate a rich diversity of microbes, typically without discernible disease symptoms. How their pathogenesis is prevented in the host remains obscure. Here, we show that the root-infecting fungus Colletotrichum fructicola of the C. gloeosporioides clade (CgE), isolated from field-grown healthy Brassicaceae plants, inhibits growth of pathogenic fungi in Arabidopsis thaliana, in a phosphate status-dependent manner. Loss of host ethylene signaling or phytoalexins, camalexin or indole glucosinolates, however, allows CgE to display pathogenesis, suggesting host contributions to endophytic CgE colonization and benefit. Compared to a closely-related C. gloeosporioides pathogen (CgP), CgE is characterized by genome expansion and >700 fungal genes (4.34%) specifically induced in the host roots when co-inoculated with CgP, including genes related to fungal secondary metabolism. This may underlie antimicrobial tolerance of CgE and its dominance over pathogenic fungi within the host, pointing to a role for fungus-fungus competition in asymptomatic fungal colonization in plants.

Pattern recognition receptors (PRRs) bind microbe- and damage-associated molecular patterns (MAMPs/DAMPs, respectively) to enhance host immunity in animals and plants. Here, we report that PRRs also confer salt tolerance in the model plant Arabidopsis thaliana following recognition of cognate ligands, such as bacterial flagellin and EF-Tu and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires the PRR-associated kinases BAK1 and BIK1, and the NADPH oxidase RBOHD. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following an exposure to immunogenic patterns. In their regulatory DNA sequences, specific binding sites for a subset of WRKY transcription factors are over-represented. Accordingly, PTST requires WRKY40 and WRKY18, which activate salt tolerance-related genes but attenuate pathogen defense-related genes, including the EDS1 immunity activator. PRR signaling leads to sustained WRKY40/WRKY18 accumulation under salt stress and utilizes both WRKYs for salt tolerance. The PRR-WRKY40/WRKY18 module also confers salt tolerance after challenge with non-pathogenic bacteria. Our findings give molecular insight into signaling plasticity underlying biotic-abiotic stress cross-tolerance in plants conferred by PRRs.

Plants, as sessile organisms, are often exposed to combined biotic stressors such as herbivores and pathogens, and abiotic stressors such as adverse climate and soil conditions. The relationships between different biotic and abiotic stresses in shaping plant responses and performance are diverged in a context-dependent manner. Trade-off is often seen in plant adaptation between different biotic and abiotic stresses, however, an initial exposure to one stress often leads to an enhanced state of the tolerance to different stresses, designated cross-tolerance. Plants often prioritize their response to one stress over that to another. This may reflect the allocation of limited resources and the hierarchical interactions between different stress signaling pathways. Here, we review the mode and mechanisms by which plants deal with their exposure to different biotic and abiotic stresses, with a particular focus on how immune responses and abiotic stress responses influence each other.

Recognition of microbe- and danger-associated molecular patterns (MAMPs and DAMPs, respectively) by pattern recognition receptors (PRRs) is central to innate immunity in both plants and animals. The plant PRRs described to date are all cell surface-localized receptors. According to their ligand-binding ectodomains, each PRR engages a specific coreceptor or adaptor kinase in its signaling complexes to regulate defense signaling. With a focus on the coreceptor RLK BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and related SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs), here we review the increasing inventory of BAK1 partners and their functions in plant immunity. We also discuss the significance of autoimmunity triggered by BAK1/SERK4 disintegration in shaping the strategies for attenuation of PRR signaling by infectious microbes and host plants.

Plants live in constantly changing and often unfavorable or stressful environments. Environmental changes induce biotic and abiotic stress, which, in turn, may cause genomic DNA damage. Hence, plants simultaneously suffer abiotic/biotic stress and DNA damage. However, little information is available on the signaling crosstalk that occurs between DNA damage and abiotic/biotic stresses. Arabidopsis thaliana SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) is a pivotal transcription factor that regulates thousands of genes in response to DNA double-strand break (DSB), and we recently reported that SOG1 has a role in immune responses. In the present study, the effects of SOG1 overexpression on the DNA damage and immune responses were examined. Results found that SOG1 overexpression enhances the regulation of numerous downstream genes. Relative to the wild type plants, then, DNA damage responses were observed to be strongly induced. SOG1 overexpression also upregulates chitin (a major components of fungal cell walls) responsive genes in the presence of DSBs, implying that pathogen defense response is activated by DNA damage via SOG1. Further, SOG1 overexpression enhances fungal resistance. These results suggest that SOG1 regulates crosstalk between DNA damage response and the immune response and that plants have evolved a sophisticated defense network to contend with environmental stress.

In mammalian cells, the transcription factor p53 plays a crucial role in transmitting DNA damage signals to maintain genome integrity. However, in plants, orthologous genes for p53 and checkpoint proteins are absent. Instead, the plant-specific transcription factor SUPPRESSOR OF GAMMA RADIATION 1 (SOG1) controls most of the genes induced by gamma irradiation and promotes DNA repair, cell cycle arrest, and stem cell death. Thus far, the genes directly controlled by SOG1 remain largely unknown, limiting the understanding of DNA damage signaling in plants. Here, we conducted a microarray analysis and chromatin immunoprecipitation (ChIP)-sequencing, and identified 146 Arabidopsis genes as direct targets of SOG1. By using the ChIP-sequencing data, we extracted the palindromic motif [CTT(N)7AAG] as a consensus SOG1-binding sequence, which mediates target gene induction in response to DNA damage. Furthermore, DNA damage-triggered phosphorylation of SOG1 is required for efficient binding to SOG1-binding sequence. Comparison between SOG1 and p53 target genes showed that both transcription factors control genes responsible for cell cycle regulation, such as CDK inhibitors, and DNA repair proteins, whereas SOG1 preferentially targets genes involved in homologous recombination. We also found that defense-related genes were enriched in the SOG1 target genes. Consistent with this, SOG1 is required for resistance against the hemi-biotrophic fungus Colletotrichum higginsianum, suggesting that SOG1 has a unique function in controlling immune response. This article is protected by copyright. All rights reserved.

P-type conversion of n−-GaN by Mg-ion implantation was successfully performed using high quality GaN epitaxial layers grown on free-standing low-dislocation-density GaN substrates. These samples showed low-temperature PL spectra quite similar to those observed from Mg-doped MOVPE-grown p-type GaN, consisting of Mg related donor–acceptor pair (DAP) and acceptor bound exciton (ABE) emission. P–n diodes fabricated by the Mg-ion implantation showed clear rectifying I–V characteristics and UV and blue light emissions were observed at forward biased conditions for the first time.

Plants solely rely on innate immunity of each individual cell to deal with a diversity of microbes in the environment. Extracellular recognition of microbe- and host damage-associated molecular patterns leads to the first layer of inducible defenses, termed pattern-triggered immunity (PTI). In plants, pattern recognition receptors (PRRs) described to date are all membrane-associated receptor-like kinases or receptor-like proteins, reflecting the prevalence of apoplastic colonization of plant-infecting microbes. An increasing inventory of elicitor-active patterns and PRRs indicates that a large number of them are limited to a certain range of plant groups/species, pointing to dynamic and convergent evolution of pattern recognition specificities. In addition to common molecular principles of PRR signaling, recent studies have revealed substantial diversification between PRRs in their functions and regulatory mechanisms. This serves to confer robustness and plasticity to the whole PTI system in natural infections, wherein different PRRs are simultaneously engaged and faced with microbial assaults. We review the functional significance and molecular basis of PRR-mediated pathogen recognition and disease resistance, and also an emerging role for PRRs in homeostatic association with beneficial or commensal microbes.

Plant defense against herbivores is modulated by herbivore-associated molecular patterns (HAMPs) from oral secretions (OS) and/or saliva of insects. Furthermore, feeding wounds initiate plant self-damage responses modulated by danger-associated molecular patterns (DAMPs) such as immune defense-promoting plant elicitor peptides (Peps). While temporal and spatial co-existence of both patterns during herbivory implies a possibility of their close interaction, the molecular mechanisms remain undetermined. Here we report that exogenous application of rice (Oryza sativa) peptides (OsPeps) can elicit multiple defense responses in rice cell cultures. Specific activation of OsPROPEP3 gene transcripts in rice leaves by wounding and OS treatments further suggests a possible involvement of the OsPep3 peptide in rice-herbivore interactions. Correspondingly, we found that simultaneous application of OsPep3 and Mythimna loreyi OS significantly amplifies an array of defense responses in rice cells, including mitogen-activated protein kinase activation, and generation of defense-related hormones and metabolites. The induction of OsPROPEP3/4 by OsPep3 points to a positive auto-feedback loop in OsPep signaling which may contribute to additional enhancement of defense signal(s). Finally, the overexpression of the OsPep receptor OsPEPR1 increases the sensitivity of rice plants not only to the cognate OsPeps but also to OS signals. Our findings collectively suggest that HAMP-DAMP signal integration provides a critical step in the amplification of defense signaling in plants.

Osmotic stress caused by drought, salt or cold decreases plant fitness. Acquired stress tolerance defines the ability of plants to withstand stress following an initial exposure1. We found previously that acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana accessions2. Here, we identify ACQOS as the locus responsible for ACQUIRED OSMOTOLERANCE. Of its five haplotypes, only plants carrying group 1 ACQOS are impaired in acquired osmotolerance. ACQOS is identical to VICTR, encoding a nucleotide-binding leucine-rich repeat (NLR) protein3. In the absence of osmotic stress, group 1 ACQOS contributes to bacterial resistance. In its presence, ACQOS causes detrimental autoimmunity, thereby reducing osmotolerance. Analysis of natural variation at the ACQOS locus suggests that functional and non-functional ACQOS alleles are being maintained due to a trade-off between biotic and abiotic stress adaptation. Thus, polymorphism in certain plant NLR genes might be influenced by competing environmental stresses.

Recognition of molecules typical of microbes or aberrant cellular states, termed microbe- or danger-associated molecular patterns (MAMPs/DAMPs), respectively, provides an important step in plant and animal innate immunity. In plants, pattern recognition receptors (PRRs) identified to date are limited to membrane-associated proteins, of which the majority has an extracellular leucine-rich repeat (LRR) or lysine-motif (LysM) domain. These PRRs undergo quality control (QC) in the Endoplasmic Reticulum (ER) that is dependent on Asn (N)-linked glycosylation (Glc

Stomatal movements are crucial for the control of plant water status and protection against pathogens. Assays on epidermal peels revealed that, similar to abscisic acid (ABA), pathogen-associated molecular pattern (PAMP) flg22 requires the AtPIP2;1 aquaporin to induce stomatal closure. Flg22 also induced an increase in osmotic water permeability (Pf) of guard cell protoplasts through activation of AtPIP2;1. The use of HyPer, a genetic probe for intracellular hydrogen peroxide (H2O2), revealed that both ABA and flg22 triggered an accumulation of H2O2 in wild-type but not pip2;1 guard cells. Pretreatment of guard cells with flg22 or ABA facilitated the influx of exogenous H2O2 Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6) were both necessary to flg22-induced Pf and both phosphorylated AtPIP2;1 on Ser121 in vitro. Accumulation of H2O2 and stomatal closure as induced by flg22 was restored in pip2;1 guard cells by a phosphomimetic form (Ser121Asp) but not by a phosphodeficient form (Ser121Ala) of AtPIP2;1. We propose a mechanism whereby phosphorylation of AtPIP2;1 Ser121 by BAK1 and/or OST1 is triggered in response to flg22 to activate its water and H2O2 transport activities. This work establishes a signaling role of plasma membrane aquaporins in guard cells and potentially in other cellular context involving H2O2 signaling.

