Functional analysis of Zinc finger proteins in the arbuscular mycorrhiza symbiosis of Medicago truncatula suggests a regulatory involvement of the PALM1 transcription factor

Abstract

The arbuscular mycorrhizal (AM) symbiosis is a widespread beneficial association of ~80% of vascular plant species with a variety of soil-dwelling fungi. Establishing and maintaining this symbiosis and in particular the arbuscule as its central intracellular interface requires major transcriptional reprogramming of the host cells in the root cortex. Although major efforts have been successful in identifying key members of signaling pathways contributing to the regulation of the AM symbiosis,mechanisms for finetuning the association with respect to the hormonal and nutritional status of the host plant are still largely unknown. As part of this study, four Medicago truncatula genes encoding Zinc finger (ZF) proteins were identified to be transcriptionally upregulated in roots colonized by Rhizophagus irregularis. Of these genes, MtZf1, MtZf2 and MtZf3 were not characterized prior to this study, while MtPalm1, encoding a C2H2-family transcription factor, had been identified to act as a regulator in M. truncatula compound leaf development. As part of a functional analysis, the activity of these genes was confirmed to be strongly correlated with the growth of intraradical AM fungal structures via transcript measurements and histochemical localizations. These studies also indicated the involvement of upstream/overlapping ORFs in the regulation of these genes, in particular MtPalm1. Additionally, three separate gene silencing experiments via RNA-interference demonstrated that roots deficient in MtPALM1, MtZF1 and MtZF2 were quantitatively hampered in AM fungal colonization, with functional disruption of MtPalm1 yielding average ~50% reductions in arbuscule formation. These phenotypes were confirmed for MtPalm1 and MtZf2 in stable Tnt1-insertion mutant lines and could be rescued through complementation via the introduction of a functional gene copy. Transcriptome profiles obtained from MtPALM1-deficient mycorrhized roots suggested this TF to act as a potential link between AM-symbiotic signaling involving strigolactones and other phytohormones, most crucially auxin. Conversely, heterologous protein interaction studies via a Yeast 2-Hybrid library mating suggested that the putative RING domain protein MtZF1 functions in targeted ubiquitination prior to AM fungal colonization. Furthermore, promoter-binding assays performed in a Yeast 1-Hybrid system gave indications of the position of the four ZFs studied in known AM regulatory networks, with protein-DNA interactions pointing to an involvement of key regulators such as RAM1, NSP2 and AMrelated NF-Y-proteins in the regulation of MtPalm1-, MtZf1- and MtZf3-expression. As a whole, the present thesis provides novel insights into the previously unknown contributions of ZF proteins to the fine-tuning of AM-symbiotic interactions, including the surprising role of the leaf developmental regulator MtPALM1 in modulating the level of AM-colonization, possibly by integrating hormonal signals during mycorrhization.