Genetic analysis of MAMP-triggered immunity in Arabidopsis

In their natural environment, plants live in a close association with a large variety of microorganisms. A number of these microorganisms can be detrimental to plants and are considered as potential pathogens. In order to ward off these pathogens, plants have developed a highly effective and dynamic immune system. As a first line of defense, plants recognize the presence of microbes through the perception of molecular structures typical of a microbial class, termed microbe-associated molecular patterns (MAMPs). In Arabidopsis, the Leu-rich repeat receptor-like kinases FLS2 and EFR recognize the bacterial MAMPs flagellin and EF-Tu (and their bioactive epitopes flg22 and elf18), respectively. Perception of these MAMPs triggers defense responses that restrict microbial invasion and growth. However, the molecular basis of MAMP-triggered immunity (MTI) is still largely unknown. As MTI functionally links to and provides an evolutionary basis for different branches of plant immunity, it is instrumental for the understanding of plant-microbe interactions. The work presented here aimed at the identification of molecular components of MTI. A forward-genetic screen revealed priority in sweet life (psl) mutants that show de-repressed anthocyanin accumulation in the presence of elf18 or flg22. PSL2 was identified as the single-copy Arabidopsis UDP-glucose:glycoprotein glucosyltranseferase (UGGT), whereas PSL25 most likely identifies the Arabidopsis endoplasmic reticulum (ER) Glucosidase I. These are components of an ER protein quality control (ERQC) pathway that ensures proper folding and maturation of membrane-resident and secreted proteins. These and other ERQC components are required for the generation of functional EFR. PSL36 was identified as a novel allele of EIN2 (ETHYLENE INSENSITIVE2), a central regulator of the ethylene(ET) pathway. Loss of EIN2 function results in pronounced defects in FLS2 and EFR signaling outputs. Whereas ET signaling is crucial for FLS2 expression, EFR steady-state levels are unaltered in ein2 plants. These data point to a role for ET in post-recognition signaling by EFR. The identification of a set of EFR-triggered genes that depend on ET-signaling for their full activation reveals possible mechanisms of signal integration during MTI

Structure-function analysis of the <em>Fusarium oxysporum</em> Avr2 effector allows uncoupling of its immune-suppressing activity from recognition

Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I-2 carrying tomato (Solanum lycopersicum). Fol strains that evade I-2 recognition carry point mutations in Avr2 (e.g. Avr2R45H), but retain full virulence. Here we investigate the virulence function of Avr2 and determine its crystal structure. Transgenic tomato and Arabidopsis expressing either wild-type ΔspAvr2 (deleted signal-peptide) or the ΔspAvr2R45H variant become hypersusceptible to fungal, and even bacterial infections, suggesting that Avr2 targets a conserved defense mechanism. Indeed, Avr2 transgenic plants are attenuated in immunity-related readouts, including flg22-induced growth inhibition, ROS production and callose deposition. The crystal structure of Avr2 reveals that the protein shares intriguing structural similarity to ToxA from the wheat pathogen Pyrenophora tritici-repentis and to TRAF proteins. The I-2 resistance-breaking Avr2V41M, Avr2R45H and Avr2R46P variants cluster on a surface-presented loop. Structure-guided mutagenesis enabled uncoupling of virulence from I-2-mediated recognition. We conclude that I-2-mediated recognition is not based on monitoring Avr2 virulence activity, which includes suppression of immune responses via an evolutionarily conserved effector target, but by recognition of a distinct epitope

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues

Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele

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 gIIα 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

Receptor quality control in the endoplasmic reticulum for plant innate immunity

Pattern recognition receptors in eukaryotes initiate defence responses on detection of microbe-associated molecular patterns shared by many microbe species. The Leu-rich repeat receptor-like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor-Tu, respectively. We describe Arabidopsis ‘priority in sweet life' (psl) mutants that show de-repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP-glucose:glycoprotein glycosyltransferase that act in concert with STT3A-containing oligosaccharyltransferase complex in an N-glycosylation pathway in the endoplasmic reticulum. However, EFR-signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid-induced, but EFR-independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N-glycosylation for different layers of plant immunity, likely through quality control of membrane-localized regulators

Pathogen‐induced pH changes regulate the growth‐defense balance in plants

Environmental adaptation of organisms relies on fast perception and response to external signals, which lead to developmental changes. Plant cell growth is strongly dependent on cell wall remodeling. However, little is known about cell wall‐related sensing of biotic stimuli and the downstream mechanisms that coordinate growth and defense responses. We generated genetically encoded pH sensors to determine absolute pH changes across the plasma membrane in response to biotic stress. A rapid apoplastic acidification by phosphorylation‐based proton pump activation in response to the fungus Fusarium oxysporum immediately reduced cellulose synthesis and cell growth and, furthermore, had a direct influence on the pathogenicity of the fungus. In addition, pH seems to influence cellulose structure. All these effects were dependent on the COMPANION OF CELLULOSE SYNTHASE proteins that are thus at the nexus of plant growth and defense. Hence, our discoveries show a remarkable connection between plant biomass production, immunity, and pH control, and advance our ability to investigate the plant growth‐defense balance.ISSN:0261-4189ISSN:1460-207

MIK2 controls root angle in a THE1- and cellulose synthase complex-dependent manner.

<p>(A-D) Nine-day-old Arabidopsis seedlings grown in an upright position (under a 10° angle relative to the direction of gravity) on MS agar medium with 1% sucrose. Pictures were taken from the front of the plate. (A-C) The growth medium contained DMSO (mock) (A), 2 nM ISX (B), or 25 μM DCB (C). (A) The white arrow indicates skewing of <i>mik2-1</i> roots relative to the vertical growth axis. (A-D) Root angle was quantified; a positive value indicates skewing to the left, while a negative value indicates skewing to the right. Error bars represent standard error of n = 15 biological replicas. Different letters indicate statistically significant differences between genotypes (ANOVA and Holm-Sidak test (<i>p</i> < 0.05)). The experiments were repeated at least three times with similar results.</p

MIK2 is required for resistance to the fungal root pathogen <i>Fusarium oxysporum</i> in a THE1-independent manner.

<p>(A,B) Percentage of chlorotic leaves per plant (A), and percentage of decayed plants (B) after infection of the roots with <i>F</i>. <i>oxysporum</i> isolate Fo5176. (A) The percentage of chlorotic leaves per plant was counted 10 days after inoculation with <i>F</i>. <i>oxysporum</i> spores. (B) The number of decayed plants was counted 3 weeks after inoculation with <i>F</i>. <i>oxysporum</i> spores. (A,B) The bars represent the average of four independent experiments, each consisting of n = 20–40 plants per genotype. Error bars represent the standard error of n = 4 experiments. Different letters indicate statistically significant differences between genotypes (ANOVA and Holm-Sidak test (<i>p</i> < 0.05)). No disease symptoms were observed on mock-inoculated plants for any of the genotypes (n = 10). (C) Representative pictures of the different genotypes in (A) and (B) after <i>F</i>. <i>oxysporum</i> infection.</p