Marker Lines Research Articles

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10265-012-0528-0) contains supplementary material, which is available to authorized users.

Zebrafish foxP2 Zinc Finger Nuclease Mutant Has Normal Axon Pathfinding

foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2nd coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

Phloem-associated auxin response maxima determine radial positioning of lateral roots in maize

In Arabidopsis thaliana, lateral-root-forming competence of pericycle cells is associated with their position at the xylem poles and depends on the establishment of protoxylem-localized auxin response maxima. In maize, our histological analyses revealed an interruption of the pericycle at the xylem poles, and confirmed the earlier reported proto-phloem-specific lateral root initiation. Phloem-pole pericycle cells were larger and had thinner cell walls compared with the other pericycle cells, highlighting the heterogeneous character of the maize root pericycle. A maize DR5::RFP marker line demonstrated the presence of auxin response maxima in differentiating xylem cells at the root tip and in cells surrounding the proto-phloem vessels. Chemical inhibition of auxin transport indicated that the establishment of the phloem-localized auxin response maxima is crucial for lateral root formation in maize, because in their absence, random divisions of pericycle and endodermis cells occurred, not resulting in organogenesis. These data hint at an evolutionarily conserved mechanism, in which the establishment of vascular auxin response maxima is required to trigger cells in the flanking outer tissue layer for lateral root initiation. It further indicates that lateral root initiation is not dependent on cellular specification or differentiation of the type of vascular tissue.

Overexpression of AtHsfB4 induces specific effects on root development of Arabidopsis

The functions of plant class B-heat shock factors (Hsfs) are not well understood. Hsfs belonging to this group differ from class A-Hsfs in structural features of the oligomerization domain and by the absence of a typical AHA motif for transcriptional activation. AtHsfB4 is expressed in different parts of the plants with highest levels in root tissue. Transgenic Arabidopsis plants overexpressing (OE) HsfB4 by CaMV-35S-promoter showed massively enhanced levels of Hsf mRNAs. The root surface of OE-plants was rough and cells became detached. Crossings with cell type specific root marker lines and confocal laser scanning microscopy provided clear evidence for a duplication of cells in the ground tissue and ectopic layers of lateral root cap (LRC) cells in HsfB4-OE plants. A duplication of endodermis cells occurs already during embryonic development, while the ectopic LRC cells are only detected during postembryonic growth. The mutant phenotypes of Hsf-OE plants are without precedence and indicate that class B-Hsfs may play an important role in root development.

The University Münster model surgery system for Orthognathic surgery. Part I – The idea behind

BackgroundWe describe a procedure for diagnosis and planning for orthognatic surgery based on international standards. A special 2D planning based on lateral cephalograms (Axis Orbital Marker Lines System) realize a transmission to the SAM 2P articulator (3D) by means of the Axis Orbital Plane.MethodsFormer intraoperative measurement of the average height of the LeFort I osteotomy plane relative to the molar occlusal plane allow to construct a virtual osteotomy plane in the lateral cephalogram. This is the basis for the development of the Axis Orbital Marker Lines System (AO-MLS).ResultsThe AO-MLS is presented graphically, and in detail, with construction guidelines. The system could be integrated into various lateral cephalometric analysis- and surgical prediction schemes. It forms the basis for a standardized transfer of the 2D planning to the 3D planning in the articulator, and vice versa. This procedure makes it possible to generate surgical planning protocols based on the model surgery, which represent the dislocations in the proximity of the real osteotomy planes.ConclusionsThe Axis Orbital Marker Lines System (software component) in conjunction with the University Münster Model Surgery System (hardware system) increases the predictability of model operations in orthognathic surgery.

A set of GFP organelle marker lines for intracellular localization studies in Medicago truncatula

Genomics advances in the model legume, Medicago truncatula, have led to an increase in the number of identified genes encoding proteins with unknown biological function. Determining the intracellular location of uncharacterized proteins often aids in the elucidation of biological function. To expedite such localization studies, we have generated a set of intracellular organelle green fluorescence protein (GFP) marker lines in M. truncatula. In addition to fluorescent detection, this set of organelle marker lines can also be used in immunohistochemical and cellular fractionation detection assays. Moreover, this set of marker lines is compatible with both transient and stable expression systems. Thus, this marker set should prove to be a useful resource for the M. truncatula research community.

Grain boundary sliding in ultrafine grained aluminum under tension at room temperature

Deformation mechanisms occurring by plastic flow of ultrafine grained aluminum processed by equal channel angular pressing at room temperature have been investigated using deformation relief on the pre-polished surface of the sample tested. The deformation behavior and the offsetting of marker lines suggest the development of grain boundary sliding in addition to intragrain dislocation slip. The contribution of grain boundary sliding to the overall deformation calculated using displacement of grains relative to each other was found to reach 24%.

