42 research outputs found
Functional and structural characterization of LysM proteins in interactions between fungi and plants
No full textPlants possess an innate immune system that recognizes various types of molecules that accurately betray microbial invasion, also known as invasion patterns (IPs), that include microbe-associated molecular patterns (MAMPs). This recognition occurs through invasion pattern receptors (IPRs) that activate a wide range of immune responses that aim to halt microbial infections. In turn, successful microbes secrete effector proteins to deregulate plant immunity. Chapter 1 introduces the significant role of the major fungal cell wall component, chitin, in the interactions between plants and fungi. On the one hand, this chapter focuses on chitin perception systems that have been characterized in detail in several plant species, while on the other hand the chapter focuses on effector proteins containing lysin motifs (LysM effectors) employed by the tomato leaf mould pathogen Cladosporium fulvum and the wheat Septoria tritici blotch pathogen Zymoseptoria tritici.To date, all chitin receptors identified in plants belong to either the LysM-containing receptor-like kinases (LysM-RLKs) or LysM-containing receptor-like proteins (LysM-RLPs). For instance, the Arabidopsis LysM-RLK AtLYK5 binds chitin with high affinity and forms a tripartite receptor complex with two further LysM-RLKs, AtLYK4 and AtCERK1, to initiate chitin signaling. Similarly, the rice chitin perception system is composed of the LysM-RLK OsCERK1 in association with the LysM-RLP OsCEBiP. In Chapter 2, by using chitin affinity-purification followed by mass spectrometry we identified two candidate chitin receptor proteins in tomato, the LysM-RLK SlLYK4 and the LysM-RLP SlCEBiP. Silencing of either SlLYK4 or SlCEBiP resulted in significantly impaired chitin responsiveness. Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR-Cas9) we generated mutants of both genes and evaluated their role in chitin signalling. While the function of SlCEBiP needs further assessment because it presently remains unclear whether the mutant that was generated truly disrupts gene function, SlLYK4 was found to play an essential role in mediating chitin signal transduction as SlLYK4 mutants displayed not only greatly compromised chitin-induced immunity but also enhanced susceptibility to C. fulvum infection. We propose that SlLYK4 is a crucial component of the chitin receptor complex of tomato.To overcome the chitin-induced tomato immunity, C. fulvum secretes the LysM effector Ecp6 to outcompete immune receptors for chitin binding. Two of its three LysMs undergo intracellular LysM dimerization, thus forming a chitin-binding groove (LysM1-LysM3) with ultra-high substrate affinity that goes beyond the affinity of host receptors. The remaining singular LysM domain of Ecp6, LysM2, also displays the capability to bind chitin, albeit with a relatively low affinity that does not permit to outcompete chitin receptors. Chapter 3 aims to investigate whether LysM2 contributes to the virulence function of Ecp6 and how it confers such contribution. Inoculation assays with C. fulvum transformants that express a suite of Ecp6 mutants in the various LysMs revealed that LysM2 contributes to C. fulvum virulence, probably through suppression of chitin-responsive gene expression. Interestingly, a physical interaction of Ecp6 with Arabidopsis AtLYK5 and with tomato SlLYK4 that was characterized in chapter could been demonstrated. Moreover, it appears that while LysM2 confers an interaction with these receptors in a chitin-independent manner, the composite LysM1-LysM3 binding groove contributes to the interaction in a chitin-dependent manner. Thus, besides competing with plant immune receptors for chitin binding, Ecp6 may perturb the assembly of functional chitin receptor complexes that are crucial for the activation of chitin-induced immunity.Many fungal LysM effectors comprise two LysMs, such as MoSlp1 from the rice blast fungus Magnaporthe oryzae, Vd2LysM from the broad host range vascular wilt fungus Verticillium dahliae, and ChElp1 and ChElp2 from the Brassicaceae anthracnose fungus Colletotrichum higginsianum. They all bind chitin, suppress chitin-triggered host immunity and contribute to fungal virulence. Chapter 4 describes the functional and structural analyses to investigate whether these fungal LysM effectors with two LysMs bind chitin through intramolecular LysM dimerization, like Ecp6, or rather through intermolecular dimerization. As our considerable efforts to obtain a crystal structure of any of these effectors by X-ray crystallography failed since crystal growth did not occur, we hypothesized that these findings could suggest the occurrence of intermolecular chitin binding for these LysM effectors. With DLS measurements and centrifugation assays we were able to confirm that the formation of chitin-induced polymeric complexes for MoSlp1, V2LysM and ChElp2 occurs, potentially mediating the elimination of chitin oligomers at infection sites by precipitation to suppress the activation of chitin-induced plant immunity.The wheat-specific pathogen Z. tritici encodes three LysM effector proteins, Mg1LysM and Mg1LysM_b that contain a single LysM, and Mg3LysM that possesses three LysMs. Previously, Mg1LysM_b was disregarded as a presumed pseudogene, while Mg1LysM and Mg3LysM were functionally characterized. Chapter 5 provides evidence to show that Mg1LysM_b is not a pseudogene and is functional during wheat colonization. We show that Mg1LysM_b binds chitin, protects fungal hyphae against chitinase hydrolysis and is able to suppress a chitin-induced ROS burst. Fungal inoculation assays reveal that while Mg3LysM confers a major contribution to Z. tritici virulence, also Mg1LysM and Mg1LysM_b contribute to virulence, albeit with smaller contributions, and that all LysM effectors display partial functional redundancy. Thus, we show that Zymoseptoria tritici utilizes three LysM effectors to disarm chitin-triggered wheat immunity.In Chapter 6, we determined a crystal structure of Z. tritici Mg1LysM to try and explain how this LysM effector protects fungal hyphae against chitinase hydrolysis. Intriguingly, the crystal structure revealed the formation of chitin-independent homodimers as well as chitin-induced dimerization of two Mg1LysM protomers. Based on DLS measurements and centrifugation assays in the presence and absence of chitin oligomers, it could be concluded that Mg1LysM forms a chitin-induced supramolecular structure that, anchored to chitin in the cell wall, may prevent hydrolysis by host chitinases. Interestingly, it could be demonstrated that Mg1LysM_b, as well as RiSLM from the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis that similarly contains a single LysM, polymerize in the presence of chitin as well, suggesting that they also undergo chitin-induced dimerization of ligand independent homodimers.Besides chitin, several other cell wall polysaccharides have previously been characterized as invasion pattern, such as β-glucan and bacterial peptidoglycan. Chapter 7 synthesizes the findings in this thesis and places them into a broader perspective to highlight the importance of chitin as well as other cell wall components in interactions between plants and microbes
The effect of EHOP on the writing of Program Analyzers
No full textEffect Handler Oriented Programming is a promising new programming paradigm, delivering separation of of concerns with regards to side effects in an otherwise functional environment.This paper discusses the applicability of this new paradigm to static code analysis programs. Different code analyzers often have many similar, if not identical pieces of code which could be abstracted away. This abstraction does not come natural to the programming paradigm of Functional Programming but are quite natural within EHOP.The current programming languages do not yet seem up to the task of rapid generalization of code and elimination of duplicate pieces of code.However, the concepts present in EHOP will almost certainly be able to eliminate much of this code reduction once the languages have matured further.The implicit passing of functionality will also allow for clearer code with less unnecessary visual clutter.CSE3000 Research ProjectComputer Science and Engineerin
Wild-type glyceraldehyde dehydrogenase from Thermoplasma acidophilum in complex with NADP
No full textThe battle for chitin recognition in plant-microbe interactions
No full textFungal cell walls play dynamic functions in interaction of fungi with their surroundings. In pathogenic fungi, the cell wall is the first structure to make physical contact with host cells. An important structural component of fungal cell walls is chitin, a well-known elicitor of immune responses in plants. Research into chitin perception has sparked since the chitin receptor from rice was cloned nearly a decade ago. Considering the widespread nature of chitin perception in plants, pathogens evidently evolved strategies to overcome detection, including alterations in the composition of cell walls, modification of their carbohydrate chains and secretion of effectors to provide cell wall protection or target host immune responses. Also non-pathogenic fungi contain chitin in their cell walls and are recipients of immune responses. Intriguingly, various mutualists employ chitin-derived signaling molecules to prepare their hosts for the mutualistic relationship. Research on the various types of interactions has revealed different molecular components that play crucial roles and, moreover, that various chitin-binding proteins contain dissimilar chitin-binding domains across species that differ in affinity and specificity. Considering the various strategies from microbes and hosts focused on chitin recognition, it is evident that this carbohydrate plays a central role in plant–fungus interactions
Computation of VaR and VaR Contribution in the Vasicek portfolio credit loss model: A comparative study
No full textElectrical Engineering, Mathematics and Computer Scienc
Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.
No full textAstrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation
