The \u3ci\u3ePseudomonas syringae\u3c/i\u3e type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9

Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis. HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI. HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity

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

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

Transcriptional dynamics driving MAMP-triggered immunity and pathogen effector-mediated immunosuppression in Arabidopsis leaves following infection with Pseudomonas syringae pv tomato DC3000

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae

Regulation of the Astaxanthin-Biosynthesis in the Green Alga Haematococcus pluvialis

Das natürliche Vorkommen der einzelligen Grünalge Haematococcus pluvialis liegt hauptsächlich in kleinen Süßwassertümpeln, Pfützen und auch in Vogeltränken. Diese Gewässer unterliegen täglichen Schwankungen der Salzkonzentration oder de Lichtintensität, die auf starke Verdunstung, Regenfälle und durch Beschattung hervorgerufen werden. Während des Lebenszyklus von Haematococcus pluvialis kann man verschiedene Stadien unterscheiden: eine grüne, freischwimmende, doppelt begeißelte Form; ein unbegeißeltes grünes Zwischenstadium, der so genannten Palmella-Form und eine Dauerform mit stark verdickter Zellwand, geißellos, mit großen Mengen an akkumuliertem rotem Pigment, dem so genannten Astaxanthin. Unter den bekannten Organismen, die Astaxantin in großen Mengen selbst herstellen können, zeigt Haematococcus pluvialis die besten Ausbeuten. Neben dem Einsatz von Astaxanthin als Farbstoff in der Fischzucht wird Astaxanthin immer wichtiger als Nahrungsergänzungsstoff mit gesundheitsfördernden Eigenschaften für den Menschen. Unter verschiedenen Stressbedingungen, wie beispielsweise stickstofflimitierende Bedingungen, unter Phosphat- oder Schwefelmangel und Salzstress kommt es zum Übergang in ein Dauerstadium und zur starken Akkumulation von Astaxanthin. Die Bedingungen, die die Astaxanthinakkumulation induzieren wurden von verschiedenen Arbeitsgruppen behandelt, die molekulare Grundlage wurde aber noch nicht detailliert untersucht und war Gegenstand meiner Doktorarbeit.Während meiner Arbeit konnte gezeigt werden, dass Haematococcus pluvialis fähig ist auf verschiedene Stressarten, wie Salzstress oder auch Lichtstress, unterschiedlich zu reagieren. Auf transkriptioneller Ebene wurden verschiedene, aber angepasste Antworten der Carotinoid- und spezifischen Astaxanthinbiosynthesegene je nach verwendetem Stress beobachtet. Darüber hinaus konnte gezeigt werden, dass die Lichtinduktion der Carotinoid- und spezifischen Astaxanthinbiosynthesegene mit dem Redoxzustand des photosynthetischen Elektronentransports korreliert. Die Verwendung verschiedener spezifischer Elektronentransporthemmstoffe half bei der Eingrenzung dieses Redoxsensors. Der Redoxzustand des Plastochinon-Pools vermittelt die transkriptionelle Aktivierung der Carotinoidbiosynthesegene deren Produkte bei der Bildung von Astaxanthin beteiligt sind. Diese Untersuchungen konnten zeigen, dass nicht nur der spezifische Astaxanthinbiosyntheseweg, sondern auch die allgemeine Carotinoidbiosynthese unter der Redoxkontrolle des photosynthetischen Elektronentransports steht.Aufgrund der biotechnologischen Relevanz von Haematococcus pluvialis für die Produktion von hohen Ausbeuten an natürlichem Astaxanthin oder anderen wichtigen Carotinoiden wurde ein System zur genetischen Veränderung etabliert. Einige der Transformanten zeigten veränderte Carotinoidzusammensetzungen, die mit einem verbesserten nicht photochemischen Quenching korrelierten. Die Chlorophyllfluoreszenzmessung wurde als Methode verwendet um nach Transformanten zu suchen, die eine veränderte Xanthophyllzusammensetzung besitzen. Die Stressinduktion der Astaxanthinsynthese durch Starklicht zeigte für eine der untersuchten Transformanten eine beschleunigte Astaxanthinakkumulation im Vergleich mit dem Wildtyp. Diese Ergebnisse zeigen eindeutig, dass das entwickelte Transformationssystem zur gezielten Veränderung der Carotinoidbiosynthese in Haematococcus pluvialis geeignet ist

Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis

A growth quantification assay for hyaloperonospora arabidopsidis isolates in Arabidopsis thaliana

There is a considerable interest in determining the role of individual oomycete effectors in promoting disease. Widely used strategies are based on manipulating effector-expression levels in the pathogen and by over-expressing particular effectors in the host by genetic transformation. In the case of the oomycete, Hyaloperonospora arabidopsidis (Hpa) genetic manipulation is not yet possible, so over-expression of predicted effectors in stably transformed Arabidopsis lines is used to investigate their capability for promoting virulence. Here, we describe a technique for quantifying pathogen growth based on the counting of asexual reproductive structures called sporangiophores in the compatible interaction between the Hpa isolate Noks1 and the Col-0 Arabidopsis accession

A growth quantification assay for hyaloperonospora arabidopsidis isolates in Arabidopsis thaliana

There is a considerable interest in determining the role of individual oomycete effectors in promoting disease. Widely used strategies are based on manipulating effector-expression levels in the pathogen and by over-expressing particular effectors in the host by genetic transformation. In the case of the oomycete, Hyaloperonospora arabidopsidis (Hpa) genetic manipulation is not yet possible, so over-expression of predicted effectors in stably transformed Arabidopsis lines is used to investigate their capability for promoting virulence. Here, we describe a technique for quantifying pathogen growth based on the counting of asexual reproductive structures called sporangiophores in the compatible interaction between the Hpa isolate Noks1 and the Col-0 Arabidopsis accession

The \u3ci\u3ePseudomonas syringae\u3c/i\u3e type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9

Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis. HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI. HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity