Peptides as sustainable tools to control plant pathogens: production of antimicrobial peptides (AMP) and development of plant elicitor peptides (Pep)
- Autores: Cristina Ruiz Ramirez
- Directores de la Tesis: Maria Pla i de Solà-Morales (dir. tes.), Anna Nadal i Matamala (codir. tes.)
- Lectura: En la Universitat de Girona ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Teresa Capell Capell (presid.), Concepció Moragrega García (secret.), Anna Coll Rius (voc.)
- Programa de doctorado: Programa de Doctorado en Tecnología por la Universidad de Girona
- Materias:
- Texto completo no disponible (Saber más ...)
- Resumen
SUMMARY Intensive agriculture produces high yields but it is highly vulnerable to disease caused by plant pathogens. This is especially relevant for fruit production. Disease control is currently based on preventive treatments using chemical compounds and antibiotics of limited efficacy and negative environmental impact. This Thesis focuses on a new strategy, based on bioactive peptides either from natural sources or tailored from naturally occurring precursors, with more favorable ecological footprint. More specifically, peptides with specific antimicrobial activity (AMP) and endogenous peptides capable of eliciting and amplifying plant defense mechanisms (Pep) were envisaged. The significant contagious diseases fire flight and bacterial spot and canker of stone fruit and almond, caused by the bacterial pathogens Erwinia amylovora (Ea) and Xanthomonas arboricola pv. pruni (Xap), respectively, were used as examples. They affect principally species within the Rosaceae family, many of which are important commercial plants broadly cultivated worldwide for fruit production or as ornamental trees and shrubs.
On design of improved AMPs based on hybrids of the Cecropin A and Melittin, BP100 (KKLFKKILKYLNH2) outstands by its high bactericidal activity against the bacterial pathogens Ea and Xap.
However, massive production in plant biofactories heavily impaired the fitness of the host. We recently developed a strategy, based on strict limitation of transgene expression through heatshock inducible promoters, that allowed producing BP100 in rice biofactories, although the yields were only 0.3% TSP. Here we used modified atmospheres, with the increased levels of CO2 commonly used in the food industry, as inductor agent, and achieved biotechnological production of phytotoxic peptides such as BP100 with higher yields. Transcriptomics analyses were used to select promoters with extremely low activity during plant development and strongly responsive to [CO2]. Five promoters were successfully used to drive expression of recombinant BP100 in rice, and in the best example, Os.hb2 resulted in yields of 1% TSP. This is within the range of other recombinant peptides in plants, making this strategy economically viable.
The experimental approach followed to identify the most suitable promoters resulted in a description of the main rice transcriptional changes triggered by 30% [CO2] in the dark. Essentially, there was a drift in the energy provision (from photosynthesis to glycolysis) and activation of the stress defense responses.
In a second approach, we addressed peptides to promote the immunity of the plant. Peps are synthesized by the host plant and, through recognition by specific receptors in plant cell membranes, modulate the early events in plant response to pathogens. Because of co-evolution of Peps and PEPRs, Peps can only be perceived by plants from the same family. Although they have been described in various Angiosperm species, there was only scarce in silico information on Peps from the Rosaceae. Here we showed the extensive presence of Pep sequences, and the corresponding precursor PROPEPs, throughout the Rosaceae family and placed this family amongst the most well-known in terms of the number of reported sequences. Analysis of 100 varieties from 36 species of the Amygdaleae and the Pyreae tribes, including the main edible and ornamental species, showed a uniform pattern. Every Amygdaleae species had two Peps, Pep1 and Pep2, and every Pyreae species had Pep3 and Pep4. Strawberry (Potentilleae tribe) presented a different sequence, Pep5. Pep2 and Pep3 were highly conserved throughout the Amygdaleae and Pyreae, respectively, while Pep1 and Pep4 sequences shared around 90% homology within each tribe and had a characteristic acidic amino acid at the N-terminal end. Complementing the Pep and PEPR system, analysis of a selection of species showed that every Rosaceae had two PEPR orthologues: PEPR1a and PEPR1b. The Rosaceae Peps and PEPRs grouped into phylogenetic clusters separate from other plant families, as has been reported for the Poaceae and Brassicaceae.
Having described the Rosaceae Pep and PEPR system, we used a selection of Peps (Pep1 and Pep2 from peach, almond and Prunus mume, apple Pep3 and pear and apple Pep4) and the Prunus spp.
and Xap pathosystem to explore their activity. Peps did not exhibit antibacterial activity. In contrast, topic application of peach and almond Peps onto leaves of the same species induced the expression of PROPEPs and a set of defense related genes (e.g. various ethylene responsive factors and pathogenesis related proteins) in a similar pattern as challenge with Xap. This indicated that Peps triggered plant defense responses. Furthermore, pre-treatment with nanomolar concentrations of Rosaceae Peps protected Prunus against Xap infection. These active doses can be explained because Peps act on the level of initial plant-pathogen signaling, where few molecules are sufficient to change the outcome of an interaction, and add to make Peps a promising tool to develop natural, targeted and environmentally friendly strategies to enhance the resistance against biotic attackers.
