Identification of receptor complex components and receptor activation mechanisms in plant innate immunity

Plants rely on an innate immune system which successfully recognizes and restricts pathogenic microbes. The key for this defense is the detection of pathogen derived non-self signatures and endogenous elicitors released during a microbial attack. Here we report the identification of PEPR2, a new receptor for endogenous elicitors in Arabidopsis (chapter 1). Together with its homologue PEPR1 it functions redundantly in the recognition of AtPep1, a plant derived peptide released during wounding and pathogen defense. Our analysis showed that the defense signaling triggered upon AtPep1 stimulation exhibits strong similarity to the response to microbe derived elicitors. For detection of pathogen derived elicitors the flagellin perception through the receptor FLS2 evolved as model system in plants. FLS2 is known to function together with an associated receptor-like kinase referred to as SERK3/BAK1. In an in vitro analysis of the FLS2-kinase and the BAK1-kinase we were able to show, that FLS2 is a substrate for the BAK1-kinase. This indicates that BAK1 acts as upstream kinase, which phosphorylates and activates the receptor upon dimerization (chapter 2). Using a mass spectrometric analysis on immunopurified FLS2 protein we identified one elicitor independent and one elicitor dependent putative phosphorylation site. The position of both sites suggests a role for phosphorylation in the regulation of ubiquitination and endocytosis. We further analyzed the impact of receptor kinase activity by a characterization of a kinase inactive version of the EF-Tu receptor EFR (chapter 3). This analysis verified that also EFR functions through BAK1 and demonstrated that kinase activity of the receptor is not required for formation of the EFR/BAK1 complex. Strikingly, kinase inactive EFR was able to initiate an elicitor dependent ethylene accumulation and conferred partial resistance to Agrobacterium tumefaciens, while other signaling events were absent. This finding revealed a diverging signaling network in which not all pathways require receptor kinase activity to get activated. By immunopurification and subsequent mass spectrometric analysis of FLS2 protein we further explored this signaling system and its components. Importantly we found not only BAK1, but also its paralogues SERK1, SERK2, SERK4 and SERK5 to co-purify with the flagellin receptor, which indicates a redundant function of these proteins (chapter 4). We also identified several isoforms of the family of 14-3-3 general regulating factors. This is in line with an in silico analysis of the FLS2 sequence, which predicted the putative phosphorylation site S 1078 to operate as 14 3 3 protein binding site. Another group of proteins which co-purified with FLS2 in an elicitor dependent manner comprises RAB-GTPases and SNARE proteins. These protein factors are known to control vesicle fusion events. Since bacterial infections trigger focal secretion, we speculate that the elicitor activated FLS2 complex might lead secretory vesicles directly to the site of infection. Taken together this works provides new insight into different levels of plant immunity. This includes not only the identification of a new receptor and receptor associated proteins, but also adds new aspects to our understanding of receptor activation and downstream signaling. Therefore these results provide a basis to further investigate plant innate immunity on the whole

Biochemical and phenotypic analysis of the p21-activated kinase DPAK3 in Drosophila melanogaster

