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Abstract Reverse genetics, facilitated by CRISPR technologies and comprehensive sequence-indexed insertion mutant collections, has advanced the identification of plants genes essential for arbuscular mycorrhizal (AM) symbiosis. However, a mutant phenotype alone is generally insufficient to reveal the specific role of the protein in AM symbiosis and in many cases, identifying interacting partner proteins is useful. To enable identification of protein:protein interactions during AM symbiosis, we established aMedicago truncatula -Diversispora epigaeayeast-two-hybrid (Y2H) library which, through Y2H-seq screening, can provide a rank-ordered list of candidate interactors of a protein of interest. We also developed a vector system to facilitate bimolecular fluorescence complementation assays (BIFC) in mycorrhizal roots so that protein interactions can be assessed in their native cell types and sub-cellular locations. We demonstrate the utility of a Y2H-seq screen coupled with BIFC in mycorrhizal roots, with a search for proteins that interact with CYCLIN DEPENDENT LIKE KINASE 2 (CKL2), a kinase essential for AM symbiosis. The Y2H-seq screen identified three 14-3-3 proteins as the highest ranked CKL2 interacting proteins. BIFC assays in mycorrhizal roots provided evidence for a CKL2:14-3-3 interaction at the periarbuscular membrane (PAM) in colonized root cells. Down-regulation of 14-3-3 by RNA interference provides initial evidence for a function in AM symbiosis. Thus, CKL2 may utilize 14-3-3 proteins to direct signaling from the PAM. The Y2H and BIFC resources will accelerate understanding of protein functions during AM symbiosis. 

The mutualistic association between plants and arbuscular mycorrhizal (AM) fungi requires intracellular accommodation of the fungal symbiont and maintenance by means of lipid provisioning. Symbiosis signaling through lysin motif (LysM) receptor-like kinases and a leucine-rich repeat receptor-like kinase DOES NOT MAKE INFECTIONS 2 (DMI2) activates transcriptional programs that underlie fungal passage through the epidermis and accommodation in cortical cells. We show that twoMedicago truncatulacortical cell–specific, membrane-bound proteins of a CYCLIN-DEPENDENT KINASE-LIKE (CKL) family associate with, and are phosphorylation substrates of, DMI2 and a subset of the LysM receptor kinases.CKL1andCKL2are required for AM symbiosis and control expression of transcription factors that regulate part of the lipid provisioning program. Onset of lipid provisioning is coupled with arbuscule branching and with the REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1) regulon for complete endosymbiont accommodation. 

Abstract Over 70% of vascular flowering plants engage in endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. VAPYRIN (VPY) is a plant protein that is required for intracellular accommodation of AM fungi but how it functions is still unclear. VPY has a large ankyrin repeat domain with potential for interactions with multiple proteins. Here we show that overexpression of the ankyrin repeat domain results in a vpy -like phenotype, consistent with the sequestration of interacting proteins. We identify distinct ankyrin repeats that are essential for intracellular accommodation of arbuscules and reveal that VPY functions in both the cytoplasm and nucleus. VPY interacts with two kinases, including DOES NOT MAKE INFECTIONS3 (DMI3), a nuclear-localized symbiosis signaling kinase. Overexpression of VPY in a symbiosis-attenuated genetic background results in a dmi3 -like phenotype suggesting that VPY negatively influences DMI3 function. Overall, the data indicate a requirement for VPY in the nucleus and cytoplasm where it may coordinate signaling and cellular accommodation processes. 

Abstract As scientists, we are at least as excited about the open questions—the things we do not know—as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such “rules” conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring. 

Summary Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in fourMedicago truncatula Tnt1‐insertion mutants affected in arbuscular mycorrhizal colonisation. We located theirTnt1insertions and identified alleles for two genes known to be involved in mycorrhization,RAM1andKIN3. We compared the effects of thekin3‐2andram1‐4mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting thatRAM1acts upstream ofKIN3.Additionally,KIN3appears to be involved in the suppression of plant defences in response to the fungal symbiont.KIN3is located on the endoplasmic reticulum of arbuscule‐containing cortical cells, andkin3‐2mutants plants hosted significantly fewer arbuscules than the wild type.KIN3plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild‐type plants, thekin3‐2mutant did not exhibit increased root colonisation under high nitrogen. 

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.