Functional analysis of the BEige and Chediak-Higashi domain gene MpSPIRRIG in Marchantia polymorpha.

BEige and Chediak-Higashi domain containing proteins (BDCPs) have been described to function in membrane-dependent processes in eukaryotes. This role was also observed for the BDCP SPIRRIG (SPI) in the model plant Arabidopsis thaliana in the context of cell morphogenesis. Additionally, AtSPI was found to control salt stress resistance by mediating mRNA stability and salt stress-dependent processing body formation. In this work, we utilize an evolutionarily comparative approach to unravel conserved, basal BDCP functions in the liverwort Marchantia polymorpha. Our phenotypic and physiological analyses show that MpSPI is involved in cell morphogenesis and salt resistance regulation, indicating that both functions are evolutionarily conserved between the two species. Co-localization was found with endosomal and P-body markers, suggesting links to membrane-dependent processes and mRNA metabolism. Finally, we present transcriptomics data showing that AtSPI and MpSPI regulate orthologous genes in A. thaliana and M. polymorpha.

Heat Stress-Dependent Association of Membrane Trafficking Proteins With mRNPs Is Selective.

The Arabidopsis AAA ATPase SKD1 is essential for ESCRT-dependent endosomal sorting by mediating the disassembly of the ESCRTIII complex in an ATP-dependent manner. In this study, we show that SKD1 localizes to messenger ribonucleoprotein complexes upon heat stress. Consistent with this, the interactome of SKD1 revealed differential interactions under normal and stress conditions and included membrane transport proteins as well as proteins associated with RNA metabolism. Localization studies with selected interactome proteins revealed that not only RNA associated proteins but also several ESCRTIII and membrane trafficking proteins were recruited to messenger ribonucleoprotein granules after heat stress.

Unravelling the molecular basis of the dominant negative effect of myosin XI tails on P-bodies.

The directional movement and positioning of organelles and macromolecules is essential for regulating and maintaining cellular functions in eukaryotic cells. In plants, these processes are actin-based and driven by class XI myosins, which transport various cargos in a directed manner. As the analysis of myosin function is challenging due to high levels of redundancy, dominant negative acting truncated myosins have frequently been used to study intracellular transport processes. A comparison of the dominant negative effect of the coiled-coil domains and the GTD domains revealed a much stronger inhibition of P-body movement by the GTD domains. In addition, we show that the GTD domain does not inhibit P-body movement when driven by a hybrid myosin in which the GTD domain was replaced by DCP2. These data suggest that the dominant negative effect of myosin tails involves a competition of the GTD domains for cargo binding sites.

A Comprehensive Toolkit for Quick and Easy Visualization of Marker Proteins, Protein-Protein Interactions and Cell Morphology in Marchantia polymorpha.

Even though stable genomic transformation of sporelings and thalli of Marchantia polymorpha is straightforward and efficient, numerous problems can arise during critical phases of the process such as efficient spore production, poor selection capacity of antibiotics or low transformation efficiency. It is therefore also desirable to establish quick methods not relying on stable transgenics to analyze the localization, interactions and functions of proteins of interest. The introduction of foreign DNA into living cells via biolistic mechanisms has been first reported roughly 30 years ago and has been commonly exploited in established plant model species such as Arabidopsis thaliana or Nicotiana benthamiana. Here, we report the fast and reliable transient biolistic transformation of Marchantia thallus epidermal cells using fluorescent protein fusions. We present a catalog of fluorescent markers which can be readily used for tagging of a variety of subcellular compartments. Moreover, we report the functionality of the bimolecular fluorescence complementation (BiFC) in M. polymorpha with the example of the p-body markers MpDCP1/2. Finally, we provide standard staining procedures for live cell imaging in M. polymorpha, applicable to visualize cell boundaries or cellular structures, to complement or support protein localizations and to understand how results gained by transient transformations can be embedded in cell architecture and dynamics. Taken together, we offer a set of easy and quick tools for experiments that aim at understanding subcellular localization, protein-protein interactions and thus functions of proteins of interest in the emerging early diverging land plant model M. polymorpha.

