Small signals lead to big changes: deciphering the mechanisms behind peptide-induced resistance
- Autores: Julia Pastor Fernández
- Directores de la Tesis: Víctor Flors (dir. tes.), Paloma Sánchez Bel (codir. tes.)
- Lectura: En la Universitat Jaume I ( España ) en 2022
- Idioma: español
- Tribunal Calificador de la Tesis: Blanca San Segundo de los Mozos (presid.), Rosa Sánchez Lucas (secret.), Juan Antonio López Ráez (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
In nature, plants are constantly challenged by environmental biotic and abiotic factors. To cope with biotic stresses, such as pest and pathogen attacks, they have evolved a broad variety of adaptative defense strategies. Sometimes plant perception of external stimuli induces an enhanced resistance state that confers protection against a future attack in local and distal tissues. This state is known as “Induced Resistance” and can be triggered by both biological and non-biological stimuli. Recently an increasing number of plant peptides were described as defense elicitors that act as secondary danger signals or phytocytokines. They are released upon pest or pathogen attack and bind to membrane receptors triggering an amplification of immune responses. However, their potential as defense elicitors is poorly studied.
In this thesis, we found that peptides from different species can induce resistance against the necrotrophic fungus Plectosphaerella cucumerina in the taxonomically distant species Arabidopsis thaliana at very low concentrations. This induction of resistance was due to the stimulation of the plant immune system since antifungal in vitro assays revealed that they do not display direct antifungal activity. An analytical method for multiple peptides identification and quantification was developed. Noteworthy, of the tested peptides Systemin conferred a high degree of protection in Arabidopsis from very low concentrations, showing an optimal threshold of action, resembling the mode of action of a phytohormone or other IR elicitors. Thus, the following analyses were focused on deciphering the mechanisms of Systemin-Induced Resistance (Sys-IR). Systemin is a short peptide that regulates the plant response against herbivores in tomato plants. It is released upon wounding or pathogen attack and induces a cascade of plant defenses that produce the accumulation of protease inhibitors in local and systemic tissue. There is also evidence of the involvement of Systemin in tomato defenses against pathogens such as the necrotrophic fungus Botrytis cinerea. However very little is known about the perception and function of Systemin in heterologous species.
Analysis of the hormonal regulation of Systemin triggered defenses in Arabidopsis revealed that, like in tomato, JA but not SA was implicated in Sys-IR. In an attempt to unveil the perception of Systemin in Arabidopsis we found that Pep1 receptor PEPR1, the homolog of Systemin in Arabidopsis, was not responsible for the Systemin signal transduction in this species. Regarding early signaling triggered by Systemin upon infection, BAK1, BIK1, AGG2, RBOHD, MPK3, and MPK6 gene expression displayed a priming profile. In addition, Systemin primed MPK3 and MPK6 phosphorylation and ROS production upon a PAMP challenge. Lost of function mutants of the mentioned genes were impaired in the enhanced resistance triggered by Systemin treatment, demonstrating their key role in Sys-IR against P. cucumerina. To understand the metabolic fingerprint of Systemin in Arabidopsis and the possible mechanisms behind Sys-IR, we performed a non-targeted metabolomic analysis of Systemin-treated plants before and after fungus infection in Arabidopsis plants. This analysis revealed, on the one hand, that phenolic compounds were overaccumulated upon Systemin treatment and, on the other hand, that Systemin primed specific indolic compounds. By gene expression and knock-out mutant analysis, we confirmed that flavonoids and tryptophan-derived compounds were essential elements in expressing functional Sys-IR.
Our next goal was to shed light on the mechanisms of Sys-IR in its species of origin, tomato, in order to find metabolic fingerprints of the Systemin mode of action as a resistance inducer. Previous studies by other authors have demonstrated how endogenous levels of the Systemin precursor, ProSystemin, influence plant resistance against pests and pathogens, however, knowledge of the effects of exogenously applied Systemin is very scarce. In the present study, we found that exogenous treatment of Systemin has a great impact on the plant metabolism at different metabolic levels and triggered enhanced resistance to the necrotrophic fungi Botrytis cinerea through priming of callose deposition. Systemin treatment strongly affected the behavior of proteins and enzymatic activities of the primary metabolism participating in the photosynthesis, carbohydrate metabolism, TCA cycle, glycolysis, and amino acid metabolism. These changes lead to the accumulation of available sugars monomers and carbon structures, including tricarboxilic acids. The overaccumulation of a starch phosphorylase, a glucan synthase-like, and callose synthase-like proteins together with a higher starch degradation in Systemin-treated plants could explain the observed priming of callose. Additionally, proteins involved in redox homeostasis and the biosynthesis of the phenolic compound were induced by Systemin. Conversely, after infection, very few changes were observed in the proteomic profile. However, these few proteomic changes were very specific for pathogen defense including pathogen-related proteins 1 and 4 (PR1 and PR4) and a 1,3-𝛃-glucanase (PR2). Unlike in the proteomic profile, major changes in the metabolome were observed after infection, showing a clear priming profile. Some amino acids, phenolic compounds, and alkaloids were identified among the metabolites induced or primed by Systemin, which correlated well with the results obtained in the proteomic analysis. On the other hand, many metabolites showed a buffering effect towards infection, while they over-accumulated in control plants upon infection, they remained at the same levels of uninfected control or even lower in Systemin-treated plants following infection. A similar pattern was observed in the enzymatic activities in infected plants, especially at late time points after pathogen challenge.
Finally, we aim to find early signaling events that enable downstream Systemin triggered responses to ensure Sys-IR against B. cinerea in tomato plants. Tomato MPK1, MPK2, and MPK3, orthologs of Arabidopsis MPK3 and MPK6, which were previously reported to be involved in tomato plants’ defense responses, were selected for further analysis. In the present work, we found that MPKs phosphorylation was primed by Systemin treatment upon a fungal PAMP challenge demonstrating its involvement in Sys-triggered defense responses. Analysis of MPK1/2 and 3 silenced plants by using the Virus-Induced Gene Silencing (VIGS) technique revealed that MPKs act upstream Systemin triggered induction of defense genes in the absence and presence of infection, including JA-related genes and genes involved in the Systemin production and release. Additionally, silenced plants in either MPK1 and 2 or MPK3, showed impaired Systemin response and protection against Botrytis cinerea, confirming that MPKs are essential signaling elements to ensure functional Sys-IR.
