A novel fluorescent protein pair facilitates FLIM-FRET analysis of plant immune receptor interaction under native conditions.

Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.

EXTRA LARGE G-PROTEIN2 mediates cell death and hyperimmunity in the chitin elicitor receptor kinase 1-4 mutant.

Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gß, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gß1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).

A Poly(A) Ribonuclease Controls the Cellotriose-Based Interaction between Piriformospora indica and Its Host Arabidopsis.

Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis (Arabidopsis thaliana) roots does not require the BAK1 coreceptor or the putative Ca2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant (cytoplasmiccalcium elevation mutant) impaired in the response to CT and various other cellooligomers (n = 2-7), but not to chitooligomers (n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica-plant mutualistic relationship.

Chitin-induced and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) phosphorylation-dependent endocytosis of Arabidopsis thaliana LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5 (LYK5).

To detect potential pathogens, plants perceive the fungal polysaccharide chitin through receptor complexes containing lysin motif receptor-like kinases (LysM-RLKs). To investigate the ligand-induced spatial dynamics of chitin receptor components, we studied the subcellular behaviour of two Arabidopsis thaliana LysM-RLKs involved in chitin signalling, CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) and LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5. We performed standard and quantitative confocal laser scanning microscopy on stably transformed A. thaliana plants expressing fluorescently tagged CERK1 and LYK5 from their native promoters. Microscopy approaches were complemented by biochemical analyses in plants and in vitro. Both CERK1 and LYK5 localized to the plasma membrane and showed constitutive endomembrane trafficking. After chitin treatment, however, CERK1 remained at the plasma membrane while LYK5 relocalized into mobile intracellular vesicles. Detailed analyses revealed that chitin perception transiently induced the internalization of LYK5 into late endocytic compartments. Plants that lacked CERK1 or expressed an enzymatically inactive CERK1 variant did not exhibit chitin-induced endocytosis of LYK5. CERK1 could phosphorylate LYK5 in vitro and chitin treatment induced CERK1-dependent phosphorylation of LYK5 in planta. Our results suggest that chitin-induced phosphorylation by CERK1 triggers LYK5 internalization. Thus, our work identifies phosphorylation as a key regulatory step in endocytosis of plant RLKs and also provides evidence for receptor complex dissociation after ligand perception.

LYM2-dependent chitin perception limits molecular flux via plasmodesmata.

Chitin acts as a pathogen-associated molecular pattern from fungal pathogens whose perception triggers a range of defense responses. We show that LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN 2 (LYM2), the Arabidopsis homolog of a rice chitin receptor-like protein, mediates a reduction in molecular flux via plasmodesmata in the presence of chitin. For this response, lym2-1 mutants are insensitive to the presence of chitin, but not to the flagellin derivative flg22. Surprisingly, the chitin-recognition receptor CHITIN ELCITOR RECEPTOR KINASE 1 (CERK1) is not required for chitin-induced changes to plasmodesmata flux, suggesting that there are at least two chitin-activated response pathways in Arabidopsis and that LYM2 is not required for CERK1-mediated chitin-triggered defense responses, indicating that these pathways are independent. In accordance with a role in the regulation of intercellular flux, LYM2 is resident at the plasma membrane and is enriched at plasmodesmata. Chitin-triggered regulation of molecular flux between cells is required for defense responses against the fungal pathogen Botrytis cinerea, and thus we conclude that the regulation of symplastic continuity and molecular flux between cells is a vital component of chitin-triggered immunity in Arabidopsis.

Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis.

Diverse stresses such as high salt conditions cause an increase in reactive oxygen species (ROS), necessitating a redox stress response. However, little is known about the signaling pathways that regulate the antioxidant system to counteract oxidative stress. Here, we show that a Glycogen Synthase Kinase3 from Arabidopsis thaliana (ASKα) regulates stress tolerance by activating Glc-6-phosphate dehydrogenase (G6PD), which is essential for maintaining the cellular redox balance. Loss of stress-activated ASKα leads to reduced G6PD activity, elevated levels of ROS, and enhanced sensitivity to salt stress. Conversely, plants overexpressing ASKα have increased G6PD activity and low levels of ROS in response to stress and are more tolerant to salt stress. ASKα stimulates the activity of a specific cytosolic G6PD isoform by phosphorylating the evolutionarily conserved Thr-467, which is implicated in cosubstrate binding. Our results reveal a novel mechanism of G6PD adaptive regulation that is critical for the cellular stress response.

