Characterization of physiological and molecular responses in common bean upon "Pseudomonas syringae" pv. "phaseolicola" infectionthe role of pectins in the defensive processes

Resumen

Plants are normally subjected to all kinds of stresses, including those caused by pathogens, which make their survival difficult. Despite being sessile organisms, plants have developed a true complete immune system to combat these attackers, which is tight regulated. Thus, during the attack of a pathogen, the plant cells trigger a series of mechanisms that, if efficient, prevent the spread of the pathogen by the plant and therefore the disease. One of the quickest responses is the release of hydrogen peroxide and other reactive oxygen species (ROS), which serve as antimicrobials but also as signaling molecules along with calcium. This signaling induces the rapid hormones release, such as salicylic acid (SA) in the case of biotrophic pathogens, which trigger a hypersensitive response (HR) that blocks the advance of the pathogen. If the plant is resistant to the pathogen, it will be very efficient and quick in activating these mechanisms, and it will quickly block the advance of the pathogen. Most of these processes have been studied in plants resistant to a certain pathogen, but little is known about what happens during an interaction where the plant is susceptible, which would be interesting in order to develop strategies to reduce the disease. Furthermore, in these immunity processes, the cell wall, being the first physical barrier, plays an important role. Whether or not the pathogen is able to penetrate the cell wall depend on the ability of the plant to remodel the architecture of the cell wall and, consequently, its physicochemical properties. One of the pathosystems that can serve as a model to learn more about pathogenicity processes is that formed by common bean (Phaseolus vulgaris L.) and Pseudomonas syringae pv. phaseolicola (Pph), which causes halo blight disease. The main symptoms are chlorosis, growth problems and characteristic spots on leaves, stems and pods, which leads to lower production. The study of this disease physiologically is very important for the León region, since nearly 50% of the national common bean production comes from its PGI. Of all its varieties, the Riñón variety is the most appreciated and profitable economically, but it is very susceptible to the bacteria attack. This thesis has aimed to obtain more information on the physiological processes that could explain the susceptibility of the common bean Riñón variety to Pph, focusing on the main role that cell wall remodeling, especially pectins, can have to obtain a solution that somehow activates the common bean immunity. Our work hypotheses are that Pph inhibits the Riñón common bean immune system at some point during the infection, probably from the start, which triggers the disease. Knowing this inhibiting mechanism or finding the point of the defensive pathway that is blocked, it would be possible to find a solution to avoid it. On the other hand, if we are able to activate or reinforce the common bean defense system, it would be possible to reduce the effects of the disease or even avoid it completely. Furthermore, we believe that the cell wall may play a key role in pathogen defense and recognition. To address these studies, we have carried out a series of in vitro experiments in order to force the infection with the bacteria, avoiding interference with other stresses, and to be able to study what happens in the first moments of the infection both at a physiological and molecular level. In addition, experiments have been carried out to elicit the immune system of the common bean, both with chemical compounds such as INA, and with plant-based preparations (PBPs), and an analysis has been made of the changes that occur in the common bean cell wall after infection and/or elicitation of the plant. Consequently, 4 partial objectives were established, which constituted each of the chapters of this thesis. Objective 1: to study the perception and first defensive responses that are activated in the common bean Riñón variety after Pph infection (chapter I). For this, some typical responses of PTI were analyzed, such as the ROS burst and the calcium influx, antioxidant activities, defense-related phytohormones, such as salicylic and abscisic acid, and the expression analysis of defense-related genes. In addition, 6 WAK receptors were identified in silico, whose expression was also evaluated. As a result, it was found that at least PvWAK1 increases its expression within minutes after infection, which could indicate a possible role in defense. In addition, there was an increase in total and apoplastic H2O2 and an increase in PR1 expression, but no activation of the antioxidant system and, above all, no SA peak was detected, which probably prevented HR activation. In our common bean variety, the lack of these defensive mechanisms led to an early cell damage and high susceptibility to Pph. The fact that PvWAK1 expression increased after Pph infection could indicate that the effectors of the bacteria did not inhibit the expression of this receptor, at least in early stages, and could be interesting to consider it for breeding or genetic engineering programs. Finally, this study also encourage to investigate whether the application of oligomers to activate defense responses in common bean Riñón variety through PvWAK receptors. Objective 2: to find out if the immune system of this common bean variety could be activated prior to infection, and if this activation rendered, such as Pph inoculation, a remodeling of the cell wall (chapter II). For this, plants pretreated or not with a structural analog of SA, called INA, were inoculated or not with Pph, and an analysis of their cell walls and activation of the defense system through ROS release was performed. As a result, the activation of the immune system with INA produced a higher amount of ROS after incubation with flg22, and INA-treated plants were more resistant to Pph infection. Pph inoculation modified the cell wall, but to a lower extent than INA-priming. In general, the Pph infection did not modify the cellulose content, but it did increase that of pectins (homogalacturonan and rhamnogalacturonan I) in pectic fractions (CDTA and Carbonate), and that of hemicelluloses (arabinoxylans) in the fraction of hemicelluloses strongly bound to the cell wall (KII fraction). The molecular weight of the polysaccharides increased mainly in the carbonate fraction, but the cell wall was found to be as enzymatically hydrolysable as the cell wall extracted from control plants. In contrast, INA treatment produced a drastic remodeling of the extracted cell walls. The cellulose content increased, there was a displacement among the fractions, as well as an increase in the molecular weight of the extracted polysaccharides in each fraction. This could be related to an increase in both pectins and extensins in all fractions, which produced a greater crosslinking between polysaccharides, rendering a cell wall more resistant to enzymatic hydrolysis. The subsequent inoculation with Pph in INA-pretreated plants did not modify the cell walls with respect to those only INA-treated. In addition, the plants primed with INA showed less leave damage than mock plants after Pph inoculation. Objective 3: to obtain information of the genes involved in the early response of common bean Riñón variety to Pph. For this, 15-day-old common bean plants were inoculated or not with Pph, and 2 or 9 hours after the infection, the RNA was extracted to perform a transcriptomic analysis by RNAseq (chapter III). At 2 hours post infection, an overexpression of defense-related genes was observed, but this increase was not sustained. This was also observed when defense-related phytohormones were quantified, such as salicylic, jasmonic and abscisic acids, and in the expression of cell wall related genes. The analysis of cell wall monosaccharides revealed an increase in the sugars content at 2 hours post-infection, but decreased again at 9 hours, except for GalA, which suggested an increase in pectin synthesis or a reduction in its degradation. This second option was confirmed by observing that most of the activities related to pectin degradation (PL, PG and PME) had decreased, while the expression of genes related to PME inhibitory enzymes (PMEIs) increased. This could be a protective strategy by the plant against the Pph pectin-degrading activities, as it has been observed in this work. Another possible strategy for protecting the integrity of the pectins, and therefore of the cell wall, could be the formation of egg-box complexes, as we have observed by immunohistochemistry. For the formation of these egg-box complexes, demethylation of pectins is necessary and although general PME activity decreased, some genes for PMEs, such as the Arabidopsis ortholog PvPME17, increased its expression. In addition, an explanation for the decreased of PME activity could be the overexpression of PMEIs. Among others, PvPMEI3, the Arabidopsis ortholog of PMEI3, was the most up-regulated. The functional analysis of this gene was evaluated in Arabidopsis pmei3 mutants, which showed susceptibility to Pph that Col-0 did not have. Finally, PvPMEI3 was shown to have PMEI activity, and it could play a key role in the resistance of common bean to Pph infection. Finally, chapter IV was spent in screening plant-based preparations for their ability to inhibit the Pph growth, as well as testing whether any of them were capable of activating the plant immune system, evaluating some of the physiological parameters and defense-related molecular factors previously studied. In summary, 4 plant-based preparations were tested, of which 2 were the most effective (grapevine pomace and nettle) in inhibiting Pph growth. Only the treatment with nettle led to the activation of some defense mechanisms, such as redox signaling processes and increased expression levels of defense-related genes, especially PR1. The common bean plants treated with these products did not decrease their final production. The first in vitro tests also suggest that the application of nettle can protect the bean from infection with Pph. All these results suggest that plant-based preparations from Urtica dioica could be used in the near future to diminish the halo blight consequences. To summarize, this thesis has deepened into the knowledge of interacting mechanisms of common bean Riñón variety-Pph pathosystem, showing some of the possible causes of susceptibility, such as the lack of SA-production shortly upon infection, which possibly prevents the common bean reinforcement of its cell wall and activates other defense mechanisms such as HR and SAR. In fact, we have shown that common bean can be elicited with a structural analog of SA, which leads to a strong cell wall reinforcement that makes the plant less susceptible to Pph and has a more reinforced cell wall with pectins and extensins, and therefore less enzymatically hydrolysable. The role of pectins in the defensive mechanisms has also been demonstrated through the transcriptome analysis that we have carried out in this work, which we have verified that some enzymes involved in the metabolism and modification of pectins play a crucial role in the defensive processes, such as PMEI3, whose mutation in Arabidopsis plants leads to the susceptibility to Pph. Finally, we have verified that the elicitation of the plant immune system is also possible through plant-based products, which opens a very interesting way for the development of ecological phytosanitary products that can be applied in field.