Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors

AbstractBackgroundPlant-pathogenic oomycetes are responsible for economically important losses on crops worldwide. Phytophthora palmivora, a broad-host-range tropical relative of the potato late blight pathogen, causes rotting diseases in many important tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus.Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped understanding of how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce.ResultsWe employed the model plant N. benthamiana to study the P. palmivora root infections at the cellular and molecular level. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features.ConclusionsThese results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

Download Full-text

Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

PLoS ONE ◽

10.1371/journal.pone.0157591 ◽

2016 ◽

Vol 11 (6) ◽

pp. e0157591 ◽

Cited By ~ 6

Author(s):

Uraiwan Khunjan ◽

Kitiya Ekchaweng ◽

Tanate Panrat ◽

Miaoying Tian ◽

Nunta Churngchow

Keyword(s):

Molecular Cloning ◽

Nicotiana Benthamiana ◽

Rubber Tree ◽

Phytophthora Palmivora ◽

Tree Expression

Download Full-text

Time-Resolved Observation of the Destination of Microinjected Potato Spindle Tuber Viroid (PSTVd) in the Abaxial Leaf Epidermal Cells of Nicotiana benthamiana

Microorganisms ◽

10.3390/microorganisms8122044 ◽

2020 ◽

Vol 8 (12) ◽

pp. 2044

Author(s):

Hyesu Seo ◽

Ying Wang ◽

Woong June Park

Keyword(s):

Nuclear Import ◽

Nicotiana Benthamiana ◽

Noncoding Rna ◽

Potato Spindle Tuber Viroid ◽

Plant Diseases ◽

Host Cells ◽

Nuclear Accumulation ◽

Time Resolved ◽

Rna Molecules ◽

High Level

Viroids are single-stranded noncoding RNA molecules of 250–400 nucleotides that cause plant diseases. One of the two families of viroids is Pospiviroidae, the members of which replicate in the nuclei of host cells. To replicate in plants, viroids of Pospiviroidae must enter the nucleus. However, the nuclear import of viroids remains understudied. In this work, we documented the time-dependent characteristics of the changes in microinjected fluorescently labeled potato spindle tuber viroid (PSTVd). The cytoplasmic fluorescence disappeared gradually, with only nuclear fluorescence remaining as the PSTVd injected in the cytoplasm was imported into the nucleus. Through this work, we determined that the time for half-maximal nuclear accumulation of the viroid was about 23 min. Interestingly, we found some cells where the nuclear import did not occur, despite the high level of cytosolic viroid injected. In some cells, the injected viroids disappeared within 10–20 min. The nuclear import of PSTVd is not a simple concentration-dependent process but was probably under the regulation of diverse factors that may be missing from some cells used for our observation.

Download Full-text

Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077402 ◽

1983 ◽

Vol 41 ◽

pp. 766-767

Author(s):

Eva-Maria Mandelkow ◽

Eckhard Mandelkow ◽

Joan Bordas

Keyword(s):

Electron Microscopy ◽

Dynamic Equilibrium ◽

Time Resolved ◽

X Ray ◽

Additional Information ◽

X Ray Scattering ◽

Low Concentrations ◽

Microtubule Growth ◽

Ray Patterns ◽

Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

Download Full-text

Electron Channeling Patterns

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077700 ◽

1977 ◽

Vol 35 ◽

pp. 12-13

Author(s):

David C. Joy

Keyword(s):

Electron Diffraction ◽

Incidence Angle ◽

Photographic Plate ◽

Diffraction Effect ◽

Angle Of Incidence ◽

Bulk Single Crystal ◽

Time Resolved ◽

Electron Channeling ◽

Spatially Resolved ◽

Large Bulk

Electron channeling patterns (ECP) were first found by Coates (1967) while observing a large bulk, single crystal of silicon in a scanning electron microscope. The geometric pattern visible was shown to be produced as a result of the changes in the angle of incidence, between the beam and the specimen surface normal, which occur when the sample is examined at low magnification (Booker, Shaw, Whelan and Hirsch 1967).A conventional electron diffraction pattern consists of an angularly resolved intensity distribution in space which may be directly viewed on a fluorescent screen or recorded on a photographic plate. An ECP, on the other hand, is produced as the result of changes in the signal collected by a suitable electron detector as the incidence angle is varied. If an integrating detector is used, or if the beam traverses the surface at a fixed angle, then no channeling contrast will be observed. The ECP is thus a time resolved electron diffraction effect. It can therefore be related to spatially resolved diffraction phenomena by an application of the concepts of reciprocity (Cowley 1969).

