A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption

Frequency drift in MR spectroscopy at 3T

Purpose: Heating of gradient coils and passive shim components is a common cause of instability in the B-0 field, especially when gradient intensive sequences are used. The aim of the study was to set a benchmark for typical drift encountered during MR spectroscopy (MRS) to assess the need for real-time field-frequency locking on MRI scanners by comparing field drift data from a large number of sites.Method: A standardized protocol was developed for 80 participating sites using 99 3T MR scanners from 3 major vendors. Phantom water signals were acquired before and after an EPI sequence. The protocol consisted of: minimal preparatory imaging; a short pre-fMRI PRESS; a ten-minute fMRI acquisition; and a long post-fMRI PRESS acquisition. Both pre- and post-fMRI PRESS were non-water suppressed. Real-time frequency stabilization/adjustment was switched off when appropriate. Sixty scanners repeated the protocol for a second dataset. In addition, a three-hour post-fMRI MRS acquisition was performed at one site to observe change of gradient temperature and drift rate. Spectral analysis was performed using MATLAB. Frequency drift in pre-fMRI PRESS data were compared with the first 5:20 minutes and the full 30:00 minutes of data after fMRI. Median (interquartile range) drifts were measured and showed in violin plot. Paired t-tests were performed to compare frequency drift pre- and post-fMRI. A simulated in vivo spectrum was generated using FID-A to visualize the effect of the observed frequency drifts. The simulated spectrum was convolved with the frequency trace for the most extreme cases. Impacts of frequency drifts on NAA and GABA were also simulated as a function of linear drift. Data from the repeated protocol were compared with the corresponding first dataset using Pearson's and intraclass correlation coefficients (ICC).Results: Of the data collected from 99 scanners, 4 were excluded due to various reasons. Thus, data from 95 scanners were ultimately analyzed. For the first 5:20 min (64 transients), median (interquartile range) drift was 0.44 (1.29) Hz before fMRI and 0.83 (1.29) Hz after. This increased to 3.15 (4.02) Hz for the full 30 min (360 transients) run. Average drift rates were 0.29 Hz/min before fMRI and 0.43 Hz/min after. Paired t-tests indicated that drift increased after fMRI, as expected (p &lt; 0.05). Simulated spectra convolved with the frequency drift showed that the intensity of the NAA singlet was reduced by up to 26%, 44 % and 18% for GE, Philips and Siemens scanners after fMRI, respectively. ICCs indicated good agreement between datasets acquired on separate days. The single site long acquisition showed drift rate was reduced to 0.03 Hz/min approximately three hours after fMRI.Discussion: This study analyzed frequency drift data from 95 3T MRI scanners. Median levels of drift were relatively low (5-min average under 1 Hz), but the most extreme cases suffered from higher levels of drift. The extent of drift varied across scanners which both linear and nonlinear drifts were observed.</p

Geochemistry and petrogenesis of Late Permian basalts from the Sichuan Basin, SW China: Implications for the geodynamics of the Emeishan mantle plume

Plume-lithosphere interactions are significant in the formation of Large Igneous Provinces (LIPs). The Permian Emeishan Large Igneous Province (ELIP) is considered to be the result of a mantle plume. The Emeishan flood basalts comprise a major part of the ELIP and they define three zones: the inner, intermediate and outer zones. Both high-Ti and low-Ti basalts are present in the inner zone, whereas only high-Ti basalts are found in the intermediate zone and outer zone. However, there are only sparse outcrops in the outer zone, and so geochemical data on basalts from the outer zone are rare and the role of plume-lithosphere interaction in the petrogenesis of volcanic rocks in the outer zone remains poorly understood. In the Sichuan basin, the Basalt Formation is found between the Permian Maokou Formation limestone and the Longtan Formation marl in some drill cores as well as in outcrops in the basin. This relationship demonstrates that the basaltic layer in the basin is part of the Emeishan flood basalts. These basalts have TiO2 contents of 3.7-4.2 wt.% and Ti/Y ratios of 604-720, being high-Ti sub-alkaline basalts. They display chondrite-normalized rare earth elements (REE) patterns enriched in light rare earth elements (LREE) relative to heavy rare earth elements (HREE) and have elevated large ion lithophile elements (LILE) and high field strength elements (HFSE). Lead isotope ratios are high (206Pb/204Pb(t)= 18.102-18.392, 207Pb/204Pb(t)= 15.578-15.606, 208Pb/204Pb(t)= 38.410-38.850), and εNd(t) values are -0.38∼1.17. Detailed petrology and geochemistry suggest that the high-Ti basalts from the Sichuan Basin did not experience significant contamination of crustal and lithospheric mantle material during the ascent of magma. We infer that these basalts resulted from low-degree melting of the plume mantle source and underwent fractional crystallization of clinopyroxene. The distribution and petrogenesis of the Sichuan Basin basalts in the outer zone are different from those of the basalts in the inner zone and there are clearly different plume-lithosphere interactions in different parts of the ELIP. In the inner zone, the temperature of the lithosphere mantle was markedly elevated due to underplating of the mantle plume, causing a substantial quantity of lithosphere mantle melting and the initial formation of low-Ti basalts. This was followed by melting of the mantle plume and the formation of high-Ti basalts. In the outer zone, lower temperatures further from the plume centre were insufficient to generate extensive melting of the lithospheric mantle. Consequently, only the mantle plume melted in the outer zone, resulting in the formation of high-Ti basalts with minimal lithospheric input

Biosynthesis of Indole Diterpene Lolitrems: Radical‐Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue

HopF2 and RIN4 co-purifty during immunoaffinity purification from high-molecular weight FPLC fractions.

