Functional analysis of arbuscular mycorrhiza-related GRAS transcription factor genes of Medicago truncatula

Abstract

The arbuscular mycorrhizal (AM) symbiosis is a widespread beneficial association of vascular plants with different mycorrhizal fungal species. It can be found in ~ 80 % of recent land plants and improves the plant’s nutrient supply, in particular with respect to phosphorus. The colonization of roots by AM fungi and the establishment of a nutrient exchange interface is conducted by a significant transcriptional reprogramming of the host cells. In the last years, transcriptomic studies on mycorrhizal roots revealed insights into this reprogramming and delivered substantial numbers of genes upregulated in AMF colonized roots. However, these studies provided only snapshots of the transcriptional program at a distinct time point in the development of the AM symbiosis. To overcome this drawback, this thesis obtained insights into the chronological development of AM-induced gene expression patterns. By using Affymetrix Medicago GeneChips, gene expression levels were monitored during the ongoing process of AMF colonization, revealing distinct gene expression patterns that reflect transcriptional regulations from 0 to 42 days post infection. Different functional classes relevant for AM function were found upregulated in the AM time course, including 12 genes encoding transcription factors (TFs) of the GRAS family. Based on their chronologic expression patterns, these GRAS TF genes were grouped in two major expression categories. Yeast Two-Hybrid approaches were used to identify protein-protein interactions of GRAS TFs with other AM-related proteins and also amongst the GRAS TFs themselves. These studies revealed interactions between MtGras1 and MtGras4, as well as MtGras4 and MtRad1. Furthermore, MtGras1 appeared to form homodimers. To functionally characterize MtGras genes strongly upregulated during the time course of AM development, promoter-gusAint studies were performed in transgenic roots of mycorrhizal wild type plants as well as MtRam1 and MtPt4 knockout mutants, showing defects in arbuscule branching and the formation of active, P-transporting arbuscules, respectively. These experiments revealed differential dependencies amongst these genes or from the developmental stages of AM formation in which they are active. While MtRad1, MtGras6, and MtGras4 are expressed independently of MtRam1 and MtPt4 in AMF-colonized roots, MtGras1 and MtGras7 are highly depending on MtRam1 and MtPt4, suggesting a role in later stages of arbuscule development. Knockdown of MtGras1 led to a downregulation of several MtGras genes as well as other genes correlated with AM formation. The identification of a GRAS TF acting in later stages of arbuscule development that nevertheless also regulates elements of the earlier stages of arbuscule formation led to the establishment of a model proposing a regulatory feedback loop. In this scenario, MtGras1 acts as a checkpoint for proper AM-related signaling, possibly supporting and accelerating arbuscule development via feedback regulation. One of the MtGras genes regulated by MtGras1 is MtGras7, being active in the later stages of arbuscule development. MtGras7 was further shown to be regulated by MtGras4 via analysis of an MtGras4 knockout line. The prerequisite for MtGras7 expression would thus be set by an MtGras4 action in the earlier stages und would be even accelerated by MtGras1 upon progression of arbuscule development. Taken together, a regulatory network of GRAS TFs is proposed, integrating different unidirectional as well as feedback regulatory mechanisms that together modulate arbuscule development.