Bikinin-like inhibitors targeting GSK3/Shaggy-like kinases: characterisation of novel compounds and elucidation of their catabolism in planta.

BACKGROUND: Plant GSK-3/Shaggy-like kinases are key players in brassinosteroid (BR) signalling which impact on plant development and participate in response to wounding, pathogens and salt stress. Bikinin was previously identified in a chemical genetics screen as an inhibitor targeting these kinases. To dissect the structural elements crucial for inhibition of GSK-3/Shaggy-like kinases by bikinin and to isolate more potent compounds we synthesised a number of related substances and tested their inhibitory activity in vitro and in vivo using Arabidopsis thaliana. RESULTS: A pyridine ring with an amido succinic acid residue in position 2 and a halogen in position 5 were crucial for inhibitory activity. The compound with an iodine substituent in position 5, denoted iodobikinin, was most active in inhibiting BIN2 activity in vitro and efficiently induced brassinosteroid-like responses in vivo. Its methyl ester, methyliodobikinin, showed improved cell permeability, making it highly potent in vivo although it had lower activity in vitro. HPLC analysis revealed that the methyl residue was rapidly cleaved off in planta liberating active iodobikinin. In addition, we provide evidence that iodobikinin and bikinin are inactivated in planta by conjugation with glutamic acid or malic acid and that the latter process is catalysed by the malate transferase SNG1. CONCLUSION: Brassinosteroids participate in regulation of many aspects of plant development and in responses to environmental cues. Thus compounds modulating their action are valuable tools to study such processes and may be an interesting opportunity to modify plant growth and performance in horticulture and agronomy. Here we report the development of bikinin derivatives with increased potency that can activate BR signalling and mimic BR action. Methyliodobikinin was 3.4 times more active in vivo than bikinin. The main reason for the superior activity of methyliodobikinin, the most potent compound, is its enhanced plant tissue permeability. Inactivation of bikinin and its derivatives in planta involves SNG1, which constitutes a novel pathway for modification of xenobiotic compounds.

A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) mutant with enhanced pathogen-induced cell death and altered receptor processing.

Plants detect pathogens by sensing microbe-associated molecular patterns (MAMPs) through pattern recognition receptors. Pattern recognition receptor complexes also have roles in cell death control, but the underlying mechanisms are poorly understood. Here, we report isolation of cerk1-4, a novel mutant allele of the Arabidopsis chitin receptor CERK1 with enhanced defense responses. We identified cerk1-4 in a forward genetic screen with barley powdery mildew and consequently characterized it by pathogen assays, mutant crosses and analysis of defense pathways. CERK1 and CERK1-4 proteins were analyzed biochemically. The cerk1-4 mutation causes an amino acid exchange in the CERK1 ectodomain. Mutant plants maintain chitin signaling capacity but exhibit hyper-inducible salicylic acid concentrations and deregulated cell death upon pathogen challenge. In contrast to chitin signaling, the cerk1-4 phenotype does not require kinase activity and is conferred by the N-terminal part of the receptor. CERK1 undergoes ectodomain shedding, a well-known process in animal cell surface proteins. Wild-type plants contain the full-length CERK1 receptor protein as well as a soluble form of the CERK1 ectodomain, whereas cerk1-4 plants lack the N-terminal shedding product. Our work suggests that CERK1 may have a chitin-independent role in cell death control and is the first report of ectodomain shedding in plants.

Broad-spectrum suppression of innate immunity is required for colonization of Arabidopsis roots by the fungus Piriformospora indica.

Piriformospora indica is a root-colonizing basidiomycete that confers a wide range of beneficial traits to its host. The fungus shows a biotrophic growth phase in Arabidopsis (Arabidopsis thaliana) roots followed by a cell death-associated colonization phase, a colonization strategy that, to our knowledge, has not yet been reported for this plant. P. indica has evolved an extraordinary capacity for plant root colonization. Its broad host spectrum encompasses gymnosperms and monocotyledonous as well as dicotyledonous angiosperms, which suggests that it has an effective mechanism(s) for bypassing or suppressing host immunity. The results of our work argue that P. indica is confronted with a functional root immune system. Moreover, the fungus does not evade detection but rather suppresses immunity triggered by various microbe-associated molecular patterns. This ability to suppress host immunity is compromised in the jasmonate mutants jasmonate insensitive1-1 and jasmonate resistant1-1. A quintuple-DELLA mutant displaying constitutive gibberellin (GA) responses and the GA biosynthesis mutant ga1-6 (for GA requiring 1) showed higher and lower degrees of colonization, respectively, in the cell death-associated stage, suggesting that P. indica recruits GA signaling to help establish proapoptotic root cell colonization. Our study demonstrates that mutualists, like pathogens, are confronted with an effective innate immune system in roots and that colonization success essentially depends on the evolution of strategies for immunosuppression.