<p>High Mr fractions containing HA immunoreactive (V_e 36–46 ml) bands were pooled (input, pool) and concentrated for one hour in a 10,000 Da Mr cutoff concentrator (input, [pool]). Concentrated pooled fractions were incubated with anti-HA magnetic resin (µMACS), immobilized, flow through (FT) collected and resin washed with low salt (LSW), no salt (NSW) buffer then eluted over four fractions with 0.1 M NH₄OH. Samples were resolved by SDS-PAGE and immunoblotted (IB) with α (RIN4) immunosera and α (HA) IgG. All bands are from the same blot exposure. Blots were cropped to remove molecular weight standards between lanes containing NSW and eluate fractions. Blots of purifications from -DEX tissue were overexposed relative to blots from +DEX purifications. Results are representative of 3 independent replicates.</p

Transgenic expression of HopF2 results in altered stomatal immunity.

<p>Four week old Col-0 or transgenic Arabdiopsis expressing HopF2 or HopF2^D175A under the control of a dexamethasone inducible promoter were sprayed with 30 µM dexamethasone. Leaf discs were prepared 24 hours after dexamethasone treatment and incubated in water or 1<b>×</b>10⁸ CFU suspensions of wild type <i>Pto</i> DC3000. Epidermal peels were performed after one (a) or three (b) hours incubation and stomatal aperture was measured. Values are means ± S.E.M of n = 60 stomata from 3<b>–</b>5 leaves. Results are representative of 3 independent replicates. Asterisks denote significant differences between means (t-test, p<0.05). (c) ABA treatment induces stomatal closure in transgenic Arabidopsis expressing HopF2. Two week old Col-0 or transgenic Arabdiopsis expressing HopF2 or HopF2^D175A under the control of a dexamethasone inducible promoter were sprayed with 30<b> </b>µM dexamethasone. First and second true leaves were detached one hour after dexamethasone treatment and incubated in buffer overnight then buffer or 10<b> </b>µM ABA for two hours. Epidermal peels were performed and stomatal aperture was measured. Values are means ± S.E.M of n = 60 stomata from 3<b>–</b>5 leaves.</p

The <i>Pseudomonas syringae</i> Type III Effector HopF2 Suppresses Arabidopsis Stomatal Immunity

<div><p><i>Pseudomonas syringae</i> subverts plant immune signalling through injection of type III secreted effectors (T3SE) into host cells. The T3SE HopF2 can disable Arabidopsis immunity through Its ADP-ribosyltransferase activity. Proteomic analysis of HopF2 interacting proteins identified a protein complex containing ATPases required for regulating stomatal aperture, suggesting HopF2 may manipulate stomatal immunity. Here we report HopF2 can inhibit stomatal immunity independent of its ADP-ribosyltransferase activity. Transgenic expression of HopF2 in Arabidopsis inhibits stomatal closing in response to <i>P. syringae</i> and increases the virulence of surface inoculated <i>P. syringae.</i> Further, transgenic expression of HopF2 inhibits flg22 induced reactive oxygen species production. Intriguingly, ADP-ribosyltransferase activity is dispensable for inhibiting stomatal immunity and flg22 induced reactive oxygen species. Together, this implies HopF2 may be a bifunctional T3SE with ADP-ribosyltransferase activity required for inhibiting apoplastic immunity and an independent function required to inhibit stomatal immunity.</p></div

Identification of HopF2/RIN4 complexes by gel filtration chromatography and immunoblotting.

<p>Clarified extracts from HopF2:HA overexpressing plants were subjected to gel filtration chromatography on a Sephacryl S-300 HR 16/60 column. Every second fraction from the void volume was resolved by SDS-PAGE and immunoblotted with anti RIN4 immunosera and anti-HA IgG. Bands were visualized with HRP-conjugated secondary anti-bodies and Amersham ECL Advance detection kit. (a) Elution profile of dexamethasone treated Arabidopsis HopF2:HA clarified extract fractionated by gel filtration chromatography. Solid Arrow indicates elution of HA and RIN4 immunoreactive bands. Dashed arrow indicates elution of HA immunoreactive bands alone. Elution volumes of six molecular weight standards are shown as triangles. (b) Immunoblots (IB) showing co-elution of RIN4 and HA immunoreactive bands at high molecular weight. Results are representative of 3 independent replicates. Arrows indicate expected band size for RIN4 (25 kDa) and HopF2 (25 kDa), respectively.</p