|dc.description.abstracteng||Poplar is a model tree to study the molecular biology of woody plant species. It also has commercial benefits as it is used as a renewable energy resource. Poplar plantations suffer from poplar rusts caused by Melampsora fungi. Fungal pathogens are recognized by plants via LysM domain-containing receptors that perceive a major component of fungal cell walls, the β-1,4-linked N-acetylglucosamine polymer chitin, which functions as a pathogen associated molecular pattern (PAMP). Chitin perception triggers defense responses that contribute to plant immunity. The components of chitin receptors have been well characterized in some plant species such as the model plants Arabidopsis thaliana (At) and Oryza sativa (Os). In A. thaliana, the perception of chitin and initiation of chitin-induced immune responses require the LysM Receptor-like Kinase (LysM-RLK) CHITIN ELICITOR RECEPTOR KINASE 1 (AtCERK1). Two additional LysM-RLKs, AtLYK4 and AtLYK5 exhibit chitin-triggered AtCERK1-dependent phosphorylation and may function as co-receptors of AtCERK1. In rice, the LysM Receptor-like Protein (LysM-RLP) CHITIN ELICITOR BINDING PROTEIN (CEBiP), associates with OsCERK1 in order to trigger chitin-induced plant defense. AtLYM2, the homolog of CEBiP in A. thaliana, is not involved in AtCERK1-mediated chitin responses, but mediates chitin-induced suppression of plasmodesmal flux independent of AtCERK1. This study analyses chitin receptor components in the hybrid poplar P. x canescens (Pc) (P. tremula x P. alba). In Chapter 1, identification and functional characterization of poplar lysin motif receptor-like kinases (LysM-RLKs) encoded by AtLYK4/AtLYK5 homologs are described. In silico analysis revealed that four homologs of AtLYK4 and two homologs of AtLYK5 are present in the Populus trichocarpa (Pt) Nisqually genome. Of these, two PcLYK4 and one PcLYK5 homologs represent pseudogenes due to internal stop codons. The homologs PcLYK4-1, PcLYK4-2 and PcLYK5-2 have an open reading frame, which code for proteins with three extracellular LysM domains, a transmembrane domain and an intracellular, inactive kinase domain. All identified PcLYK4/PcLYK5 genes are expressed in roots, wood, developing xylem, bark and leaves. Subcellular localization studies by transient expression of the poplar PcLYK4/PcLYK5 genes in Nicotiana benthamiana as well as analyses of stably-transformed Arabidopsis thaliana showed that PcLYK4/PcLYK5 proteins localize at the cell periphery, indicating plasma membrane localization similar to their homologs in Arabidopsis. Functional characterization was carried out by testing the complementation of the Arabidopsis double knock-out mutant, Atlyk4-1 Atlyk5-2, which does not show chitin triggered reactive oxygen species (ROS) production and mitogen-activated kinase (MAPK) phosphorylation. Transgenic expression of PcLYK4-1, PcLYK4-2 and PcLYK5-2 partially restored the chitin sensitivity of Atlyk4-1 Atlyk5-2. Upon chitin treatment, PcLYK4/PcLYK5 proteins are internalized into endosomes. The chitin-triggered endocytotic removal of PcLYK5-2 was shown to be AtCERK1-dependent.
In Chapter 2, the role of poplar LYM2 homologs in chitin-triggered responses was analysed. Two AtLYM2 homologs, PcLYM2-1 and PcLYM2-2, were identified in poplar. The second homolog, PcLYM2-2, is subject to mutually exclusive splicing of the first exon, which generates two protein variants with distinct N-teminal LysM domains, designated as PcLYM2-2.1 and PcLYM2-2.2. All identified PcLYM2 proteins are likely GPI-anchored proteins. Analysis of the transcript abundance showed that all PcLYM2 genes are expressed in roots, wood, developing xylem, bark and leaves. PcLYM2-1 exhibits higher mRNA levels in all analysed tissues compared to PcLYM2-2. The differential splicing of PcLYM2-2 pre-mRNA is tissue-specific, resulting in high levels of PcLYM2-2.1 in wood and developing xylem in contrast to the high levels of PcLYM2-2.2 in bark and leaves. A chitin pull-down assay showed that all PcLYM2 homologs can bind the PAMP chitin, with PcLYM2-1 having the highest binding capacity compared to both PcLYM2-2 splicing variants. To analyze the role of PcLYM2 proteins in chitin signaling, poplar knock-out lines were generated through a CRISPR/Cas9 approach. Knocking out PcLYM2 genes in poplar did not affect chitin-induced ROS production and MAPK activation, suggesting that PcLYM2 is not involved in these two chitin-induced responses. Subcellular localization studies revealed that all identified PcLYM2 proteins localize at the plasma membrane and plasmodesmal-PM, indicating a plasmodesmata-related function. Plasmodesmal flux analysis with the Pclym2-1 Pclym2-2 double knock-out mutants suggests that PcLYM2 proteins mediate chitin-triggered plasmodesmal closure. Moreover, knocking out PcLYM2-1 is sufficient to abolish this response, suggesting that PcLYM2-1 plays a major role in poplar in particular for mediating this response. Individual functions of the two splicing variants PcLYM2-2.1 and PcLYM2-2.2 in the regulation of plasmodesmal flux or other ligand-induced processes remain to be identified. Expression levels in different tissues suggest that PcLYM2 proteins may have a potential to form tissue-specific complexes through homo- or heterodimerization depending on their abundance in the corresponding tissues.||de