Download Full-text

Time-resolved high-resolution electron microscopy of structural stability in mgo clusters

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100165823 ◽

1996 ◽

Vol 54 ◽

pp. 672-673

Author(s):

T. Kizuka ◽

N. Tanaka

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Magnesium Oxide ◽

Structural Stability ◽

High Resolution Electron Microscopy ◽

Video Tape ◽

Solid State Physics ◽

Time Resolved ◽

Resolution Electron ◽

Structural And Functional Properties

Structure and stability of atomic clusters have been studied by time-resolved high-resolution electron microscopy (TRHREM). Typical examples are observations of structural fluctuation in gold (Au) clusters supported on silicon oxide films, graphtized carbon films and magnesium oxide (MgO) films. All the observations have been performed on the clusters consisted of single metal element. Structural stability of ceramics clusters, such as metal-oxide, metal-nitride and metal-carbide clusters, has not been observed by TRHREM although the clusters show anomalous structural and functional properties concerning to solid state physics and materials science.In the present study, the behavior of ceramic, magnesium oxide (MgO) clusters is for the first time observed by TRHREM at 1/60 s time resolution and at atomic resolution down to 0.2 nm.MgO and gold were subsequently deposited on sodium chloride (001) substrates. The specimens, single crystalline MgO films on which Au particles were dispersed were separated in distilled water and observed by using a 200-kV high-resolution electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder system.

Download Full-text

Cryo-TEM of amphiphilic polymer and amphiphile/polymer solutions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100150216 ◽

1993 ◽

Vol 51 ◽

pp. 876-877

Author(s):

Yeshayahu Talmon

Keyword(s):

Microstructural Characterization ◽

Building Blocks ◽

Quantitative Information ◽

Physical Models ◽

Specimen Preparation ◽

Time Resolved ◽

Temperature Changes ◽

Temperature And Humidity ◽

Microstructured Fluids ◽

Air Streams

To achieve complete microstructural characterization of self-aggregating systems, one needs direct images in addition to quantitative information from non-imaging, e.g., scattering or Theological measurements, techniques. Cryo-TEM enables us to image fluid microstructures at better than one nanometer resolution, with minimal specimen preparation artifacts. Direct images are used to determine the “building blocks” of the fluid microstructure; these are used to build reliable physical models with which quantitative information from techniques such as small-angle x-ray or neutron scattering can be analyzed.To prepare vitrified specimens of microstructured fluids, we have developed the Controlled Environment Vitrification System (CEVS), that enables us to prepare samples under controlled temperature and humidity conditions, thus minimizing microstructural rearrangement due to volatile evaporation or temperature changes. The CEVS may be used to trigger on-the-grid processes to induce formation of new phases, or to study intermediate, transient structures during change of phase (“time-resolved cryo-TEM”). Recently we have developed a new CEVS, where temperature and humidity are controlled by continuous flow of a mixture of humidified and dry air streams.

Download Full-text

The promises and perfidy of cryomicroscopy and analysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016964x ◽

1994 ◽

Vol 52 ◽

pp. 382-383

Author(s):

Patrick Echlin

Keyword(s):

Low Temperature ◽

High Energy ◽

Short Paper ◽

List Type ◽

Time Resolved ◽

Energy Beam ◽

Cellular Analysis ◽

Dissolved Ions ◽

Embedding Medium ◽

Low Temperature Microscopy

The unusual title of this short paper and its accompanying tutorial is deliberate, because the intent is to investigate the effectiveness of low temperature microscopy and analysis as one of the more significant elements of the less interventionist procedures we can use to prepare, examine and analyse hydrated and organic materials in high energy beam instruments. The promises offered by all these procedures are well rehearsed and the litany of petitions and responses may be enunciated in the following mantra.Vitrified water can form the perfect embedding medium for bio-organic samples.Frozen samples provide an important, but not exclusive, milieu for the in situ sub-cellular analysis of the dissolved ions and electrolytes whose activities are central to living processes.The rapid conversion of liquids to solids provides a means of arresting dynamic processes and permits resolution of the time resolved interactions between water and suspended and dissolved materials.The low temperature environment necessary for cryomicroscopy and analysis, diminish, but alas do not prevent, the deleterious side effects of ionizing radiation.Sample contamination is virtually eliminated.

Download Full-text

Electron-beam damage of oxides at high current densities

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155141 ◽

1989 ◽

Vol 47 ◽

pp. 634-635

Author(s):

M. R. McCartney ◽

J. K. Weiss ◽

David J. Smith

Keyword(s):

Electron Beam ◽

Electron Microscope ◽

Current Density ◽

Transition Metal Oxides ◽

Time Resolved ◽

Rapid Acquisition ◽

Resolution Electron ◽

Current Densities ◽

Electron Stimulated Desorption ◽

Channel Analyzer

It is well-known that electron-beam irradiation within the electron microscope can induce a variety of surface reactions. In the particular case of maximally-valent transition-metal oxides (TMO), which are susceptible to electron-stimulated desorption (ESD) of oxygen, it is apparent that the final reduced product depends, amongst other things, upon the ionicity of the original oxide, the energy and current density of the incident electrons, and the residual microscope vacuum. For example, when TMO are irradiated in a high-resolution electron microscope (HREM) at current densities of 5-50 A/cm2, epitaxial layers of the monoxide phase are found. In contrast, when these oxides are exposed to the extreme current density probe of an EM equipped with a field emission gun (FEG), the irradiated area has been reported to develop either holes or regions almost completely depleted of oxygen. ’ In this paper, we describe the responses of three TMO (WO3, V2O5 and TiO2) when irradiated by the focussed probe of a Philips 400ST FEG TEM, also equipped with a Gatan 666 Parallel Electron Energy Loss Spectrometer (P-EELS). The multi-channel analyzer of the spectrometer was modified to take advantage of the extremely rapid acquisition capabilities of the P-EELS to obtain time-resolved spectra of the oxides during the irradiation period. After irradiation, the specimens were immediately removed to a JEM-4000EX HREM for imaging of the damaged regions.

Download Full-text

Multiple phase transitions investigated by high-speed TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100175880 ◽

1990 ◽

Vol 48 (4) ◽

pp. 550-551

Author(s):

Oleg Bostanjoglo ◽

Peter Thomsen-Schmidt

Keyword(s):

Phase Transitions ◽

High Speed ◽

Short Exposure ◽

Semiconductor Film ◽

Time Resolved ◽

Short Exposure Time ◽

Multiple Phase ◽

Model Substance ◽

History Of ◽

Electron Image

Thin GexTe1-x (x = 0.15-0.8) were studied as a model substance of a composite semiconductor film, in addition being of interest for optical storage material. Two complementary modes of time-resolved TEM were used to trace the phase transitions, induced by an attached Q-switched (50 ns FWHM) and frequency doubled (532 nm) Nd:YAG laser. The laser radiation was focused onto the specimen within the TEM to a 20 μm spot (FWHM). Discrete intermediate states were visualized by short-exposure time doubleframe imaging /1,2/. The full history of a transformation was gained by tracking the electron image intensity with photomultiplier and storage oscilloscopes (space/time resolution 100 nm/3 ns) /3/. In order to avoid radiation damage by the probing electron beam to detector and specimen, the beam is pulsed in this continuous mode of time-resolved TEM,too.Short events ( <2 μs) are followed by illuminating with an extended single electron pulse (fig. 1c)

Download Full-text