Recognition of molecules typical of microbes or aberrant cellular states, termed microbe- or danger-associated molecular patterns (MAMPs/DAMPs), respectively, provides an important step in plant and animal innate immunity. In plants, pattern recognition receptors (PRRs) identified to date are limited to membrane-associated proteins, of which the majority has an extracellular leucine-rich repeat (LRR) or lysine-motif (LysM) domain. These PRRs undergo quality control (QC) in the Endoplasmic Reticulum (ER) that is dependent on Asn (N)-linked glycosylation (Glc3Man9GlcNAc2 conjugation) of their extracellular domain. In Arabidopsis, genetic studies have revealed that a subset of these PRRs require an intact N-glycosylation pathway in the ER for their biogenesis and function. Here, we describe methods for immunoblot-based detection of protein glycosylation states in plants.

Recognition of microbial challenges leads to enhanced immunity at both the local and systemic levels. In Arabidopsis, EFR and PEPR1/PEPR2 act as the receptor for the bacterial elongation factor EF-Tu (elf18 epitope) and for the endogenous PROPEP-derived Pep epitopes, respectively. The PEPR pathway has been described to mediate defence signalling following microbial recognition. Here we show that PROPEP2/PROPEP3 induction upon pathogen challenges is robust against jasmonate, salicylate, or ethylene dysfunction. Comparative transcriptome profiling between Pep2- and elf18-treated plants points to co-activation of otherwise antagonistic jasmonate- and salicylate-mediated immune branches as a key output of PEPR signalling. Accordingly, as well as basal defences against hemibiotrophic pathogens, systemic immunity is reduced in pepr1 pepr2 plants. Remarkably, PROPEP2/PROPEP3 induction is essentially restricted to the pathogen challenge sites during pathogen-induced systemic immunity. Localized Pep application activates genetically separable jasmonate and salicylate branches in systemic leaves without significant PROPEP2/PROPEP3 induction. Our results suggest that local PEPR activation provides a critical step in connecting local to systemic immunity by reinforcing separate defence signalling pathways.

Recognition of molecular patterns characteristic of microbes or altered-self leads to immune activation in multicellular eukaryotes. In Arabidopsis thaliana , the leucine-rich-repeat receptor kinases FLAGELLIN-SENSING2 ( FLS2 ) and EF-TU RECEPTOR ( EFR ) recognize bacterial flagellin and elongation factor EF-Tu (and their elicitor-active epitopes flg22 and elf18), respectively. Likewise, PEP1 RECEPTOR1 ( PEPR1 ) and PEPR2 recognize the elicitor-active Pep epitopes conserved in Arabidopsis ELICITOR PEPTIDE PRECURSOR s ( PROPEP s). Here we reveal that loss of ETHYLENE-INSENSITIVE2 ( EIN2 ), a master signaling regulator of the phytohormone ethylene (ET), lowers sensitivity to both elf18 and flg22 in different defense-related outputs. Remarkably, in contrast to a large decrease in FLS2 expression, EFR expression and receptor accumulation remain unaffected in ein2 plants. Genome-wide transcriptome profiling has uncovered an inventory of EIN2-dependent and EFR-regulated genes. This dataset highlights important aspects of how ET modulates EFR-triggered immunity: the potentiation of salicylate-based immunity and the repression of a jasmonate-related branch. EFR requires ET signaling components for PROPEP2 activation but not for PROPEP3 activation, pointing to both ET-dependent and -independent engagement of the PEPR pathway during EFR-triggered immunity. Moreover, PEPR activation compensates the ein2 defects for a subset of EFR-regulated genes. Accordingly, ein2 pepr1 pepr2 plants exhibit additive defects in EFR-triggered antibacterial immunity, compared with ein2 or pepr1 pepr2 plants. Our findings suggest that the PEPR pathway not only mediates ET signaling but also compensates for its absence in enhancing plant immunity.

