<i>OsRAM2</i> Function in Lipid Biosynthesis Is Required for Arbuscular Mycorrhizal Symbiosis in Rice

Arbuscular mycorrhiza (AM) is a mutualistic symbiosis formed between most land plants and Glomeromycotina fungi. During symbiosis, plants provide organic carbon to fungi in exchange for mineral nutrients. Previous legume studies showed that the <i>required for arbuscular mycorrhization2</i> (<i>RAM2</i>) gene is necessary for transferring lipids from plants to AM fungi (AMF) and is also likely to play a "signaling" role at the root surface. To further explore <i>RAM2</i> functions in other plant lineages, in this study, two rice (<i>Oryza sativa</i>) genes, <i>OsRAM2</i> and <i>OsRAM2L</i>, were identified as orthologs of legume <i>RAM2</i>. Examining their expression patterns during symbiosis revealed that only <i>OsRAM2</i> was strongly upregulated upon AMF inoculation. CRISPR/Cas9 mutagenesis was then performed to obtain three <i>Osram2</i> mutant lines (<i>-1</i>, <i>-2</i>, and <i>-3</i>). After inoculation by AMF <i>Rhizophagus irregularis</i> or <i>Funneliformis mosseae</i>, all of the mutant lines showed extremely low colonization rates and the rarely observed arbuscules were all defective, thus supporting a conserved "nutritional" role of <i>RAM2</i> between monocot and dicot lineages. As for the signaling role, although the hyphopodia numbers formed by both AMF on <i>Osram2</i> mutants were indeed reduced, their morphology showed no abnormality, with fungal hyphae invading roots successfully. Promoter activities further indicated that <i>OsRAM2</i> was not expressed in epidermal cells below hyphopodia or outer cortical cells enclosing fungal hyphae but instead expressed exclusively in cortical cells containing arbuscules. Therefore, this suggested an indirect role of <i>RAM2</i> rather than a direct involvement in determining the symbiosis signals at the root surface.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.