Abstract text: Cell adhesion, enabled by pectic polysaccharides, is critical for plant survival. Knockout mutations of genes involved in synthesising these polysaccharides are lethal, hindering their characterisation. A key polysaccharide essential for viability is rhamnogalacturonan-II (RG-II). RG-II, which forms borate diester-linked dimers, contains twelve sugar units and at least twenty unique glycosidic bonds. Recently, our group developed a gene-editing strategy in Arabidopsis thaliana callus to study these mutants, bypassing embryo lethality. Using this method, we identified the gene responsible for transferring 3-deoxy-D-octulosonic acid (Kdo) into RG-II, RG-II CMP-β-Kdo transferase (RCKT1), formerly known as Male Gametophyte Defective 2 (MGP2) or Sialyltransferase-like protein 1 (SIA1).

We are now applying this approach to characterise other genes predicted to participate in RG-II biosynthesis. We are combining heterologous expression and crystallisation of the candidate glycosyltransferases with the generation of these novel gene-edited mutant lines. To test heterologous protein activity, we are screening acceptor specificity using microscale thermophoresis (MST). To characterise gene-edited lines, we are analysing the cell wall composition of the mutants, with a focus on RG-II dimerisation and structure. We will present data describing our progress on these novel candidates, contributing to the dissection of RG-II assembly.