Studies of plant innate immunity provide new functional insights on class IIa WRKY transcription factors and reveals a role for two Glucan Synthase-Like genes in gametophyte development

Chapter one: Plants have evolved a sophisticated innate immune system that is composed of multiple layers. The integration of signals derived from these layers constitutes a crucial prerequisite for efficient defence. Resistance (R) proteins serve as direct or indirect recognition receptors for pathogen-derived isolate-specific effector proteins. Members of the superfamily of WRKY transcription factors regulate plant responses towards pathogens either as activators or repressors. The barley (Hordeum vulgare) R protein MLA confers resistance towards the powdery mildew Blumeria gramins f. sp. hordeii. MLA physically interacts through its N-terminal coiled coil (CC) domain with the transcriptional repressors HvWRKY1 and HvWRKY2 in an effector-dependent manner. This effector-stimulated interaction provides a mechanistic model how plants can integrate defence-related signals from different recognition layers and thereby modulate expression of defence-associated genes. Arabidopsis thaliana lacks a functional homologue of MLA and is susceptible towards the powdery mildew Golovinomyces orontii. Similar to barley, mutations of the functional homologues of HvWRKY1 and HvWRKY2, namely AtWRKY18 and AtWRKY40, in Arabidopsis confer resistance towards G. orontii. In this work I analyzed structural and functional conservation between the transcriptional repressors from barley (HvWRKY1 and HvWRKY2) and their homologues in Arabidopsis (AtWRKY18 and AtWRKY40). My results revealed that AtWRKY18 and AtWRKY40 can associate via a conserved C-terminal motif with selective R-gene encoded proteins. Identification of the previously characterized R protein HRT, as putative interactor of AtWRKY18 and AtWRKY40, provides a suitable model for further studies. In addition, genetic studies using the Atwrky18 AtwrkyY40 double mutant identified differential requirements for the defence-related genes EDS1, CYP81F2, PEN2, PEN1 and PAD3 in pre- and post-invasive resistance towards G. orontii. The results support the central role of EDS1 in plant immunity and indicate a novel PEN2-independened function for CYP81F2 in post-invasive resistance. The solution structure of the MLA CC domain was used as a basis to further investigate MLA-dependent associations with HvWRKY1 and HvWRKY2. The crystal structure predicts homo-dimerization of the receptor in vivo. Analysis of structure-guided targeted amino acid substitution variants of MLA in yeast provided the first evidence for receptor self-association in vivo. Chapter two: Members of the Glucan Synthase-Like (GSL) family are believed to be involved in the synthesis of the cell wall component callose in specialized locations throughout the plant. I identified two members of the Arabidopsis GSL gene family, GSL8 and GSL10, that are independently required for male gametophyte development and plant growth. Analysis of gsl8 and gsl10 mutant pollen during development revealed specific malfunctions associated with asymmetric microspore division. GSL8 and GSL10 are not essential for normal microspore growth and polarity, but have a novel role in entry of microspores into mitosis. Impaired function of GSL10 also leads to perturbation of microspore division symmetry, irregular callose deposition and failure of generative cell engulfment by the vegetative cell cytoplasm. Silencing of GSL8 or GSL10 in transgenic lines expressing gene-specific dsRNAi constructs resulted in a dwarfed growth habit, thereby revealing additional and independent wild-type gene functions for normal plant growth.