Structure and Function of the TIR Domain from the Grape NLR Protein RPV1

The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices ("AE" interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.This research was supported by the Australian Research Council (ARC) Discovery Projects DP120100685 and DP160102244. BK is a NHMRC Research Fellow (1003325 and 1110971). SW is funded by ARC DECRA (DE160100893)

Multiple functional self-association interfaces in plant TIR domains

Toll/interleukin-1 receptor/resistance protein (TIR) domains are present in plant and animal innate immunity receptors and appear to play a scaffold function in defense signaling. In both systems, self-association of TIR domains is crucial for their function. In plants, the TIR domain is associated with intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs). Previous studies from several plant NLRs have identified two distinct interfaces that are required for TIR:TIR dimerization in different NLRs. We show that the two interfaces previously identified are both important for self-association and defense signaling of multiple TIR–NLR proteins. Collectively, this work suggests that there is a common mechanism of TIR domain self-association in signaling across the TIR–NLR class of receptor proteins.This research was supported by the Australian Research Council (ARC) Discovery Projects (DP120100685, DP120103558, and DP160102244) and the National Science Foundation (NSF-IOS-1146793 to B.J.S.). B.K. is a National Health and Medical Research Council Research Fellow (1003325 and 1110971). M.B. and S.J.W. are recipients of ARC Discovery Early Career Research Awards (DE130101292 and DE160100893, respectively)

ガーネット、サマータウン、グローバル・イシューズ案内 : 英語教材のフィクション対ノンフィクション

For the sake of the Japanese learners of English who love reading, this paper introduces three different series of readers: Garnet Oracle Readers, Summertown Readers, and National Geographic's Global Issues. Garnet Oracle and Summertown are both so-called graded readers. The former are for highschool and university students who study English as a foreign or second language while the latter are written for business people who have to learn English as a lingua franca. Both are,however, original fictional stories, some of which are quite enjoyable and really worth reading. Peter Viney, Garnet's main author, can write a variety of genres: for example, Space Romance is a romantic sci-fi story in an impressive setting; A Tidy Ghost is a witty ghost story whose terror dramatically changes into sheer humor at the ending; but,above all,his Underground is highly recommended because of the unforgettable character Tommy, a mute elderly man who lives in the London underground, saving the protagonist in big trouble. Summertown's counterpart must be James Schofield. Although his amateurish suspense stories tend to be rather boring, his humorous stories such as Room Service and Double Trouble are readable with a lot of laughter. National Geographic's Global Issues may seem to be no comparison with these interesting stories since they are serious nonfiction pamphlets edited for American high school students. Despite the foreign language, Japanese students can also appreciate the discussed, grave environmental problems of our planet Earth where the population explosion has been causing disastrous situations. In a sense, fact is truly stranger than fiction. So, which is more interesting, fiction or nonfiction? I hope you read the three series and decide for yourself

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Small-Angle X-Ray Scattering for the Discerning Macromolecular Crystallographer

The role of small-angle X-ray scattering (SAXS) in structural biology is now well established, and its usefulness in combination with macromolecular crystallography is clear. However, the highly averaged SAXS data present a significant risk of over-interpretation to the unwary practitioner, and it can be challenging to frame SAXS results in a manner that maximises the reliability of the conclusions drawn. In this review, a series of recent examples are used to illustrate both the challenges for interpretation and approaches through which these can be overcome

Time-resolved laboratory µ-XRF reveals silicon distribution in relation to manganese toxicity in soybean and sunflower

Synchrotron- and laboratory-based micro-X-ray fluorescence (µ-XRF) is a powerful technique to quantify the distribution of elements in physically large intact samples, including live plants, at room temperature and atmospheric pressure. However, analysis of light elements with atomic number (Z) less than that of phosphorus is challenging due to the need for a vacuum which of course is not compatible with live plant material, or the availability of a helium environment.A new laboratory µ-XRF instrument was used to examine the effects of silicon (Si) on the manganese (Mn) status of soybean (Glycine max) and sunflower (Helianthus annuus) grown at elevated Mn in solution. The use of a helium environment allowed for highly sensitive detection of both Si and Mn to determine their distribution.The µXRF analysis revealed that when Si was added to the nutrient solution, the Si also accumulated in the base of the trichomes, being co-located with the Mn and reducing the darkening of the trichomes. Like soybean, the addition of Si did not reduce the concentrations of Mn in accumulations despite seeming to reduce its adverse effects.The ability to gain information on the dynamics of the metallome or ionome within living plants or excised hydrated tissues can offer valuable insights into their ecophysiology, and laboratory µ-XRF is likely to become available to more plant scientists for use in their research

X-ray fluorescence spectroscopy (XRF) for metallome analysis of herbarium specimens

Background: “Herbarium X-ray Fluorescence (XRF) Ionomics” is a new quantitative approach for extracting the elemental concentrations from herbarium specimens using handheld XRF devices. These instruments are principally designed for dense sample material of infinite thickness (such as rock or soil powder), and their built-in algorithms and factory calibrations perform poorly on the thin dry plant leaves encountered in herbaria. While empirical calibrations have been used for ‘correcting’ measured XRF values post hoc, this approach has major shortcomings. As such, a universal independent data analysis pipeline permitting full control and transparency throughout the quantification process is highly desirable. Here we have developed such a pipeline based on Dynamic Analysis as implemented in the GeoPIXE package, employing a Fundamental Parameters approach requiring only a description of the measurement hardware and derivation of the sample areal density, based on a universal standard. Results: The new pipeline was tested on potassium, calcium, manganese, iron, cobalt, nickel, and zinc concentrations in dry plant leaves. The Dynamic Analysis method can correct for complex X-ray interactions and performs better than both the built-in instrument algorithms and the empirical calibration approach. The new pipeline is also able to identify and quantify elements that are not detected and reported by the device built-in algorithms and provides good estimates of elemental concentrations where empirical calibrations are not straightforward. Conclusions: The new pipeline for processing XRF data of herbarium specimens has a greater accuracy and is more robust than the device built-in algorithms and empirical calibrations. It also gives access to all elements detected in the XRF spectrum. The new analysis pipeline has made Herbarium XRF approach even more powerful to study the metallome of existing plant collections

A statistical approach to removing diffraction from X-ray fluorescence spectra

Diffraction peaks can lead to inaccurate elemental abundances in X-ray fluorescence maps. However, manually removing diffraction peaks is laborious while existing automated methods unnecessarily remove significant volumes of fluorescence data. Here we propose a new automated method to remove diffraction from multiple-detector spectra based on a statistical threshold. This method eliminates only the diffraction peaks from the spectra, retaining more of the fluorescence data and increasing the information content available for diffraction-free elemental quantification and mapping. By retaining the majority of the fluorescence data, the proposed method does not require an increase in dwell times or additional data to be collected to compensate for the removed fluorescence data. This method is therefore particularly valuable in instances where measurement time and data volumes are highly restricted, such as for instruments on planetary exploration missions like the Planetary Instrument for X-ray Lithochemistry on NASA's Mars 2020 mission Perseverance Rover.</p

Assessing radiation dose limits for X-ray fluorescence microscopy analysis of plant specimens

Background and Aims: X-ray fluorescence microscopy (XFM) is a powerful technique to elucidate the distribution of elements within plants. However, accumulated radiation exposure during analysis can lead to structural damage and experimental artefacts including elemental redistribution. To date, acceptable dose-limits have not been systematically established for hydrated plant specimens. Methods: Here we systematically explore acceptable dose-rate limits for investigating fresh sunflower (Helianthus annuus) leaf and root samples and investigate the time-dose damage in leaves attached to live plants. Key results: We find that dose-limits in fresh roots and leaves are comparatively low (4.1 kGy), based on localised disintegration of structures and element-specific redistribution. In contrast, frozen-hydrated samples did not incur any apparent damage even at doses as high as 587 kGy. Furthermore, we find that for living plants subjected to XFM measurement in vivo and grown for a further 9 d before being reimaged with XFM, the leaves display elemental redistribution at doses as low as 0.9 kGy and they continue to develop bleaching and necrosis in the days after exposure. Conclusions: The suggested radiation dose limits for studies using XFM to examine plants are important for the increasing number of plant scientists undertaking multidimensional measurements such as tomography and repeated imaging using XFM