Dueling for sugars Bacteria thrive on sugar. So do plant cells. Yamada et al. now show how the fight for sugar plays out in the extracellular spaces around plant cells when pathogenic bacteria are invading the plant (see the Perspective by Dodds and Lagudah). In the model plant Arabidopsis , part of the defense response incited by the presence of pathogenic bacteria includes transcriptional and posttranscriptional regulation of sugar transporters. The resulting uptake of monosaccharides from the extracellular space makes life a little bit more difficult for the invading bacteria. Science , this issue p. 1427 ; see also p. 1377

In this chapter we describe methods for long-term preservation of ascomycete genus Colletotrichum species. Colletotrichum species employ a hemibiotrophic infection strategy and cause clear anthracnose diseases on various host plants including the model plant Arabidopsis thaliana. Their infection proceeds in a highly synchronized manner, which is helpful for the dissection of the fungus-plant interactions at the molecular level. Gene engineering methods, including efficient protocols for targeted gene disruption, and whole-genome sequences are available for several Colletotrichum species. Thus, these pathogens provide us with model systems to address the molecular mechanisms underlying hemibiotrophic fungal pathogenicity.We describe how to prepare glycerol stocks or filter paper fungal stocks for long-term preservation of Colletotrichum species. These two methods are easily handled, and provide a stable preservation for at least a few years.

This chapter describes how to apply microbe-associated molecular pattern (MAMP) or damage-associated molecular pattern (DAMP) solutions to Arabidopsis roots to trace defense responses in the root. Plants sense the presence of microbes via the perception of MAMPs or DAMPs by surface-localized pattern recognition receptors. The mechanisms governing plant root immunity are poorly characterized compared with those underlying plant immunity in the leaf, despite the fact that plant roots constantly interact with countless microbes living in soils that carry potential MAMPs and could stimulate the production of plant-derived DAMPs during colonization. To understand how a plant root immune system detects and reacts to the potential sources of a stimulus, we describe a simple method to monitor activation of root immunity upon MAMP/DAMP treatment by measuring relative expression of defense-related genes by RT-qPCR.

After Colletotrichum storage methods in Chapter 23 , we describe here experimental methods for the inoculation of Colletotrichum higginsianum (C.h.) on Arabidopsis leaves. We put a particular focus on the methods for lesion measurements after the drop-inoculation of the leaves and on C.h. biomass measurements in the leaves by RT-qPCR analysis. As an option, we also briefly describe methods for counting C.h. entry ratio.

This chapter describes how to apply microbe-associated molecular pattern (MAMP) or damage-associated molecular pattern (DAMP) solutions to Arabidopsis roots to trace defense responses in the root. Plants sense the presence of microbes via the perception of MAMPs or DAMPs by surface-localized pattern recognition receptors. The mechanisms governing plant root immunity are poorly characterized compared with those underlying plant immunity in the leaf, despite the fact that plant roots constantly interact with countless microbes living in soils that carry potential MAMPs and could stimulate the production of plant-derived DAMPs during colonization. To understand how a plant root immune system detects and reacts to the potential sources of a stimulus, we describe a simple method to monitor activation of root immunity upon MAMP/DAMP treatment by measuring relative expression of defense-related genes by RT-qPCR.

In this chapter we describe methods for long-term preservation of ascomycete genus Colletotrichum species. Colletotrichum species employ a hemibiotrophic infection strategy and cause clear anthracnose diseases on various host plants including the model plant Arabidopsis thaliana. Their infection proceeds in a highly synchronized manner, which is helpful for the dissection of the fungus-plant interactions at the molecular level. Gene engineering methods, including efficient protocols for targeted gene disruption, and whole-genome sequences are available for several Colletotrichum species. Thus, these pathogens provide us with model systems to address the molecular mechanisms underlying hemibiotrophic fungal pathogenicity.We describe how to prepare glycerol stocks or filter paper fungal stocks for long-term preservation of Colletotrichum species. These two methods are easily handled, and provide a stable preservation for at least a few years.

