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

Abstract

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method using CRISPR/Cas9-mediated gene to generate callus carrying loss-of-function mutations in the MPG2 gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We show that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno- octulosonic acid (Kdo) and thus cannot form the α-L-Rha p -(1→5)-α-D-kdo p -(1→ sidechain. We suggest that MGP2 encodes an inverting CMP-β -K do transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization.