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"Schulze, Sebastian"
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A role for calcium-dependent protein kinases in differential CO₂- and ABA-controlled stomatal closing and low CO₂-induced stomatal opening in Arabidopsis
• Low concentrations of CO₂ cause stomatal opening, whereas [CO₂] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO₂-induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+-binding proteins that function in the stomatal CO₂ response remain unknown.
• Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed.
• We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO₂, light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO₂-induced stomatal closure and, unexpectedly, also in low CO₂-induced stomatal opening. Furthermore, K⁺-uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO₂-regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants.
• Our findings describe combinatorial cpk mutants that function in CO₂ control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.
Journal Article
A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture
Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.
Journal Article
Activation of defense against Phytophthora infestans in potato by down-regulation of syntaxin gene expression
The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed.
Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane-localized SYNTAXIN-RELATED 1 (StSYR1) and SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively.
Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1-RNAi plants, but not StSNAP33-RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans-infected StSYR1-RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines.
The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1-RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose-containing papillae.
Journal Article
Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity
• The pathogenicity of the Gram‐negative plant‐pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. • In this study, we analyzed eight T3Es from Xcv strain 85‐10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen‐associated molecular pattern (PAMP)‐triggered plant defense gene expression. • In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP‐triggered immunity (PTI) and effector‐triggered immunity (ETI). • XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI‐related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB‐mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.
Journal Article
Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1
Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (
) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of
genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from
pv.
(
), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86
lines mainly of the genus
were screened for phenotypical reactions after
-mediated transient expression of 21 different
effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative
genes and (iii) identify promising plant lines as repertoire for
gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential
genes. In some cases, putative
genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several
spp. lines, and
infection assays revealed that XopQ is a host range determinant in many
species. Non-host resistance against
and XopQ recognition in
required
, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing R
as candidate for targeted crop improvement.
Journal Article
Thermal Effects in Dissimilar Magnetic Pulse Welding
Magnetic pulse welding (MPW) is often categorized as a cold welding technology, whereas latest studies evidence melted and rapidly cooled regions within the joining interface. These phenomena already occur at very low impact velocities, when the heat input due to plastic deformation is comparatively low and where jetting in the kind of a distinct material flow is not initiated. As another heat source, this study investigates the cloud of particles (CoP), which is ejected as a result of the high speed impact. MPW experiments with different collision conditions are carried out in vacuum to suppress the interaction with the surrounding air for an improved process monitoring. Long time exposures and flash measurements indicate a higher temperature in the joining gap for smaller collision angles. Furthermore, the CoP becomes a finely dispersed metal vapor because of the higher degree of compression and the increased temperature. These conditions are beneficial for the surface activation of both joining partners. A numerical temperature model based on the theory of liquid state bonding is developed and considers the heating due to the CoP as well as the enthalpy of fusion and crystallization, respectively. The time offset between the heat input and the contact is identified as an important factor for a successful weld formation. Low values are beneficial to ensure high surface temperatures at the time of contact, which corresponds to the experimental results at small collision angles.
Journal Article
Correction: A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture
[This corrects the article DOI: 10.1371/journal.ppat.1003121.].
Journal Article
Suppression of the AvrBs1-specific hypersensitive response by the YopJ effector homolog AvrBsT from Xanthomonas depends on a SNF1-related kinase
Pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) depends on a type III secretion system that translocates a cocktail of > 25 type III effector proteins into the plant cell. In this study, we identified the effector AvrBsT as a suppressor of specific plant defense. AvrBsT belongs to the YopJ/AvrRxv protein family, members of which are predicted to act as proteases and/or acetyltransferases. AvrBsT suppresses the hypersensitive response (HR) that is elicited by the effector protein AvrBs1 from Xcv in resistant pepper plants. HR suppression occurs inside the plant cell and depends on a conserved predicted catalytic residue of AvrBsT. Yeast two-hybrid based analyses identified plant interaction partners of AvrBs1 and AvrBsT, including a putative regulator of sugar metabolism, SNF1-related kinase 1 (SnRK1), as interactor of AvrBsT. Intriguingly, gene silencing experiments revealed that SnRK1 is required for the induction of the AvrBs1-specific HR. We therefore speculate that SnRK1 is involved in the AvrBsT-mediated suppression of the AvrBs1-specific HR.
Journal Article
Towards Data-Efficient Deployment of Reinforcement Learning Systems
A fundamental concern in the deployment of artificial agents in real-life is their capacity to quickly adapt to their surroundings. Traditional reinforcement learning (RL) struggles with this requirement in two ways. Firstly, iterative exploration of unconstrained environment dynamics yields numerous uninformative updates and consequently slow adaptation. Secondly, final policies have no capacity to adapt to future observations and have to either slowly learn indefinitely or retrain entirely as observations occur. This thesis explores two formulations aimed at addressing these issues. The consideration of entire task distributions in meta-RL evolves policies quickly adapting to specific instances on their own. By forcing agents to specifically request feedback, Active RL enforces selective observations and updates. Both of these formulations reduce to a Bayes-Adaptive setting in which a probabilistic belief over possible environments is maintained. Many existing solutions only provide asymptotic guarantees that are of limited use in practical contexts. We develop a variational approach to approximate belief management and support its validity empirically through a broad range of ablations. We then consider recently successful planning approaches but uncover and discuss obstacles in their application to the discussed settings. An important factor influencing the data requirements and stability of RL systems is the choice of appropriate hyperparameters. We develop a Bayesian optimisation approach exploiting the iterative structure of training processes whose empiric performance exceeds that of existing baselines. A final contribution of this thesis concerns increasing the scalability and expressiveness of Gaussian Processes (GPs). While we make no direct use of the presented framework, GPs have been used to model probabilistic beliefs in closely related settings.
Dissertation