Análisis de las proteínas RIN4-like de Arabidopsis thaliana y de su interacción con proteínas involucradas en defensa frente a estreses bióticos

Abstract

Elevated CO2 and temperature as a consequence of climate change are altering interactions between plants and pests with important implications for ecology and agriculture. This is due to a positive effect on pest appearance as a result of a shortening of their life cycle and an increment on their host range. An example is the spider mite Tetranychus urticae, which is becoming a model of arthropod pest as it is able to feed on more than 1,100 plant species and has its entire genome sequenced and annotated. On the other hand, plants have evolved a sophisticated innate immune system to recognize these invading organisms and induce host defenses to achieve resistance. A component of this system in Arabidopsis thaliana is RIN4, an intrinsically disordered protein anchored to the plasma membrane. Its regions lacking stable structure function as a protein hub promoting protein interactions. It is also a post translational modification target of multiple bacterial effectors, which allows indirect recognition of a wide variety of pathogenic molecules and activates plant disease resistance (R) proteins. A. thaliana has 15 RIN4-like paralogous genes, four of which (AT2G17660, AT3G07195, AT3G48450 and AT5G48657) were found significantly overexpressed in A. thaliana rosettes infested with T. urticae in a RNAseq experiment performed in the laboratory. In this work, the sequences of these proteins were analyzed and compared. As RIN4, the proteins AT3G07195 and AT5G48657 have intrinsically disordered regions and two NOI (NO3-induced) domains, which play an important role in effector recognition and R protein activation. By contrast, AT2G17660 and AT3G48450 are shorter and only possess one NOI domain. Several defence related RIN4 interacting proteins were identified in an interaction repository. Four of these, R proteins RPM1, RPS2, RIPK and NDR1, were also overexpressed upon T. urticae infestation and were chosen to study their interactions with the RIN4-like proteins. Selected proteins were amplified and cloned into the yeast expression vectors pDEST22 and pDEST32 with the aim of performing a yeast two hybrid assay. Depending on the results, additional experiment went to be proposed regarding the consequences on the interactions of phosophorylations, partial deletions or in planta assays. Restrictions due to Covid-19 impeded bring to completion these experiments.