Functional analysis of PR-1RK receptors: novel genes associated to combat against pathogens

Abstract

Receptors-like kinases (RLKs) are proteins located on the cell membrane having an extracellular domain that captures the signals coming from outside the cell and transmitting them to the kinase domain, which in turn transduces this signaling to the intracellular environment. In plants, many of these receptors have the function of signaling to an attack of pathogens, helping the plant to defend itself. Among the proteins that act directly at the plant-pathogen interface, inhibiting the growth of invading microorganisms or reestablishing plant homeostasis, we highlight the PR proteins (pathogenicity related), a heterogeneous group of proteins whose PR-1 family is the most studied. PR-1 proteins are part of the SCP / TAPS superfamily present in all eukaryotes. For a long time, the biochemical activity of PR-1 was not known. However, it was recently found that they have the ability to bind to lipids, such as sterols and fatty acids. Interestingly, among the fourteen PR-1 genes found in the cacao genome, two genes (TcPR-1f and TcPR-1g) encode larger proteins whose C-terminal has high similarity with protein kinases. Between the PR-1 (SCP / TAPS) and kinase domains of TcPR-1f and TcPR-1g the existence of hydrophobic region was verified, strongly indicating the architecture of a RLK. These proteins were then termed PR-1RKs. From the analysis of gene expression, we detected that TcPR-1g was highly expressed during infection by Moniliophthora perniciosa, the fungus that causes Witches´ Broom Disease (WBD) of the cacao. Because of the importance of both PR-1 proteins and RLKs in relation to plant defense, the discovery of new RLKs in cacao (PR-1Ks) and the increase of expression of one of them during the progression of WBD, we attempted a functional and biochemical characterization of these proteins. To this end, transgenic Micro-Tom tomato plants overexpressing TcPR-1f and TcPR-1g will be produced, and will be submitted to resistance analysis against pathogens such as M. perniciosa S-type. In addition, recombinant proteins will be produced by overexpression in E. coli cells, separately representing the PR-1 and kinase domains. Isothermal titration calorimetry (ITC) experiments will be performed to determine possible ligands and autophosphorylation, and, mass spectrometry analyses will verify the autophosphorylative capacity of these proteins. (AU)