Gegenstand dieser Arbeit ist das Drosophila melanogaster Protein DPAK3, ein Vertreter der hochkonservierten Familie der p21-aktivierten Kinasen (PAK). DPAK3 und seine Homologen aus anderen Insektenarten und C. elegans können aufgrund eines Vergleichs der Proteinsequenz und struktureller Merkmale in eine eigenen Untergruppe 1* innerhalb der Gruppe 1 der PAK-Proteine eingeordnet werden. Das Genom von Drosophila kodiert noch für zwei weitere PAK-Proteine, das zur Gruppe 1 gehörende DPAK1 und das Gruppe 2 PAK-Protein Mbt. Wie die klassischen Gruppe 1 PAK-Proteine bildet DPAK3 im inaktiven Zustand Dimere. DPAK3 interagiert mit den GTP-gebundenen Formen der RhoGTPasen Rac1, Rac2 und Cdc42. Durch die Bindung dieser Proteine geht DPAK3 aus dem dimeren in den monomeren Zustand über und seine Kinaseaktivität wird durch diese Bindung gesteigert. DPAK3 ist für die Ausbildung der korrekten Morphologie kultivierter Drosophila Zellen erforderlich und beeinflußt die Regulation des Aktinzytoskeletts. Weiterhin konnte CK2beta, die regulatorische Untereinheit der Casein Kinase 2, als neuer Regulator von p21-aktivierten Kinasen identifiziert werden. Das Genom von Drosophila besitzt drei Transkriptionseinheiten, die für CK2beta', CK2betatestes und fünf verschiedene Isoformen von CK2beta kodieren. Eine vergleichende Analyse zeigt, daß alle CK2beta-Proteine mit DPAK1, DPAK3 und in geringerem Maß auch mit Mbt interagieren und in der Lage sind, die Aktivität der PAK-Proteine in vitro zu hemmen. Die Bindung von CK2beta an DPAK3 wird, wie bei allen anderen Serin- / Threoninkinasen, die bisher als Interaktionspartner von CK2beta identifiziert wurden, über die Kinasedomäne von DPAK3 vermittelt. Die Bildung des aus zwei katalytischen CK2a und zwei CK2beta Untereinheiten bestehenden CK2-Holoenzyms hängt von der Fähigkeit von CK2beta ab, Dimere zu bilden. Es konnte gezeigt werden, daß die Bildung eines b-b Dimers für die Interaktion mit und Regulation von DPAK3 nicht erforderlich ist. In vivo wurden die bisher bekannten Dpak3 Allele untersucht, wobei kein gesichertes Nullallel identifiziert werden konnte. Durch enzymatisch katalysierte Rekombination wurde eine neue Deletion hergestellt, die das komplette Leseraster von Dpak3 entfernt. Mit Hilfe von genetischen Mosaiken wurde die Rolle von DPAK3 in der Augenentwicklung untersucht. Durch den Verlust der Genfunktion von Dpak3 wird die Ausbildung der korrekten Struktur der Komplexaugen nur leicht beeinträchtigt. Bei der Analyse einer Dpak1 Mutante wurde dasselbe Ergebnis erzielt. Gleichzeitiger Verlust der Genfunktion von Dpak1 und Dpak3 hingegen führt zu massiven strukturellen Defekten. DPAK1 und DPAK3 erfüllen somit zumindest teilweise redundante Funktionen in der Augenentwicklung. Es wird Gegenstand zukünftiger Studien sein müssen, die gemeinsamen und getrennten Funktionen dieser PAK-Proteine in Drosophila aufzuklären.Subject of this work is the Drosophila melanogaster protein DPAK3, a member of the highly conserved family of p21-activated kinases. Based on the comparison of the amino acid sequence and structural features, DPAK3 and its homologues from other insect species and C. elegans can be assigned to a distinct subgroup 1* within the group 1 PAK proteins. The genome of Drosophila encodes for two additional PAK proteins, DPAK1 and Mbt, which belong to the group 1 and group 2 p21-activated kinases, respectively. Like the classical group 1 PAK proteins, DPAK3 forms dimers in its inactive conformation. DPAK3 binds to and is activated by the Rho GTPases Rac1, Rac2 and Cdc42. The interaction with these proteins leads to the disruption of the DPAK3 dimer and an increase in the kinase activity of DPAK3. DPAK3 is necessary for the development of the normal morphology of cultured Drosophila cells and influences the regulation of the actin cytoskeleton. CK2b the regulatory subunit of casein kinase 2 was identified as a new regulator of p21 activated kinases. The genome of Drosophila possesses three different transcriptional units that encode the proteins CK2b', CK2btestes and five different isoforms of CK2b. A comparative analysis shows that all CK2b proteins interact with DPAK1, DPAK3 and Mbt and negatively regulate the activity of these kinases in vitro. CK2b binds to the kinase domain of DPAK3 which is consistent with previous results obtained from other serine/threonine kinases interacting with CK2b. The CK2 holoenzyme consists of two catalytically active CK2a subunits and two regulatory CK2b subunits. My results show that the ability of CK2b to form dimers, which is essential for the formation of the CK2 holoenzyme, is not necessary for the regulation of p21 activated kinases. The analysis of the available Dpak3 alleles in vivo revealed the necessity to create a new bona fide loss of function allele. To accomplish this goal, a new deletion which removes the entire Dpak3 open reading frame was created by enzymatically catalysed recombination. Genetic mosaics were used to study the role of DPAK3 in eye development. The morphology of the complex eyes was only slightly impaired by the loss of Dpak3 function. The same result was obtained when analysing Dpak1 mutants, but removal of the gene function of both Dpak1 and Dpak3 leads to massive structural defects. This shows that Dpak1 and Dpak3 have at least partially redundant functions in eye development. Further studies will be necessary to reveal the common and distinct functions of these p21-activated kinases in Drosophila