Neighboring plants divergently modulate effects of loss-of-function in maize mycorrhizal phosphate uptake on host physiology and root fungal microbiota.

Maize, a main crop worldwide, establishes a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi providing nutrients to the roots from soil volumes which are normally not in reach of the non-colonized root. The mycorrhizal phosphate uptake pathway (MPU) spans from extraradical hyphae to root cortex cells housing fungal arbuscules and promotes the supply of phosphate to the mycorrhizal host in exchange for photosynthetic carbon. This symbiotic association with the mycobiont has been shown to affect plant host nutritional status and growth performance. However, whether and how the MPU affects the root microbial community associated with mycorrhizal hosts in association with neighboring plants, remains to be demonstrated. Here the maize germinal Mu transposon insertion mutant pht1;6, defective in mycorrhiza-specific Pi transporter PHT1;6 gene, and wild type B73 (wt) plants were grown in mono- and mixed culture and examined under greenhouse and field conditions. Disruption of the MPU in pht1;6 resulted in strongly diminished growth performance, in reduced P allocation to photosynthetic source leaves, and in imbalances in leaf elemental composition beyond P. At the microbial community level a loss of MPU activity had a minor effect on the root-associated fungal microbiome which was almost fully restricted to AM fungi of the Glomeromycotina. Moreover, while wt grew better in presence of pht1;6, pht1;6 accumulated little biomass irrespective of whether it was grown in mono- or mixed culture and despite of an enhanced fungal colonization of its roots in co-culture with wt. This suggested that a functional MPU is prerequisite to maintain maize growth and that neighboring plants competed for AM fungal Pi in low P soil. Thus future strategies towards improving yield in maize populations on soils with low inputs of P fertilizer could be realized by enhancing MPU at the individual plant level while leaving the root-associated fungal community largely unaffected.

Mycorrhizal phosphate uptake pathway in maize: vital for growth and cob development on nutrient poor agricultural and greenhouse soils.

Arbuscular mycorrhizal fungi (AMF) form a mutually beneficial symbiosis with plant roots providing predominantly phosphorus in the form of orthophosphate (Pi) in exchange for plant carbohydrates on low P soils. The goal of this work was to generate molecular-genetic evidence in support of a major impact of the mycorrhizal Pi uptake (MPU) pathway on the productivity of the major crop plant maize under field and controlled conditions. Here we show, that a loss-of-function mutation in the mycorrhiza-specific Pi transporter gene Pht1;6 correlates with a dramatic reduction of above-ground biomass and cob production in agro-ecosystems with low P soils. In parallel mutant pht1;6 plants exhibited an altered fingerprint of chemical elements in shoots dependent on soil P availability. In controlled environments mycorrhiza development was impaired in mutant plants when grown alone. The presence of neighboring mycorrhizal nurse plants enhanced the reduced mycorrhiza formation in pht1;6 roots. Uptake of (33)P-labeled orthophosphate via the MPU pathway was strongly impaired in colonized mutant plants. Moreover, repression of the MPU pathway resulted in a redirection of Pi to neighboring plants. In line with previous results, our data highlight the relevance of the MPU pathway in Pi allocation within plant communities and in particular the role of Pht1;6 for the establishment of symbiotic Pi uptake and for maize productivity and nutritional value in low-input agricultural systems. In a first attempt to identify cellular pathways which are affected by Pht1;6 activity, gene expression profiling via RNA-Seq was performed and revealed a set of maize genes involved in cellular signaling which exhibited differential regulation in mycorrhizal pht1;6 and control plants. The RNA data provided support for the hypothesis that fungal supply of Pi and/or Pi transport across Pht1;6 affects cell wall biosynthesis and hormone metabolism in colonized root cells.