GSK3-mediated phosphorylation of DEK3 regulates chromatin accessibility and stress tolerance in Arabidopsis.

Chromatin dynamics enable the precise control of transcriptional programmes. The balance between restricting and opening of regulatory sequences on the DNA needs to be adjusted to prevailing conditions and is fine-tuned by chromatin remodelling proteins. DEK is an evolutionarily conserved chromatin architectural protein regulating important chromatin-related processes. However, the molecular link between DEK-induced chromatin reconfigurations and upstream signalling events remains unknown. Here, we show that ASKß/AtSK31 is a salt stress-activated glycogen synthase kinase 3 (GSK3) from Arabidopsis thaliana that phosphorylates DEK3. This specific phosphorylation alters nuclear DEK3 protein complex composition and affects nucleosome occupancy and chromatin accessibility that is translated into changes in gene expression, contributing to salt stress tolerance. These findings reveal that DEK3 phosphorylation is critical for chromatin function and cellular stress response and provide a mechanistic example of how GSK3-based signalling is directly linked to chromatin, facilitating a transcriptional response.

The lysin motif receptor-like kinase (LysM-RLK) CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation.

Plants detect potential pathogens by sensing microbe-associated molecular patterns via pattern recognition receptors. In the dicot model plant Arabidopsis, the lysin motif (LysM)-containing chitin elicitor receptor kinase 1 (CERK1) has been shown to be essential for perception of the fungal cell wall component chitin and for resistance to fungal pathogens. Recent in vitro studies with CERK1 protein expressed heterologously in yeast suggested direct chitin binding activity. Here we show in an affinity purification approach that CERK1 is a major chitin-binding protein of Arabidopsis cells, along with several known and putative chitinases. The ectodomain of CERK1 harbors three distinct LysM domains with potential ligand binding capacity. We demonstrate that the CERK1 ectodomain binds chitin and partially deacetylated chitosan directly without any requirement for interacting proteins and that all three LysM domains are necessary for chitin binding. Ligand-induced phosphorylation events are a general feature of animal and plant signal transduction pathways. Our studies show that chitin, chitin oligomers, and chitosan rapidly induce in vivo phosphorylation of CERK1 at multiple residues in the juxtamembrane and kinase domain. Functional analyses with a kinase dead variant provide evidence that kinase activity of CERK1 is required for its chitin-dependent in vivo phosphorylation, as well as for early defense responses and downstream signaling. Collectively, our data suggest that in Arabidopsis, CERK1 is a major chitin, chitosan, and chito-oligomer binding component and that chitin signaling depends on CERK1 post-translational modification and kinase activity.

ASKtheta, a group-III Arabidopsis GSK3, functions in the brassinosteroid signalling pathway.

Brassinosteroids (BRs) are plant hormones that regulate many processes including cell elongation, leaf development, pollen tube growth and xylem differentiation. GSK3/shaggy-like kinases (GSK) are critical regulators of intracellular signalling initiated by the binding of BR to the BRI1 receptor complex. Three GSKs have already been shown to relay BR responses, including phosphorylation of the transcriptional regulator BES1. However, recent studies indicate that one or more yet unidentified protein kinases are involved in BR signalling. Here, we show that the in vivo protein kinase activity of the group-III GSK, ASKtheta, was negatively regulated by BRI1. Arabidopsis thaliana plants with enhanced ASKtheta activity displayed a bri1-like phenotype. ASKtheta overexpressors accumulated high levels of brassinolide, castasterone and typhasterol, and were insensitive to BR. ASKtheta localized to the nucleus and directly phosphorylated BES1 and BZR1. Moreover, the BES1/BZR1-like transcription factor BEH2 was isolated as an ASKtheta interaction partner in a yeast two-hybrid screen. ASKtheta phosphorylated BEH2 both in vitro and in vivo. Overall, these data provide strong evidence that ASKtheta is a novel component of the BR signalling cascade, targeting the transcription factors BES1, BZR1 and BEH2.

Identification of cis- and trans-acting elements in pHW126, a representative of a novel group of rolling circle plasmids.

pHW126, pIGRK, pIGMS31 and pRAO1 are the only known members of a novel and as yet uncharacterised family of rolling circle plasmids. pHW126 contains only two open reading frames, of which one shows homology to pMV158-family mobilisation proteins. Here we provide evidence that the second open reading frame encodes a replication protein (Rep). Mutation or deletion of this gene resulted in replication deficient constructs, but providing functional Rep from a compatible vector rescued these constructs, indicating that Rep acts in trans. An approximately 300 bp cis-acting region representing the origin of replication was identified upstream of the rep gene. The origin was identified to be composed of three parts: an accessory region, a conserved stretch and four perfect tandem repeats. The two latter elements were essential for replication. Constructs with a deletion of the accessory region could still replicate, but their loss rate was high, indicating that the accessory region is necessary for plasmid maintenance under non-selective conditions. Interestingly, pHW126 could replicate in all Enterobacteriaceae tested while Agrobacterium tumefaciens and Pseudomonas syringae were inappropriate hosts. Thus, pHW126 seems to have a rather limited host range.

Mixed Linkage ß-1,3/1,4-Glucan Oligosaccharides Induce Defense Responses in Hordeum vulgare and Arabidopsis thaliana.

Plants detect conserved microbe-associated molecular patterns (MAMPs) and modified "self" molecules produced during pathogen infection [danger associated molecular patterns (DAMPs)] with plasma membrane-resident pattern recognition receptors (PRRs). PRR-mediated MAMP and/or DAMP perception activates signal transduction cascades, transcriptional reprogramming and plant immune responses collectively referred to as pattern-triggered immunity (PTI). Potential sources for MAMPs and DAMPs are microbial and plant cell walls, which are complex extracellular matrices composed of different carbohydrates and glycoproteins. Mixed linkage ß-1,3/1,4-glucan (ß-1,3/1,4-MLG) oligosaccharides are abundant components of monocot plant cell walls and are present in symbiotic, pathogenic and apathogenic fungi, oomycetes and bacteria, but have not been detected in the cell walls of dicot plant species so far. Here, we provide evidence that the monocot crop plant H. vulgare and the dicot A. thaliana can perceive ß-1,3/1,4-MLG oligosaccharides and react with prototypical PTI responses. A collection of Arabidopsis innate immunity signaling mutants and >100 Arabidopsis ecotypes showed unaltered responses upon treatment with ß-1,3/1,4-MLG oligosaccharides suggesting the employment of a so far unknown and highly conserved perception machinery. In conclusion, we postulate that ß-1,3/1,4-MLG oligosaccharides have the dual capacity to act as immune-active DAMPs and/or MAMPs in monocot and dicot plant species.

Frequency and diversity of small cryptic plasmids in the genus Rahnella.

BACKGROUND: Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids. RESULTS: In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified. CONCLUSIONS: For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to different groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the importance of plasmids for lateral gene transfer (including chromosomal sequences) to distinct genera.

Nucleoporin NUP88/MOS7 is required for manifestation of phenotypes associated with the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 mutant cerk1-4.

Arabidopsis nucleoporin MOS7/NUP88 was identified in a forward-genetic screen for components that contribute to auto-immunity of the deregulated Resistance (R) gene mutant snc1, and is required for immunity to biotrophic and hemi-biotrophic pathogens. In a recent study, we showed that MOS7 is also essential to mount a full defense response against the necrotrophic fungal pathogen Botrytis cinerea, suggesting that MOS7 modulates plant defense responses to different types of pathogenic microbes. Here, we extend our analyses of MOS7-dependent plant immune responses and report the genetic requirement of MOS7 for manifestation of phenotypes associated with the CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) mutant cerk1-4.

Live and let die--Arabidopsis nonhost resistance to powdery mildews.

