Constitutive overexpression of RAM1 increases arbuscule density during arbuscular mycorrhizal symbiosis in Brachypodium distachyon

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of plants and fungi of the sub-phylum Glomeromycotina. The endosymbiotic AM fungi colonize the inner cortical cells of the roots, where they form branched hyphae called arbuscules that function in nutrient exchange with the plant. To support arbuscule development and subsequently bidirectional nutrient exchange, the root cortical cells undergo substantial re-programming, including major alterations in transcription. REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1), studied in several dicot plant species, is a major regulator of this cortical cell transcriptional program. Here, we generated ram1 mutants and RAM1 overexpressors in a monocot, Brachypodium distachyon. The AM phenotypes of two ram1 lines revealed that RAM1 is only partly required to enable arbuscule development in B. distachyon. Transgenic lines constitutively overexpressing BdRAM1 showed constitutive expression of AM-inducible genes even in the shoots. Following inoculation with AM fungi, BdRAM1-overexpressing roots showed higher arbuscule densities relative to controls, indicating the potential to directly manipulate the relative proportion of symbiotic interfaces via modulation of RAM1. However, the overexpressors also show altered expression of hormone biosynthesis genes and aberrant growth patterns including stunted bushy shoots and poor seed set. While these phenotypes possibly provide additional clues about BdRAM19s scope of influence, they also indicate that directed approaches to increase the density of symbiotic interfaces will require a more focused, potentially cell-type specific manipulation of transcription factor gene expression.