A genetically encoded biosensor reveals spatiotemporal variation in cellular phosphate content in Brachypodium distachyon mycorrhizal roots

Arbuscular mycorrhizal (AM) symbiosis is accompanied by alterations to root cell metabolism and physiology, and to the pathways of orthophosphate (Pi) entry into the root, which increase with Pi delivery to cortical cells via arbuscules. How AM symbiosis influences the Pi content and Pi response dynamics of cells in the root cortex and epidermis is unknown. Using fluorescence resonance energy transfer (FRET)-based Pi biosensors, we mapped the relative cytosolic and plastidic Pi content of Brachypodium distachyon mycorrhizal root cells, analyzed responses to extracellular Pi and traced extraradical hyphae-mediated Pi transfer to colonized cells. Colonized cortical cells had a higher cytosolic Pi content relative to noncolonized cortical and epidermal cells, while plastidic Pi content was highest in cells at the infection front. Pi application to the entire mycorrhizal root resulted in transient changes in cytosolic Pi that differed in direction and magnitude depending on cell type and arbuscule status; cells with mature arbuscules showed a substantial transient increase in cytosolic Pi while those with collapsed arbuscules showed a decrease. Directed Pi application to extraradical hyphae resulted in measurable changes in cytosolic Pi of colonized cells 18 h after application. Our experiments reveal that cells within a mycorrhizal root vary in Pi content and Pi response dynamics

Constitutive Overexpression of RAM1 Leads to an Increase in Arbuscule Density in Brachypodium distachyon

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of plants and fungi of the subphylum Glomeromycotina. 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 subsequent bidirectional nutrient exchange, the root cortical cells undergo substantial transcriptional reprogramming. REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1), previously 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 plants showed higher arbuscule densities relative to controls, indicating the potential to 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 the scope of influence of BdRAM1, 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