LYM2 mediates chitin-induced plasmodesmal flux reduction in Populus x canescens

Introduction Lysin motif domain-containing glycosylphosphatidyl inositol-anchored protein 2 (LYM2) is a GPI-anchored LysM receptor-like protein (LysM-RLP) that harbors three extracellular LysM-domains and a GPI moiety at the C- terminus. In rice, the LYM2 homolog CEBiP is part of the canonical chitin receptor complex mediating chitin triggered MAPK activation and ROS burst. In the annual plant Arabidopsis thaliana , AtLYM2 is specifically involved in chitin-mediated plasmodesmal flux regulation. The aim of this study is to analyse components of chitin perception in the perennial plant poplar and to explore if LYM2 proteins in dicots are generally involved in chitin mediated PD closure. Methods A BLAST search identified LYM2 orthologs in the genomes of Populus trichocarpa and Populus x canescens . The poplar paralogs PcLYM2–1 and PcLYM2–2 were subsequently characterized on the molecular and functional level using chitin affinity purification and protein-tagging with fluorescent labels. The chitin response of knockout lines generated via the CRISPR/Cas9 approach was examined with particle bombardment assays. Results PcLYM2–2 exhibits tissue-specific alternative splicing, resulting in variants that differ exclusively at the first exon encoding the three LysM domains. Chitin-binding assays showed that all identified PcLYM2 proteins bind chitin. Subcellular localization studies in Nicotiana benthamiana indicated that all identified PcLYM2 proteins localize at plasmodesmata, suggesting a specific subcellular function of these proteins. Plasmodesmal flux analysis of wildtype poplar and Pclym2–1 Pclym2–2 double knockout lines demonstrated that PcLYM2 proteins mediate chitin-triggered PD closure. Loss-of-function of PcLYM2–1 is sufficient to abolish chitin-induced PD closure, suggesting that PcLYM2-1 is an essential LysM-RLP involved in this process. Conclusion Poplar regulates plasmodesmal flux after chitin detection through LYM2 orthologs. The expression of LYM2 variants may modify chitin perception and signaling in different tissues of poplar.