set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula

• Published in: The Plant journal : for cell and molecular biology 2014-12, Vol.80 (6), p.1151-1163
• Main Authors: Ivanov, Sergey, Harrison, Maria J
• Format: Article
• Language: English
• Subjects: actin; apoplast; Biological Transport; Biomarkers - metabolism; Cell Membrane - metabolism; Cytoskeleton; Cytoskeleton - metabolism; endoplasmic reticulum; endosymbionts; fluorescence; fluorescent proteins; fungi; Gene Expression Regulation, Plant; GFP; Golgi apparatus; hyphae; Hyphae - genetics; legume; Legumes; mCherry; Medicago truncatula; Medicago truncatula - genetics; Medicago truncatula - microbiology; Membranes; microtubules; Microtubules - metabolism; mitochondria; Mycorrhizae - physiology; nodulation; Organelles - metabolism; periarbuscular membrane; peroxisomes; Phosphate Transport Proteins - genetics; Plant biology; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Roots - genetics; Plant Roots - metabolism; Plants, Genetically Modified; plasma membrane; plasmodesmata; Plastids; Promoter Regions, Genetic - physiology; Proteins; root; secretion; Symbiosis; technical advance; tonoplast; trans-Golgi Network - metabolism; vesicular arbuscular mycorrhizae
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.12706

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.