Titolo della tesi: The role of Berberine Bridge Enzyme-like proteins in the homeostasis of plant Damage-Associated Molecular Patterns (DAMPs) in Immunity and Development
Plants have an efficient and complex innate immune system capable of responding to biotic and abiotic stresses. Plant immunity relies on the recognition of danger signals through the action of specialized transmembrane receptors, known as PRRs (Pattern Recognition Receptors), which can recognize a plethora of pathogen-associated signals known as Pathogen/Microbe-Associated Molecular Patterns (PAMPs/MAMPs) i.e. “non-self” molecules that activate defense responses. Immune responses can also be triggered by Damage-Associated Molecular Patterns (DAMPs) i.e. endogenous molecules, released upon pathogen infection of mechanical injury, recognized as signals of an “altered-self”. Some DAMPs derive from the degradation of the cell wall polysaccharides, for example, the oligogalacturonides (OGs) from the pectic component homogalacturonan, and cellodextrins (CDs) from cellulose.
The recognition of both PAMPs/MAMPs and DAMPs activate the so-called Pattern-Triggered Immunity (PTI), which is considered the first level of plant immunity. Moreover, plants have evolved a second layer of immunity, the so-called ETI (Effector-Triggered Immunity), an accelerated, amplified and genotype-specific response that in most cases results in a hypersensitive response. Plants also possess the ability to induce, prime and amplify immune responses at distal sites, after a local attack.
Maintenance of immunity is costly and immune responses are typically counterbalanced by decreasing the biomass production. This effect likely reflects the natural trade-off existing between growth and defense. It is known that high levels of OGs negatively affect plant growth, likely as a consequence of an extensive activation of defense responses. The accumulation of OGs in vivo is favored by the interaction of microbial enzymes, polygalacturonases (PGs), with plant-derived inhibitors (PGIPs). In order to study the OGs biology in vivo, a specific biological tool has been generated in the laboratory where I performed my work, namely transgenic plants expressing an inducible PGIP-PG chimera, indicated as “OG-machine”, which allows the release on command of OGs in planta. An exaggerated immune response induced by OGs may be lethal for the plant, pointing to the need of homeostatic mechanisms which could prevent deleterious effects of high accumulation of OGs. A possible homeostatic control of DAMPs, and more specifically of OGs and CDs, has been recently identified in the laboratory where I carried out my thesis. Elicitor activity of OGs and CDs is strongly decreased by specific oxidases, namely OGOX1-4 and CELLOX1, respectively, belonging to the Berberine-Bridge Enzyme-like (BBE-like) family, which in Arabidopsis thaliana comprises 27 members. In particular, the OGOX1 gene encodes for two isoforms: the long isoform (OGOX1.1) and the short isoform (OGOX1.2), likely arising from an alternative splicing event.
The work of my thesis has been divided into two different parts. The first part aimed at defining the role of OGOX1 and CELLOX1 in the homeostasis of DAMPs and their involvement in immunity in A. thaliana. Moreover, the action of OGs and CDs in plant defense was investigated. By a reverse genetic approach, my results showed that over expression of OGOX1 decreases the OG-induced defense genes expression. CELLOX1 over-expressing plants also show some differences in response to defense gene expression induced by CD3, suggesting some alterations in the perception/transduction elements involved in the response to DAMPs. In addition, a possible synergic effect between OGs and CD3 on the activation of immune responses was investigated; however, only an additive action was observed when the two elicitors were simultaneously used for treatment. Histochemical analysis on POGOX1:GUS lines established that OGOX1 is induced, in basal condition, in the primary root as well as upon pathogen attacks, elicitor treatment and wounding in adult leaves. These results point to a role of OGOX1 in Arabidopsis immunity and development.
Moreover, I attempted to determine the sub-cellular localization of both the long and short OGOX1 isoform, upon GFP tagging at the N-terminus and using the native promoter (POGOX1::GFP-OGOX1). Unfortunately, confocal microscopy analysis, on stably transformed Arabidopsis lines, displayed a very low fluorescence pattern.
Finally, I have analyzed a still unexplored aspect of OGs biology: systemic OG-induced resistance (OG-IR), a systemic response induced upon treatment with OGs of few leaves and leading to pathogens resistance at distal, untreated plant tissue. I found that local OG treatment leads to protection against necrotrophic bacteria P. carotovorum at the local and systemic level in both wild-type and OGOX1-OE plants, and only at the systemic levels to B. cinerea in both wild-type and OGM plants.
In the second part of my thesis, I attempted to further elucidate the role of OGOX1 in immunity induced by OGs, by crossing plants expressing the OGM chimera to plants overexpressing OGOX1. It is expected that the simultaneous over expression of OGOX1, by oxidizing OGs and abolishing their elicitor action, could revert negative effects of the OGM on growth. No recovery of the root phenotype was observed in OGOX1-OE#11.8xOGM plants, while root length appeared recovered in OGOX1-OE#1.9xOGM line. Unfortunately, the observed recovery was due to lower OGM transcripts and protein levels in the double transgenic line compared to OGM seedlings, suggesting an apparent silencing of transgenes in the crossed plants.
Taken together my results demonstrate that OGOX1 is a regulator of OG homeostasis, and it is involved in plant growth and defense. Moreover, they reveal a role of exogenous and endogenous OGs in local and systemic protections against pathogens.
Further analyses are necessary to fully elucidate the role of other BBE-like enzymes in cell wall mediating signaling in plants and to clarify molecular mechanisms that take place in growth-defense trade-off. These results could open new perspectives to obtain plants more resistant to microbial diseases, maintaining normal growth.