After Colletotrichum storage methods in Chapter 23 , we describe here experimental methods for the inoculation of Colletotrichum higginsianum (C.h.) on Arabidopsis leaves. We put a particular focus on the methods for lesion measurements after the drop-inoculation of the leaves and on C.h. biomass measurements in the leaves by RT-qPCR analysis. As an option, we also briefly describe methods for counting C.h. entry ratio.

Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch.

SummaryPlants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling.

Like in animals, cell surface and intracellular receptors mediate immune recognition of potential microbial intruders in plants. Membrane-localized pattern recognition receptors (PRRs) initiate immune responses upon perception of cognate microbe-associated molecular patterns (MAMPs). MAMP-triggered immunity provides a first line of defence that restricts the invasion and propagation of both adapted and non-adapted pathogens. The Leu-rich repeat (LRR) receptor protein kinases (RKs) define a major class of trans-membrane receptors in plants, of which some members are engaged in MAMP recognition and/or defence signalling. The endoplasmic reticulum (ER) quality control (QC) systems monitor N-glycosylation and folding states of the extracellular, ligand-binding LRR domains of LRR-RKs. Recent progress reveals a critical role of evolutionarily conserved ERQC components for different layers of plant immunity. N-glycosylation appears to play a role in ERQC fidelity rather than in ligand binding of LRR-RKs. Moreover, even closely related PRRs show receptor-specific requirements for N-glycosylation. These findings are reminiscent of the earlier defined function of the cytosolic chaperon complex for LRR domain-containing intracellular immune receptors. QC of the LRR domains might provide a basis not only for the maintenance but also for diversification of recognition specificities for immune receptors in plants.

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR(N143Q) lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR(N143Q) in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.

Recognition of microbe-associated molecular patterns (MAMPs), conserved structures typical of a microbial class, triggers immune responses in eukaryotes. This is accompanied by a diverse set of physiological responses that are thought to enhance defense activity in plants. However, the extent and mechanisms by which MAMP-induced events contribute to host immunity are poorly understood. Here we reveal Arabidopsis priority in sweet life4 ( psl4 ) and psl5 mutants that are insensitive to the bacterial elongation factor (EF)-Tu epitope elf18 but responsive to flagellin epitope flg22. PSL4 and PSL5, respectively, identify β- and α-subunits of endoplasmic reticulum-resident glucosidase II, which is essential for stable accumulation and quality control of the elf18 receptor EFR but not the flg22 receptor FLS2. We notice that EFR signaling is partially and differentially impaired without a significant decrease of the receptor steady-state levels in 2 weakly dysfunctional gII α alleles, designated psl5 -1 and rsw3 . Remarkably, rsw3 plants exhibit marked supersusceptibility against a virulent bacterial phytopathogen despite nearly intact coactivation of MAPKs, reactive oxygen species, ethylene biosynthesis, and callose deposition in response to elf18, demonstrating that these signaling outputs alone are insufficient to mount effective immunity. However, rsw3 plants fail to maintain high transcript levels of defense-promoting WRKY , PR1, and PR2 genes at late time points (4 to 24 h) after elf18 elicitation. This points to an unexpected separation between initial and sustained activation of EFR-mediated signaling in the absence of proper glucosidase II-mediated endoplasmic reticulum quality control. Our findings strongly suggest the importance of sustained MAMP receptor signaling as a key step in the establishment of robust immunity.

Pathogenic bacteria inject a diverse set of effectors into eukaryotic host cells and manipulate the cellular environment to enhance bacterial fitness. There is mounting evidence that the transcription machinery in the nucleus of plant cells is a target of various bacterial effectors. These effectors seem to mimic the actions of host nuclear components. To monitor and counteract such virulence-promoting strategies, plants have acquired unique immune-sensing mechanisms.