The cytochrome bc1 complex inhibitor Ametoctradin has an unusual binding mode

Ametoctradin is an agricultural fungicide that selectively inhibits the cytochrome bc1 complex of oomycetes. Previous spectrophotometric studies using the purified cytochrome bc1 complex from Pythium sp. showed that Ametoctradin binds to the Qo-site of the enzyme. However, as modeling studies suggested a binding mode like that of the substrate ubiquinol, the possibility for a dual Qo- and Qi-site binding mode was left open. In this work, binding studies and enzyme assays with mitochondrial membrane preparations from Pythium sp. and an S. cerevisiae strain with a modified Qi-site were used to investigate further the binding mode of Ametoctradin. The results obtained argue that the compound could bind to both the Qo- and Qi-sites of the cytochrome bc1 complex and that its position or binding pose in the Qi-site differs from that of Cyazofamid and Amisulbrom, the two Qi-site-targeting, anti-oomycetes compounds. Furthermore, the data support the argument that Ametoctradin prefers binding to the reduced cytochrome bc1 complex. Thus, Ametoctradin has an unusual binding mode and further studies with this compound may offer the opportunity to better understand the catalytic cycle of the cytochrome bc1 complex

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

Perception of the Arabidopsis Danger Signal Peptide 1 Involves the Pattern Recognition Receptor AtPEPR1 and Its Close Homologue AtPEPR2*

Plasma membrane-borne pattern recognition receptors, which recognize microbe-associated molecular patterns and endogenous damage-associated molecular patterns, provide the first line of defense in innate immunity. In plants, leucine-rich repeat receptor kinases fulfill this role, as exemplified by FLS2 and EFR, the receptors for the microbe-associated molecular patterns flagellin and elongation factor Tu. Here we examined the perception of the damage-associated molecular pattern peptide 1 (AtPep1), an endogenous peptide of Arabidopsis identified earlier and shown to be perceived by the leucine-rich repeat protein kinase PEPR1. Using seedling growth inhibition, elicitation of an oxidative burst and induction of ethylene biosynthesis, we show that wild type plants and the pepr1 and pepr2 mutants, affected in PEPR1 and in its homologue PEPR2, are sensitive to AtPep1, but that the double mutant pepr1/pepr2 is completely insensitive. As a central body of our study, we provide electrophysiological evidence that at the level of the plasma membrane, AtPep1 triggers a receptor-dependent transient depolarization through activation of plasma membrane anion channels, and that this effect is absent in the double mutant pepr1/pepr2. The double mutant also fails to respond to AtPep2 and AtPep3, two distant homologues of AtPep1 on the basis of homology screening, implying that the PEPR1 and PEPR2 are responsible for their perception too. Our findings provide a basic framework to study the biological role of AtPep1-related danger signals and their cognate receptors

An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery.

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley-Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14α-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy

(A-E) The fungal silencing machinery is required for efficient SIGS in distal leaf parts.

<p><b>(A,B)</b> The fungal <i>dicer-like-1</i> mutant Fg-IFA65_Δdcl-1 heavily infected barley leaves despite a prior spray-treatment with <i>CYP3</i>-dsRNA. Photographs were taken at 6 dpi. <b>(C)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts in the wild type Fg-IFA65 and the mutant Fg-IFA65_Δdcl-1 at 6 dpi in the distal, semi-systemic leaf areas. <b>(D)</b> Inhibition of <i>CYP51</i> gene expression upon <i>CYP3</i>-dsRNA treatment of axenically grown Fg-IFA65_- Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in samples treated with <i>CYP3</i>-dsRNA compared with mock-treated controls was statistically significant (*P < 0.05, **P < 0.01; Student´s t test). <b>(E-G)</b> Profiling of <i>CYP3</i>-dsRNA-derived sRNAs in axenically grown Fg-IFA65. (E) Scaffold of the 791 nt long <i>CYP3</i>-dsRNA. The fragments of <i>CYP51</i> genes are indicated. (F,G) Total sRNAs were isolated from axenically-cultured Fg-IFA65. sRNA reads of fungal sRNAs from untreated (F) and <i>CYP3</i>-dsRNA-treated (G) fungal cultures are mapped to the sequence of <i>CYP3</i>-dsRNA.</p

(A,B) Defense-related salicylate- and jasmonate-responsive genes are not induced by <i>CYP3</i>-dsRNA.

<p>Detached second leaves of three-week-old barley were sprayed with 20 ng μL^-1 <i>CYP3</i>-dsRNA or TE (control), respectively, and 48 h later drop-inoculated with Fg-IFA65. Leaves were harvested 6 dpi and analyzed for gene expression by qPCR: <b>(A)</b> <i>Pathogenesis-related</i> 1 (<i>HvPR1</i>) and <b>(B)</b> <i>S-adenosyl-l-methionine</i>:<i>jasmonic acid carboxyl methyltransferase</i> (<i>HvJMT</i>). Both genes are highly responsive to Fg-IFA65 but not to <i>CYP3</i>-dsRNA or TE treatment. Please note that a combined treatment of <i>CYP3</i>-dsRNA followed by Fg-IFA65 48 h later also did not induce these marker genes, which shows independently that fungal development on <i>CYP3</i>-dsRNA-treated leaves is strongly inhibited.</p

(A-E) The fungal silencing machinery is required for efficient SIGS in distal leaf parts.

<p><b>(A,B)</b> The fungal <i>dicer-like-1</i> mutant Fg-IFA65_Δdcl-1 heavily infected barley leaves despite a prior spray-treatment with <i>CYP3</i>-dsRNA. Photographs were taken at 6 dpi. <b>(C)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts in the wild type Fg-IFA65 and the mutant Fg-IFA65_Δdcl-1 at 6 dpi in the distal, semi-systemic leaf areas. <b>(D)</b> Inhibition of <i>CYP51</i> gene expression upon <i>CYP3</i>-dsRNA treatment of axenically grown Fg-IFA65_- Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in samples treated with <i>CYP3</i>-dsRNA compared with mock-treated controls was statistically significant (*P < 0.05, **P < 0.01; Student´s t test). <b>(E-G)</b> Profiling of <i>CYP3</i>-dsRNA-derived sRNAs in axenically grown Fg-IFA65. (E) Scaffold of the 791 nt long <i>CYP3</i>-dsRNA. The fragments of <i>CYP51</i> genes are indicated. (F,G) Total sRNAs were isolated from axenically-cultured Fg-IFA65. sRNA reads of fungal sRNAs from untreated (F) and <i>CYP3</i>-dsRNA-treated (G) fungal cultures are mapped to the sequence of <i>CYP3</i>-dsRNA.</p

(A,B) Northern gel blot analysis of <i>CYP3</i>-dsRNA and <i>CYP3</i>-dsRNA-derived siRNA accumulation in local and distal (semi-systemic) barley leaf areas.

<p><b>(A)</b> Detection of 791 nt long <i>CYP3</i>-dsRNA precursor in pooled leaf tissue from non-infected leaves using [α-32P]-dCTP labeled <i>CYP3</i>-dsRNA as probe. Local (L) and distal (semi-systemic [S]) leaf segments were sampled separately at the indicated times after spraying with <i>CYP3-</i>dsRNA. No signal was detected in samples from TE-sprayed plants. <b>(B)</b> Recording <i>CYP3</i>-dsRNA-derived small RNAs in local and distal (semi-systemic) leaf areas using [α-32P]-dCTP labeled <i>CYP3</i>-dsRNA as probe. In this experiment, small RNAs could not be detected in distal (non-sprayed) tissues. siRNA generated <i>in vitro</i> by a commercial Dicer preparation from <i>CYP3</i>-dsRNA was used as positive control. No signal was detected in samples from TE-sprayed plants. Ethidium bromide-stained rRNA served as the loading control. Signals originate from the same membrane but different exposure times.</p