The term "nonhost resistance" (NHR) describes the phenomenon that an entire plant species is resistant to all genetic variants of a non-adapted pathogen species. In nature, NHR represents the most robust form of plant immunity and is therefore of scientific as well as economic importance. Due to its highly complex nature, NHR has previously not been studied in detail. Recently, the establishment of model interaction systems utilizing Arabidopsis and non-adapted powdery mildews allowed the identification of several key components and conceptual conclusions. It is now generally accepted that NHR of Arabidopsis to powdery mildews comprises two distinct layers of defence: pre-invasion entry control at the cell periphery and post-invasion resistance based on cell death execution. The timely production and localised discharge of toxic compounds at sites of fungal attack appear to be pivotal for entry control. This process requires proteins involved in secretion and trans-membrane transport, synthesis and activation of indolic glucosinolates as well as gene regulation and post-translational protein modification. Post-invasion defence relies on lipase-like proteins and salicylic acid signalling. To what extent pathogen-associated molecular pattern- or effector-triggered immunity contribute to NHR remains to be investigated and is likely to depend on the model system studied.

AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants.

Plant innate immunity relies on a set of pattern recognition receptors (PRRs) that respond to ligands known as pathogen-associated molecular patterns (PAMPs). To overcome such immunity, phytopathogenic bacteria deliver virulence molecules called effector proteins into the plant cell that collectively promote pathogenesis. The vast majority of PRRs controlling PAMP-triggered immunity (PTI) and the mechanisms used by specific effectors to suppress these pathways are mostly unknown. Here, we show that the Arabidopsis LysM receptor kinase CERK1, which is critical for chitin elicitor signaling and resistance to fungal pathogens, plays an essential role in restricting bacterial growth on plants. This is supported by the fact that CERK1 is a target of the bacterial type III effector protein AvrPtoB, which blocks all defense responses through this receptor. AvrPtoB ubiquitinates the CERK1 kinase domain in vitro and targets CERK1 for degradation in vivo. We show that CERK1 is a determinant of bacterial immunity, but its contribution is overcome by bacteria expressing AvrPtoB. Our results reveal a new pathway for plant immunity against bacteria and a role for AvrPtoB E3-ligase activity in suppressing PTI.

Isolation and characterization of pHW15, a small cryptic plasmid from Rahnella genomospecies 2.

A small cryptic plasmid designated pHW15 was isolated from Rahnella genomospecies 2 WMR15 and its complete nucleotide sequence was determined. The plasmid contained 3002 bp with a G+C content of 47.4%. The origin of replication was identified by deletion analysis as a region of about 600 bp. This region had an identity of 70% to the replication origin of the ColE1 plasmid at the nucleotide level. Sequence analysis revealed the typical elements: RNA I, RNA II and their corresponding promoters, a sequence allowing hybridisation of RNA II to the DNA and favouring processing by RNaseH, a single-strand initiation determinant (ssi) that allows initiation of lagging-strand synthesis, and a terH sequence required for termination of lagging-strand synthesis. The plasmid contained three expressed open reading frames, one of which showed homology to a ColE1 plasmid-encoded protein. Furthermore, a multimer resolution site was identified by sequence analysis. Its deletion resulted in formation of plasmid multimers during growth leading to an increased plasmid loss rate.

Quantification of free and total salicylic acid in plants by solid-phase extraction and isocratic high-performance anion-exchange chromatography.

Salicylic acid (SA) is an important signaling compound in plants and is involved in various defense responses. Here we report a new method for quantification of free and total soluble SA in Arabidopsis thaliana with 5-fluorosalicylic acid (5-FSA) as internal standard. The SA was isolated from leaf extracts by solid-phase extraction with phenyl-phase cartridges and selectively eluted as the cationic iron(III)-complex. Recoveries of SA and 5-FSA were equal and exceeded 90%. Free SA was subsequently released from the iron(III)-complex by addition of 2,2'-bipyridyl and high-performance anion-exchange chromatography was performed on an NH2 column. The SA appeared as last peak with a retention time of 15 min, baseline-separated from other substances. On-line detection was performed fluorimetrically for both SA and 5-FSA at an excitation wavelength of 300 nm and an emission wavelength of 410 nm, because both substances give similar fluorescence spectra. The detection limit for SA was 5 ng g(-1) FW for a sample size of 100 mg. Thus the main advantages of the method are highly selective sample preparation, increased sensitivity, reduced analysis time compared with reversed-phase HPLC, and use of a novel internal standard detectable under the same conditions as SA. The techniques described are applicable to other plant materials.