Fine tuning of light signaling is crucial to plant development. Following light-triggered nuclear translocation, the photoreceptor phytochrome A (phyA) regulates gene expression under continuous far-red light and is rapidly destabilized upon red light irradiation by E3 ubiquitin ligases, including COP1. Here we provide evidence that the light signaling repressors SPA proteins contribute to COP1-mediated phyA degradation and that a COP1/SPA1 protein complex is tightly associated with phyA ubiquitination activity. Furthermore, a phosphorylated phyA form accumulates in the nucleus and preferentially associates with the COP1/SPA1 complex. In contrast, underphosphorylated phyA predominantly associates with the phyA-signaling intermediates FHY3 and FHY1. However, COP1 associates with underphosphorylated phyA in the absence of FHY3 or FHY1, suggesting that phyA associations with FHY3 and FHY1 protect underphosphorylated phyA from being recognized by the COP1/SPA complex. We propose that light-induced phyA phosphorylation acts as a switch controlling differential interactions of the photoreceptor with signal propagation or attenuation machineries.

Pathogens infect a host by suppressing defense responses induced upon recognition of microbe‐associated molecular patterns (MAMPs). Despite this suppression, MAMP receptors mediate basal resistance to limit host susceptibility, via a process that is poorly understood. The Arabidopsis leucine‐rich repeat (LRR) receptor kinase BAK1 associates and functions with different cell surface LRR receptors for a wide range of ligands, including MAMPs. We report that BAK1 depletion is linked to defense activation through the endogenous PROPEP peptides (Pep epitopes) and their LRR receptor kinases PEPR1/PEPR2, despite critical defects in MAMP signaling. In bak1‐knockout plants, PEPR elicitation results in extensive cell death and the prioritization of salicylate‐based defenses over jasmonate‐based defenses, in addition to elevated proligand and receptor accumulation. BAK1 disruption stimulates the release of PROPEP3, produced in response to Pep application and during pathogen challenge, and renders PEPRs necessary for basal resistance. These findings are biologically relevant, since specific BAK1 depletion coincides with PEPR‐dependent resistance to the fungal pathogen Colletotrichum higginsianum. Thus, the PEPR pathway ensures basal resistance when MAMP‐triggered defenses are compromised by BAK1 depletion.

Tandem affinity purification (TAP) strategies constitute an efficient approach for protein complex purification from many different organisms. However, the application of such strategies for purifying endogenous Arabidopsis multi-protein complexes has not yet been reported. Here, we describe an alternative TAP (TAPa) system that successfully allows protein complex purification from Arabidopsis. In our newly generated TAPa tag we have replaced the tobacco etch virus (TEV) protease cleavage site with the more specific and low-temperature active rhinovirus 3C protease site. In addition, the second purification step can now be performed through two different affinity tags: a six His repeat or nine copies of a myc repeat. To examine our purification procedure we generated a C-terminal fusion between the TAPa tag and CSN3, a component of the multi-protein COP9 signalosome (CSN) complex. Subsequent analysis showed that CSN3-TAPa could rescue a csn3 mutant, and that the components of the CSN complex could be co-purified with CSN3-TAPa. As part of our long running interest in light signaling in Arabidopsis we have generated Arabidopsis transgenic lines harboring, both N-terminal and C-terminal TAPa fusions of many different light signaling pathway regulators. Molecular characterization of these transgenic lines showed fusion expression in 88% of the genes analyzed and that this expression is largely independent of the fusion orientation. Mutant complementation analysis showed that most of the TAPa fusions analyzed retained function of the wild-type proteins. Taken together, the data demonstrate the suitability of the TAPa system to allow efficient multi-protein complex isolation from stably transformed Arabidopsis.

COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to repress photomorphogenesis. Here, we demonstrate that COP10 interacts with ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in vitro, an activity distinct from previous characterized UEVs such as MMS2 and UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically interacts with COP1 and the CSN. Purified CDD (COP10, DDB1, DET1) complex also shows enhancement of E2 activity (UEA) similar to that observed with COP10 itself. Our data suggests that COP10, along with COP1 and the CSN, promotes the degradation of positive regulators of photomorphogenesis, such as the transcription factor HY5, via the ubiquitin/26S proteasome system. Thus, the CDD complex may act as a ubiquitylation-promoting factor to regulate photomorphogenesis.