Computational explosion mechanics and related progress

Explosion mechanics is the theoretical basis for the design of highly efficient arms and ammunition and industrial explosion safety. Because it involves the complex physical and mechanical behaviors of multi-materials under extreme conditions, such as high speed, high temperature and high pressure, it is almost impossible to give exact solutions for explosion problems. As explosion occurs in a very short time and has a strong destructive effect, there will be limited amounts of experimental data obtained during the explosion process. With the continuous development of numerical methods and computer performance, computational explosion mechanics has become a new interdisciplinary branch of explosion mechanics, material dynamics, computational mathematics and computer technology, and greatly promoted the development of explosion mechanics and weapons equipment. Since the late 1960s, US-led western developed countries have developed more than one hundred calculation codes of explosion mechanics. Based on the simulation software for explosion mechanics, calculations about three-dimensional physical processes on a system scale during the course of weapon system development have been performed, which resulted in the development of a number of high efficiency arms and ammunition. Such research institutions as the Beijing Institute of Technology, the Chinese Academy of Engineering Physics, the Institute of Applied Physics and Computational Mathematics, the Institute of Mechanics of the Chinese Academy of Sciences, Peking University, the University of Science and Technology of China and other research institutions have developed different numerical methods for explosion mechanics, dynamic constitutive models and software development. Finite Difference Method and Finite Element Method are the most common methods of the discrete methods adopted in computational explosion mechanics. The former is a representative method by which time and space are covered with cells to gain approximate numerical solutions after partial differential equations (governing equations) are established. The latter is a representative method by which continuous space is decomposed into finite elements. Classed by coordinates, Eulerian method and Lagrangrian Method are two common methods utilized in computational explosion mechanics. On the base of these two methods, two hybrid methods, ALE (Arbitrary Lagrange Euler) and CLE (Coupled Lagrange Euler), are developed. Eulerian method has an advantage in treating the large deformation of materials, which always occurs in explosion mechanics. However, it is difficult to handle mixed cells using this method if the studied system includes multi-materials because a transition region forms along the interface which becomes fuzzy between materials. How to determine the position of interfaces and how to calculate the physical quantities in the mixed cells have been difficult problems in Eulerian codes. PIC (Particle in Cell) method and VOF (Volume of Fluid) method are the traditional methods to solve the interface problems in Eulerian codes. Youngs method can determine the interface clearly by transporting fluid of cells around the interface, becoming the mainstream of VOF methods. The original Youngs method just determines the interface in a mixed cell, and cannot determine the transport order and quantity of material. That is a problem of Eulerian methods. A modified Youngs method is adopted in [1], in which the material’s fractions are adopted to determine the transport order of material. Ref. [2] proposes an interface treatment method, in which the interface curve of material is traced by a series of straight-line segments which connect head and tails, and the material interface is determined by marker line located on the grid lines. Ref. [3] proposed a hybrid VOF and PIC multi-material interface treatment method, in which PIC method is utilized in the important regions, and continuous method is used in the other, resulting in high precision and high efficiency of algorithm by which the deformation of material is tracked successfully during penetration. Another method, Level Set, has been proposed and widely used in recent years. In [4], Level Set method combined with Ghost Fluid method is employed to track the

Ethylene‐induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion

• Hyponastic growth is an upward petiole movement induced by plants in response to various external stimuli. It is caused by unequal growth rates between adaxial and abaxial sides of the petiole, which bring rosette leaves to a more vertical position. The volatile hormone ethylene is a key regulator inducing hyponasty in Arabidopsis thaliana. Here, we studied whether ethylene-mediated hyponasty occurs through local stimulation of cell expansion and whether this involves the reorientation of cortical microtubules (CMTs). • To study cell size differences between the two sides of a petiole in ethylene and control conditions, we analyzed epidermal imprints. We studied the involvement of CMT orientation in epidermal cells using the tubulin marker line as well as genetic and pharmacological means of CMT manipulation. • Our results demonstrate that ethylene induces cell expansion at the abaxial side of the- petiole and that this can account for the observed differential growth. At the abaxial side, ethylene induces CMT reorientation from longitudinal to transverse, whereas, at the adaxial side, it has an opposite effect. The inhibition of CMTs disturbed ethylene-induced hyponastic growth. • This work provides evidence that ethylene stimulates cell expansion in a tissue-specific manner and that it is associated with tissue-specific changes in the arrangement of CMTs along the petiole.

High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development

Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P(2)) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P(2) reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P(2) significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P(2) in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport.

