Molisso, Donata (2018) FUNCTIONAL CHARACTERIZATION OF TOMATO PROSYSTEMIN AND PROSYSTEMIN REGIONS: NOVEL TOOLS FOR PLANT DEFENSE. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: FUNCTIONAL CHARACTERIZATION OF TOMATO PROSYSTEMIN AND PROSYSTEMIN REGIONS: NOVEL TOOLS FOR PLANT DEFENSE
Creators:
CreatorsEmail
Molisso, Donatadonata.molisso@unina.it
Date: 18 December 2018
Number of Pages: 129
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Biotecnologie
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Rao, RosaUNSPECIFIED
Monti, Simona MariaUNSPECIFIED
Date: 18 December 2018
Number of Pages: 129
Uncontrolled Keywords: plant defense, Prosystemin, IDP, functional characterization
Settori scientifico-disciplinari del MIUR: Area 07 - Scienze agrarie e veterinarie > AGR/07 - Genetica agraria
Additional Information: Dottorato di ricerca svolto presso il Dipartimento di Agraria
Date Deposited: 15 Jan 2019 09:14
Last Modified: 16 Jun 2020 09:15
URI: http://www.fedoa.unina.it/id/eprint/12541

Abstract

Prosystemin (ProSys) is a pro-hormone of 200 aminoacidic residues which releases a bioactive peptide hormone of 18 amino acids called Systemin (Sys) involved in the activation of a complex signaling cascade that leads to the production of defense compounds. The tomato genome contains only one copy of Prosys gene; it is composed of 4176 bp and is structured into 11 exons, of which the last one codes for Sys. Sys peptide was traditionally considered as the principal actor that confers protection against both biotic and abiotic environmental challenges observed in tomato plant overexpressing the ProSys. Thus, a single peptide hormone is capable of eliciting multiple defense pathways to counteract a wide range of unfavourable conditions for the plant. So far, it was unknown whether ProSys had any biological function other than being an intermediate in the synthesis of Sys. However, recent evidences suggest that Prosys devoid of the Sys sequence contributes to defense responses. This observation prompted us to investigate the biochemical and structural features of the ProSys protein. To this purpose ProSys has been expressed in BL21 (DE3) E. coli cells and purified. A detailed characterization of this pro-hormone by means of multidisciplinary approach revealed for the first time that this precursor behaves like an intrinsically disordered protein (IDP) possessing intrinsically disordered regions (IDRs) within the sequence. However, to find out an alternative delivery strategy not relying on transgenic plants, we decided to investigate the effects of exogenous application of the recombinant pro-hormone on the defense responses and its potential use as a plant protection tool in tomato. In particular, plant assays revealed that ProSys direct treatment of leaves is biologically active being very effective in the induction, both locally and systemically, of tomato defense-related genes, conferring protection against different pests. To our knowledge, this is the first biotic stress related IDP identified in plants, suggesting new interesting insights on the role of IDPs. into plant response against biotic stressors. IDPs are functionally important proteins lacking a stable or ordered three-dimensional structure. Despite being highly flexible, it has been demonstrated that IDPs have crucial roles in signal transduction process, cell-cycle regulation, gene expression and molecular recognition. The role of IDPs in these processes has been systematically studied in the animal kingdom. In contrast, less reports of these proteins from the plant kingdom are available in the scientific literature. In plant biology, IDPs play crucial roles among plant stress responses, signaling, and molecular recognition pathways, that resemble the functional roles of ProSys in the tomato defense pathways activated upon several biotic and abiotic stresses. These evidences aimed our study focused on the establishment of a relationship between ProSys structure and its biological activity. To this purpose different regions of ProSys have been expressed in BL21 (DE3) E. coli cells, purified and then characterized by a biophysical and biochemical point of view. Results showed that the recombinant fragments are disordered in agreement with what previously shown for the whole precursor. It was subsequently investigated whether the recombinant ProSys Fragments had any biological activity in activating defense responses upon biotic or abiotic attacks. In particular, by using a combination of gene expression analysis and bioassays, we proved that the exogenous supply of the recombinant ProSys Fragments to tomato plants promotes early and late plant defense genes, but only two fragments (namely Fragment I and III, encompassing the N-terminal part of the protein) were found to be the most promising. In addition, it was observed that the latter ones counteracted the development of Spodoptera littoralis larvae and the fungal leaf colonization. These results suggest that the direct application of these recombinant products, which are safe to humans and no-target organisms, may represent an exploitable tool for crop protection.

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