Comparison of the defensive elicitation activity of cerato-populin and cerato-platanin: a structural model

Plants can defend themselves from potential pathogenic microorganisms relying on a complex interplay of signaling pathways: activation of the MAPK cascade, transcription of defense related genes, production of reactive oxygen species, nitric oxide and synthesis of other defensive compounds such as phytoalexins. These events are triggered by the recognition of pathogen’s effectors (effector-triggered immunity) or PAMPs (PAMP-triggered immunity). The Cerato Platanin Family (CPF) members are Cys-rich proteins secreted and localized on fungal cell walls, involved in several aspects of fungal development and pathogen-host interactions. Although more than hundred genes of the CPF have been identified and analyzed, the structural and functional characterization of the expressed proteins has been restricted only to few members of the family. Interestingly, those proteins have been shown to bind chitin with diverse affinity and after foliar treatment they elicit defensive mechanisms in host and non-host plants. This property turns cerato platanins into interesting candidates, worth to be studied to develop new fungal elicitors with applications in sustainable agriculture. This study focus on cerato-platanin (CP), core member of the family and on the orthologous cerato-populin (Pop1). The latter shows an identity of 62% and an overall homology of 73% with respect to CP. Both proteins are able to induce MAPKs phosphorylation, production of reactive oxygen species and nitric oxide, overexpression of defense’s related genes, programmed cell death and synthesis of phytoalexins. CP, however, when compared to Pop1, induces a faster response and, in some cases, a stronger activity on plane leaves. Aim of the present research is to verify if the dissimilarities observed in the defense elicitation activity of these proteins can be associated to their structural and dynamic features. Taking advantage of the available CP NMR structure, Pop1’s 3D one was obtained by homology modeling. Experimental residual dipolar couplings and 1H, 15N, 13C resonance assignments were used to validate the model. Previous works on CPF members, addressed the highly conserved random coil regions (loops b1-b2 and b2-b3) as sufficient and necessary to induce necrosis in plants’ leaves: that region was investigated in both Pop1 and CP. In the two proteins the loops differ, in their primary sequence, for few mutations and an insertion with a consequent diversification of the proteins’ electrostatic surface. A set of 2D and 3D NMR experiments was performed to characterize both the spatial arrangement and the dynamic features of the loops. NOE data revealed a more extended network of interactions between the loops in Pop1 than in CP. In addition, in Pop1 we identified a salt bridge Lys25/Asp52 and a strong hydrophobic interaction between Phe26/Trp53. These structural features were expected not only to affect the loops’ spatial arrangement, but also to reduce the degree of their conformational freedom. Relaxation data and the order parameter S2 indeed highlighted reduced flexibility, in particular for loop b1-b2 of Pop1. In vitro NMR experiments, where Pop1 and CP were titrated with oligosaccharides, supported the hypothesis that the loops structural and dynamic differences may be responsible for the different chitin-binding properties of the two proteins: CP selectively binds tetramers of chitin in a shallow groove on one side of the barrel defined by loops b1-b2, b2-b3 and b4-b5, Pop1, instead, interacts in a non-specific fashion with oligosaccharides. Because the region involved in chitin-binding is also responsible for the defense elicitation activity, possibly being recognized by plant's receptors, it is reasonable to expect that those structural and dynamic modifications may also justify the different extent of defense elicitation. To test that hypothesis, the initial steps of a protocol aimed to the identify a receptor for CP, in silico, are presented.

Numerical modelling of unsteady mixed flows

This thesis concerns mixed flows (which are characterized by the simultaneous occurrence of free-surface and pressurized flow in sewers, tunnels, culverts or under bridges), and contributes to the improvement of the existing numerical tools for modelling these phenomena. The classic Preissmann slot approach is selected due to its simplicity and capability of predicting results comparable to those of a more recent and complex two-equation model, as shown here with reference to a laboratory test case. In order to enhance the computational efficiency, a local time stepping strategy is implemented in a shock-capturing Godunov-type finite volume numerical scheme for the integration of the de Saint-Venant equations. The results of different numerical tests show that local time stepping reduces run time significantly (between −29% and −85% CPU time for the test cases considered) compared to the conventional global time stepping, especially when only a small region of the flow field is surcharged, while solution accuracy and mass conservation are not impaired. The second part of this thesis is devoted to the modelling of the hydraulic effects of potentially pressurized structures, such as bridges and culverts, inserted in open channel domains. To this aim, a two-dimensional mixed flow model is developed first. The classic conservative formulation of the 2D shallow water equations for free-surface flow is adapted by assuming that two fictitious vertical slots, normally intersecting, are added on the ceiling of each integration element. Numerical results show that this schematization is suitable for the prediction of 2D flooding phenomena in which the pressurization of crossing structures can be expected. Given that the Preissmann model does not allow for the possibility of bridge overtopping, a one-dimensional model is also presented in this thesis to handle this particular condition. The flows below and above the deck are considered as parallel, and linked to the upstream and downstream reaches of the channel by introducing suitable internal boundary conditions. The comparison with experimental data and with the results of HEC-RAS simulations shows that the proposed model can be a useful and effective tool for predicting overtopping and backwater effects induced by the presence of bridges and culverts.