The occurrence of ‘bulbs’, a complex configuration of the vacuolar membrane, is affected by mutations of vacuolar SNARE and phospholipase in Arabidopsis

The plant vacuole fulfills a variety of functions, and is essential for plant growth and development. We previously identified complex and mobile structures on the continuous vacuolar membrane, which we refer to as 'bulbs'. To ascertain their biological significance and function, we searched for markers associated with bulbs, and mutants that show abnormalities with respect to bulbs. We observed bulb-like structures after expression of non-membranous proteins as well as the functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecules VAM3 and VTI11. Bulbs are formed in more tissues than previously reported, including flowering organs, suspension culture cells, endodermal cells in the flowering stem, and at very early stages of seed germination. Using existing and newly developed marker lines, we found that the frequency of bulb occurrence is significantly decreased in multiple shoot gravitropism (sgr) mutants, which are known to have a defect in vacuolar membrane properties in endodermal cells. Based on results with new marker lines, which enabled us to observe the process of bulb biogenesis, and analysis of the phenotypes of these mutants, we propose multiple mechanisms for bulb formation, one of which may be that used for formation of transvacuolar strands.

Reliability-guided phase-unwrapping algorithm for the measurement of discontinuous three-dimensional objects

Accurate and robust phase unwrapping is an important procedure for three dimensional (3D) profilometry measurement. The mask cut phase-unwrapping algorithm merges the characteristics of Goldstein's branch cut and reliability-guided path-following algorithms, so that it has higher accuracy and stability. However, it cannot handle discontinuous phase regions because it completely relies on the phase quality map to guide the placement of mask cuts, thereby easily causing phase error propagation. To overcome these drawbacks, we propose a new phase-unwrapping method that merges the residue check principle and a Laplace phase derivative variance quality map that we developed. First, the entire phase was divided into several isolated regions in accordance with the quality map. Then, each pixel of the absolute phase marker line was taken as the starting point to unwrap the discontinuous phase regions based on reliability guidance. In addition, a new list-trimming algorithm was employed to guarantee a speedy phase-unwrapping procedure. The entire phase unwrapping and accurate 3D measurement of discontinuous objects was successfully completed. The simulated and experimental data both demonstrate the validity of the proposed phase-unwrapping algorithm.

Using maize as a model to study pollen tube growth and guidance, cross-incompatibility and sperm delivery in grasses

In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and require transportation to the female gametes via the vegetative pollen tube cell to achieve double fertilization. The path of the pollen tube towards the female gametophyte (embryo sac) has been intensively studied in many intra- and interspecific crossing experiments with the aim of increasing the gene pool of crop plants for greater yield, improved biotic and abiotic stress resistance, and for introducing new agronomic traits. Many attempts to hybridize different species or genotypes failed due to the difficulty for the pollen tubes in reaching the female gametophyte. Detailed studies showed that these processes are controlled by various self-incompatible (intraspecific) and cross-incompatible (interspecific) hybridization mechanisms. Understanding the molecular mechanisms of crossing barriers is therefore of great interest in plant reproduction, evolution and breeding research. In particular, pre-zygotic hybridization barriers related to pollen tube germination, growth, guidance and sperm delivery, which are considered the major hybridization controls in nature and thus also contribute to species isolation and speciation, have been intensively investigated. Despite this general interest, surprisingly little is known about these processes in the most important agronomic plant family, the Gramineae, Poaceae or grasses. Small polymorphic proteins and their receptors, degradation of sterility locus proteins and general compounds such as calcium, γ-aminobutyric acid or nitric oxide have been shown to be involved in progamic pollen germination, adhesion, tube growth and guidance, as well as sperm release. Most advances have been made in the Brassicaceae, Papaveraceae, Linderniaceae and Solanaceae families including their well-understood self-incompatibility (SI) systems. Grass species evolved similar mechanisms to control the penetration and growth of self-pollen to promote intraspecific outcrossing and to prevent fertilization by alien sperm cells. However, in the Poaceae, the underlying molecular mechanisms are still largely unknown. We propose to develop maize (Zea mays) as a model to investigate the above-described processes to understand the associated intra- and interspecific crossing barriers in grasses. Many genetic, cellular and biotechnological tools including the completion of a reference genome (inbred line B73) have been established in the last decade and many more maize inbred genomes are expected to be available soon. Moreover, a cellular marker line database as well as large transposon insertion collections and improved Agrobacterium transformation protocols are now available. Additionally, the processes described above are well studied at the morphological level and a number of mutants have been described already, awaiting disclosure of the relevant genes. The identification of the first key players in pollen tube growth, guidance and burst show maize to be an excellent grass model to investigate these processes in more detail. Here we provide an overview of our current understanding of these processes in Poaceae with a focus on maize, and also include relevant discoveries in eudicot model species.

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.

