Funktionale Analyse von Proteinen mit Dirigent- und Jacalin-ähnlichen Lektin-Domänen aus Reis und Arabidopsis thaliana im Rahmen der Pathogenresistenz
Esch, Lara; Schaffrath, Ulrich (Thesis advisor); Panstruga, Ralph (Thesis advisor)
Aachen (2019, 2020)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2019
Due to their sessile lifestyle plants are exposed to numerous biotic and abiotic stress factors. To react to the different environmental influences, plants have developed differentiated defense mechanisms. The initiation of a defense response is based, amongst others, on the specific recognition of conserved microbe-associated or damage-associated molecules that are released upon pathogen infection. Usually membrane bound receptors recognize these molecules. Lectins are proteins that specifically bind carbohydrates and proteins. Lectin domains can be part of theses membrane bound receptors. Recently mainly nucleocytoplamsatic lectins were associated with defense reactions. The jacalin-related lectin OsJAC1 of rice, as well as its orthologous proteins TaJA1 of wheat and HvJAC1 of barley, are involved in a broadspectrum resistance to various pathogens. These proteins that were found exclusively in the family of Poaceae, consist of an N-terminal dirigent and a C-terminal jacalin-related lectin (JRL) domain and were termend MonocotJRLs. Fusion proteins, like these MonocotJRLs, have specific functions that are based on the properties of the two domains. In this study the function of MonocotJRLs in pathogen resistance considering potential interaction partners of the lectin domain was characterized. Moreover, it was tested, whether proteins with a MonocotJRL-like function exist in the dicotyledonous model plant Arabidopsis thaliana. This study showed that the domain function of the three MonocotJRLs OsJAC1, TaAJ1 and HvJAC1 is conserved regarding the resistance to powdery mildew in barley even after domain swap. In a yeast two hybrid (Y2H) assay it was confirmed that the artificially separated dirigent and JRL domains of OsJAC1 physically interact with each other. To further characterize the function of the rice protein OsJAC1 in the barley powdery mildew resistance a split ubiquitin based Y2H experiment revealed putative interaction partners form barley and Blumeria graminis f. sp. hordei. The barley protein HORVU7Hr1H072050 was identified several times. Therefore, it was tested in an independent bimolecular fluorescence complementation assay as a putative OsJAC1 interaction partner. Different splicing varieties of this gene were tested. Nevertheless, there was no indication for an interaction of this protein with OsJAC1. Whether this is true for other potential OsJAC1 interaction partners that were identified in the Y2H experiment has yet to be tested. The majority of eukaryotic proteins consist of more than one domain. The combination of a dirigent domain and a JRL domain is specific for the MonocotJRLs and have not yet been found for example in Arabidopsis thaliana. Based on the Rosetta Stone theory, that states that there is the possibility of a functional cooperation of two proteins in one organism, when a fusion protein of these proteins exists in another organism, it is suspected that there are single domain proteins that functionally cooperate to function similar to MonocotJRLs. Therefore, in this study, numerous dirigent and JRL proteins from Arabidopsis thaliana were tested. Experiments where Arabidopsis candidate genes were overexpressed in barley showed that the protein pairs AtJAX1 and AtDIR19, AtJAL39 and AtDIR11 as well as AtJAL2 and AtDIR11 increase resistance of barley to powdery mildew similar to OsJAC1. AtJAX1 and AtDIR19 are localized directly neighbouring on the Arabidopsis genome. Nevertheless, in a BiFC experiment in Nicotiana benthamina no physical interaction of the proteins was detected. Moreover, the complementation of the Arabidopsis ecotype Col-0 that expresses AtJAX1 with a prematureIXStop codon, with the full-length AtJAX1 did not influence the resistance of these plants to powdery mildew. The AtJAX1-AtDIR19-overexpression-mediated powdery mildew resistance in barley is therefore probably subjected to different mechanisms than the MonocotJRL-mediated resistance. This is supported by the result of an experiment where the MonocotJRLs OsJAC1, TaJA1 and HvJAC1 were overexpressed in Arabidopsis thaliana, but resistance to powdery mildew was not increased. To analyse the candidate pair AtJAL39 and AtDIR11, the proteins were localized. The proteins localize in the cytoplasm of the cells. To further characterize the function of the dirigent and JRL proteins of Arabidopsis thaliana und confirm the hypothetical functional cooperation of the proteins the identification of interaction partners is an important step.