Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissues

Arabidopsis encodes ten ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21‐22 nucleotide (nt) long small RNAs (sRNA) to mediate post‐transcriptional‐gene‐silencing (PTGS) or 24nt sRNAs to promote RNA‐directed‐DNA‐methylation. Using full‐locus constructs, we characterized the expression, biochemical properties, and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 down‐regulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5’‐adenosine bias, but unlike Arabidopsis AGO2, it binds most efficiently 24nt sRNAs. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci‐of‐origin. AGO3 localizes to the cytoplasm and co‐fractionates with polysomes to possibly mediate PTGS via translation inhibition

Distinct ankyrin repeat subdomains control VAPYRIN locations and intracellular accommodation functions during arbuscular mycorrhizal symbiosis

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

Origin and evolution of the octoploid strawberry genome.

Cultivated strawberry emerged from the hybridization of two wild octoploid species, both descendants from the merger of four diploid progenitor species into a single nucleus more than 1 million years ago. Here we report a near-complete chromosome-scale assembly for cultivated octoploid strawberry (Fragaria × ananassa) and uncovered the origin and evolutionary processes that shaped this complex allopolyploid. We identified the extant relatives of each diploid progenitor species and provide support for the North American origin of octoploid strawberry. We examined the dynamics among the four subgenomes in octoploid strawberry and uncovered the presence of a single dominant subgenome with significantly greater gene content, gene expression abundance, and biased exchanges between homoeologous chromosomes, as compared with the other subgenomes. Pathway analysis showed that certain metabolomic and disease-resistance traits are largely controlled by the dominant subgenome. These findings and the reference genome should serve as a powerful platform for future evolutionary studies and enable molecular breeding in strawberry

Parton distribution benchmarking with LHC data

We present a detailed comparison of the most recent sets of NNLO PDFs from the ABM, CT, HERAPDF, MSTW and NNPDF collaborations. We compare parton distributions at low and high scales and parton luminosities relevant for LHC phenomenology. We study the PDF dependence of LHC benchmark inclusive cross sections and differential distributions for electroweak boson and jet production in the cases in which the experimental covariance matrix is available. We quantify the agreement between data and theory by computing the χ 2 for each data set with all the various PDFs. PDF comparisons are performed consistently for common values of the strong coupling. We also present a benchmark comparison of jet production at the LHC, comparing the results from various available codes and scale settings. Finally, we discuss the implications of the updated NNLO PDF sets for the combined PDF+α s uncertainty in the gluon fusion Higgs production cross section

Parton distributions for the LHC run II

We present NNPDF3.0, the first set of parton distribution functions (PDFs) determined with a methodology validated by a closure test. NNPDF3.0 uses a global dataset including HERA-II deep-inelastic inclusive cross-sections, the combined HERA charm data, jet production from ATLAS and CMS, vector boson rapidity and transverse momentum distributions from ATLAS, CMS and LHCb, W+c data from CMS and top quark pair production total cross sections from ATLAS and CMS. Results are based on LO, NLO and NNLO QCD theory and also include electroweak corrections. To validate our methodology, we show that PDFs determined from pseudo-data generated from a known underlying law correctly reproduce the statistical distributions expected on the basis of the assumed experimental uncertainties. This closure test ensures that our methodological uncertainties are negligible in comparison to the generic theoretical and experimental uncertainties of PDF determination. This enables us to determine with confidence PDFs at different perturbative orders and using a variety of experimental datasets ranging from HERA-only up to a global set including the latest LHC results, all using precisely the same validated methodology. We explore some of the phenomenological implications of our results for the upcoming 13 TeV Run of the LHC, in particular for Higgs production cross-sections.Comment: 151 pages, 69 figures. More typos corrected: published versio

Cell Biology Of The Arbuscular Mycorrhizal Symbiosis: Localization And Functional Studies Of Medicago Truncatula Phosphate Transporters And Vapyrin

The arbuscular mycorrhizal (AM) symbiosis is an ancient, widespread and ecologically important mutualism between plants and fungi. This symbiosis is characterized by highly-branched hyphae called arbuscules that form within plant cells to facilitate mutually beneficial nutrient exchange. These structures are surrounded by a specialized plant membrane termed the periarbuscular membrane. In the legume Medicago truncatula, phosphate is taken up across this membrane by the phosphate transporter MtPT4, which localizes around arbuscule branches. The experiments described in this dissertation employ molecular genetic approaches together with a system to study fluorescent fusion proteins in live cells hosting arbuscules. In the second chapter, the periarbuscular membrane is revealed to contain distinct, complementary protein domains. In the third chapter, the mechanism of polar targeting of MtPT4 to the periarbuscular membrane is dissected by studying the localization of fluorescent protein tagged-membrane proteins expressed during different stages of arbuscule development. These experiments revealed that asymmetric localization of MtPT4 is mediated by precise temporal expression, coupled with significant and temporary changes in the direction and cargo selection of the plant secretory pathway for newly synthesized proteins. Together, these data point towards a surprising but logical mechanism for polar protein localization during the AM symbiosis and highlight the extent to which cells can become specialized during an intracellular biotic interaction. In addition, a mutant MtPT4 protein with a single amino acid change fails to localize in the periarbuscular membrane and instead is retained in the Trans-Golgi Network (TGN). This mutation causes a loss-of-function phenotype, confirming that localization of MtPT4 at the host-symbiont interface is required for its function. The fourth chapter describes additional M. truncatula phosphate transporters, one of which localizes specifically to the periarbuscular membrane. The fifth chapter reports discovery and characterization of a gene named Vapyrin. Knockdown of this gene impairs root penetration by AM fungi and abolishes arbuscule formation. Vapyrin is induced coincident with fungal infection, and encodes a novel protein containing a VAP (VAMP Associated Protein) domain and eight ankyrin repeats, both established protein-protein interaction domains. We hypothesize that Vapyrin functions in secretion during fungal infection through recruitment to an endosomal compartment

Live-Cell Imaging Reveals Periarbuscular Membrane Domains and Organelle Location in Medicago truncatula Roots during Arbuscular Mycorrhizal Symbiosis1[W][OA]

In the arbuscular mycorrhizal symbiosis, the fungal symbiont colonizes root cortical cells, where it establishes differentiated hyphae called arbuscules. As each arbuscule develops, the cortical cell undergoes a transient reorganization and envelops the arbuscule in a novel symbiosis-specific membrane, called the periarbuscular membrane. The periarbuscular membrane, which is continuous with the plant plasma membrane of the cortical cell, is a key interface in the symbiosis; however, relatively little is known of its composition or the mechanisms of its development. Here, we used fluorescent protein fusions to obtain both spatial and temporal information about the protein composition of the periarbuscular membrane. The data indicate that the periarbuscular membrane is composed of at least two distinct domains, an “arbuscule branch domain” that contains the symbiosis-specific phosphate transporter, MtPT4, and an “arbuscule trunk domain” that contains MtBcp1. This suggests a developmental transition from plasma membrane to periarbuscular membrane, with biogenesis of a novel membrane domain associated with the repeated dichotomous branching of the hyphae. Additionally, we took advantage of available organelle-specific fluorescent marker proteins to further evaluate cells during arbuscule development and degeneration. The three-dimensional data provide new insights into relocation of Golgi and peroxisomes and also illustrate that cells with arbuscules can retain a large continuous vacuolar system throughout development

Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissue

Arabidopsis encodes ten ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22nt small RNAs (sRNA) to mediate post-transcriptional-gene- silencing (PTGS) or 24nt sRNAs to promote RNA-directed-DNA-methylation. Using full- locus constructs, we characterized the expression, biochemical properties, and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression differs drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 down-regulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5’-adenosine bias, but unlike most Arabidopsis AGOs - AGO2 included - it binds efficiently both 24nt and 21nt sRNAs. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci-of-origin. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition