A putative rhamnogalacturonan-II CMP-β-Kdo transferase identified using CRISPR/Cas9 gene edited callus to circumvent embryo lethality

Abstract

The pectin rhamnogalacturonan II (RG-II) is an extraordinarily complex plant carbohydrate, crucial for developmental processes. RG-II contains over 20 distinct glycosidic linkages involving 12 distinct sugars; its biosynthesis is predicted to require numerous glycosyltransferases (GTs). RG-II’s low abundance in the plant cell wall belies its vital role in plant development. Minor structural modifications lead to lethality or severe growth impairment, posing significant challenges for GT identification via reverse genetics. Here we developed a novel method to generate viable loss-of-function Arabidopsis mutants in callus tissue via gene editing. We combined this with a candidate gene approach to characterize the GT29 RCKT1/MGP2. Analysis ofrckt1callus revealed a loss of 3-deoxy-D-manno-octulosonic acid (Kdo) from RG-II sidechain C, suggesting RCKT1’s role as theRG-IICMP-β-Kdo transferase1. RCKT1 becomes only the second confirmed GT implicated in RG-II biosynthesis. This discovery provides insight into RG-II’s structural impact on plant cell walls, as well as a method to further uncover the machinery required for the synthesis of this enigmatic polymer.