Functional analysis of arbuscular mycorrhiza-related membrane transporter and defensin genes of Medicago truncatula

Abstract

The arbuscular mycorrhiza (AM) symbiosis, an interaction of ~80% of terrestrial plants and Glomeromycota fungi, dating back ~450 million years and promoting the colonization of land by plants, improves the nutrient supply of the host. Vice versa the mutualistic interaction itself is influenced by nutrient availability, which alters the cost-benefit-ratio. Thus, the expression of AM-related membrane transporter and selected marker genes was studied in Medicago truncatula roots, mycorrhized with Rhizophagus irregularis, supplied with different phosphate (P) and nitrogen (N) amounts. Here, the root colonization, as well as the rate of arbuscule formation, were enhanced by P depletion, implying a high impact of nutrient allocation on the symbiosis. More than 100 membrane transporter genes were induced in the first two weeks of the AM-interaction and their accumulated induction increased further during the next four weeks. Five representative candidate genes from highly AM-induced gene families, encoding copper (MtCopMd1), oligopeptide (MtOliMd1), ABC (MtABCG3), and nitrogen (MtAMT2;4) membrane transporters as well as a defensin (MtDefMd1) were investigated to study processes at the plant-fungal interface. Arbuscule-harboring cells displayed different spacio-temporal levels of MtCopMd1-, MtOliMd1-, and MtABCG3 promoter activities, indicating a diverging regulation during AM. In this context, MtCopMd1 and MtAMT2;4 promoters were also activated by a strong nitrogen depletion, whereas MtOliMd1 and MtABCG3 were solely expressed in a P-dependent manner. Furthermore, the selected membrane transporter genes were differentially repressed in mutant or RNAi knockdown roots, lacking key regulators of AM symbioses. In Medicago truncatula roots expressing artificial micro-RNAs that target MtOliMd1 and MtABCG3, MtRam1, encoding a key transcription factor regulating arbuscular branching was significantly less expressed. Contrasting this, a strong knock-down of MtAMT2;4 expression by RNA-interference did not change the transcription of selected AM marker genes. Nevertheless, MtAMT2;4 localization correlated with arbuscule structures. The heterologous expression of MtAMT2;4 in frog oocytes indicated that ammonia, rather than ammonium, might be the transported substrate. Finally, the expression of MtDefMd genes, encoding defensins with specific structural properties, were studied. Here, a core set of five defensin genes was induced over the time of fungal colonization of Medicago truncatula roots. MtDefMd1 and MtDefMd2 activation was placed relative to arbuscule formation and degradation, using mutants in key AM-activated regulator genes. Since cells with fully developed arbuscules displayed different levels of MtDefMd1 and MtDefMd2 promoter activities, they indicated an up-regulation towards later stages of arbuscule formation. Co-localization of an MtDefMd1-mGFP6 fusion with subcellular fluorescence markers revealed that this defensin is associated with arbuscules about to collapse, and ultimately is located in vacuolar compartments. Consecutively, two EXO70 genes, associated with vesicle targeting, were found to be repressed in MtDefMd1-overexpressing roots. By monitoring the expression and localization of different AM-related membrane transporter genes and their encoded gene products, this thesis provides novel insights on processes that occur in either active or degrading arbuscules. These are starting points for further studies of the cellular targeting towards symbiotic membranes during the biogenesis and in particular during the degradation of arbuscule structures.