Evaluation of Lighted Pavement Marker Stop Bars at Intersections with Light Rail Lines

This paper presents an evaluation of the use of lighted pavement markers parallel to an intersection stop bar for three treatment intersections in Houston, Texas. The line of pavement markers was active, with a red light displayed from each marker during the red interval of the traffic signal. Each lighted pavement marker contained multiple LEDs, which operated such that each marker displayed an alternating wigwag pattern. The intended impact of the pavement markers was to reduce red light running violations and crashes on the intersection approach to an arterial that had an at-grade light rail line within its median. A before-and-after study was conducted to determine changes in crashes, red light running violations, and right turn on red violations at the study sites (right turns on red are prohibited at all the study sites). The analysis results showed that the lighted pavement markers reduced red light running violations at all three treatment sites, with two of the three reductions being statistically significant. Right turn on red violations were also reduced, and the reduction was statistically significant at all three treatment sites. A fourth site was chosen as a comparison (nontreatment) site, and the before-and-after study showed no notable change in the number of red light running violations or right turn on red violations at this site.

Reproductive Meristem22 is a unique marker for the early stages of stamen development

Stamens undergo a very elaborate development program that gives rise not only to many specific tissue types, but also to the male gametes. The specification of stamen identity is coordinated by a group of homeotic genes such as APETALA3 (AP3) and PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA (SEP1-4) genes. Genome-wide transcriptomic comparisons between floral buds of wild-type and ap3 mutants led to the identification of the REM22 gene, which is expressed in the early stages of stamen development. This gene is member of the plant-specific B3 DNA-binding superfamily. In this work, we dissect the spatio-temporal expression pattern of REM22 during the early stages of stamen development. To this end, both in situ hybridization analyses as well as in vivo fluorescence strategies were employed. At stage 4 of flower development, REM22 is expressed exclusively in those undifferentiated cells of the floral meristem that will give rise to the stamen primordia. At stage 5, REM22 expression is restricted to the epidermal and the subepidermal layers of anther primordia. Later, this expression is confined to the middle layer and the differentiating tapetal cells. After stage 10 when all the tissues of the anther have differentiated, REM22 expression is no longer detectable. Furthermore, we examined the pREM22::GUS-GFP marker line in an inducible system where the ectopic AG function is used to promote microsporogenesis. The data support the idea that REM22 expression is a useful marker to study the early stages of stamen development.

SOS3 mediates lateral root development under low salt stress through regulation of auxin redistribution and maxima in Arabidopsis

• The SOS signaling pathway plays an important role in plant salt tolerance. However, little is known about how the SOS pathway modulates organ development in response to salt stress. Here, the involvement of SOS signaling in NaCl-induced lateral root (LR) development in Arabidopsis was assessed. • Wild-type and sos3-1 mutant seedlings on iso-osmotic concentrations of NaCl and mannitol were analyzed. The marker lines for auxin accumulation, auxin transport, cell division activity and stem cells were also examined. • The results showed that ionic effect alleviates the inhibitory effects of osmotic stress on LR development. LR development of the sos3-1 mutant showed increased sensitivity specifically to low salt. Under low-salt conditions, auxin in cotyledons and LR primordia (LRP) of the sos3-1 mutant was markedly reduced. Decreases in auxin polar transport of mutant roots may cause insufficient auxin supply, resulting in defects not only in LR initiation but also in cell division activity in LRP. • Our data uncover a novel role of the SOS3 gene in modulation of LR developmental plasticity and adaptation in response to low salt stress, and reveal a new mechanism for plants to sense and adapt to small changes of salt.

A Host-Factor Interaction and Localization Map for a Plant-Adapted Rhabdovirus Implicates Cytoplasm-Tethered Transcription Activators in Cell-to-Cell Movement

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

CULLIN 4-RING FINGER-LIGASE plays a key role in the control of endoreplication cycles in Arabidopsis trichomes

One of the predominant cell-cycle programs found in mature tissues is endoreplication, also known as endoreduplication, that leads to cellular polyploidy. A key question for the understanding of endoreplication cycles is how oscillating levels of cyclin-dependent kinase activity are generated that control repeated rounds of DNA replication. The APC/C performs a pivotal function in the mitotic cell cycle by promoting anaphase and paving the road for a new round of DNA replication. However, using marker lines and plants in which APC/C components are knocked down, we show here that outgrowing and endoreplicating Arabidopsis leaf hairs display no or very little APC/C activity. Instead we find that RBX1-containing Cullin-RING E3 ubiquitin-Ligases (CRLs) are of central importance for the progression through endoreplication cycles; in particular, we have identified CULLIN4 as a major regulator of endoreplication in Arabidopsis trichomes. We have incorporated our findings into a bio-mathematical simulation presenting a robust two-step model of endoreplication control with one type of cyclin-dependent kinase inhibitor function for entry and a CRL-dependent oscillation of cyclin-dependent kinase activity via degradation of a second type of CDK inhibitor during endoreplication cycles.