Image_1_Exopolysaccharide is required for motility, stress tolerance, and plant colonization by the endophytic bacterium Paraburkholderia phytofirmans PsJN.JPEG

Paraburkholderia phytofirmans PsJN is an endophytic bacterium and has been shown to promote the growth and health of many different plants. Exopolysaccharide (EPS) plays important roles in plant-bacteria interaction and tolerance to environmental stresses. However, the function of EPS in PsJN and its interaction with plants remain largely unknown. In this study, a deletion mutation of bceQ gene, encoding a putative flippase for the EPS biosynthesis, was introduced in the genome of PsJN. The ΔbceQ mutant produced a significantly lower level of EPS than the wild type strain in culture media. Compared to the wild type PsJN, the ΔbceQ mutant was more sensitive to desiccation, UV damage, salt (NaCl) and iron (FeCl3) stresses, and bacteriophage infection. More importantly, the mutation of bceQ decreased the endophytic colonization of PsJN in camelina (Camelina sativa) and pea (Camelina sativa) under plant drought stress conditions. To the best of our knowledge, this is the first report that EPS production is required for the maximal colonization of an endophytic bacterium in the plant tissues under stress conditions.</p

westerlies_past1000_supplementary – Supplemental material for Variation of the summer Asian westerly jet over the last millennium based on the PMIP3 simulations

Supplemental material, westerlies_past1000_supplementary for Variation of the summer Asian westerly jet over the last millennium based on the PMIP3 simulations by Nanxuan Jiang, Qing Yan and Huijun Wang in The Holocene</p

The trafficking of preRTA-EGFP in <i>png1</i>Δ.

<p>The <i>png1</i>Δ and isogenic wild type (BY4743) harboring preRTA-EGFP was visualized at 2, 4, 6 and 24 hpi with an Olympus BX41 fluorescence microscope. Yeast cells were treated with FM4-64 to stain the vacuole. Merged images show localization of preRTA-EGFP relative to the vacuole.</p

Wild Type RTA and Less Toxic Variants Have Distinct Requirements for Png1 for Their Depurination Activity and Toxicity in <i>Saccharomyces cerevisiae</i>

<div><p>Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: <i>N</i>-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome.</p></div

Ribosome depurination of mature (A) and precursor (B) forms of EGFP-tagged RTA variants compared with wild type RTA-EGFP <i>in vivo</i>.

<p>Ribosomes were isolated from yeast (W303) expressing wild type RTA or each variant at 0, 2, 4, 6 and 8 hpi and depurination was analyzed by qRT-PCR. Two pairs of primers designed to amplify the target amplicon (depurinated SRL) and the reference amplicon (25S rRNA) were used. The data was analyzed by the comparative ΔC_T method (ΔΔC_T). The y-axis indicates the fold change in depurination in yeast harboring the precursor and mature forms of wild type RTA or each variant relative to yeast harboring the empty vector. The tables below each figure show the level of depurination. The data are shown as mean and standard deviation calculated from three replicates. Differences in mean depurination by the RTA variants relative to matRTA (A) or preRTA (B) at each time point were analyzed using two sided, two-sample t tests (*P<0.05, **P<0.01, ***P<0.001).</p

The viability and expression of yeast cells expressing the precursor and mature forms of RTA variants fused to EGFP at their C-termini.

<p>(A)Yeast cells were first grown in SD-Leu supplemented with 2% glucose and then transferred to SD-Leu supplemented with 2% galactose. At 10 hpi, a series of ten-fold dilutions were plated on media containing 2% glucose and grown at 30°C for approximately 48 h. The precursor forms are shown on the left and the mature forms are shown on the right. The mutations of RTA are indicated on the right. VC represents empty vector. EGFP represents EGFP open reading frame in the empty vector. Membrane fractions isolated from cells expressing the EGFP tagged mature (B) and precursor (C) forms of wild type RTA and RTA mutants were separated on a 12% SDS-polyacrylamide gel and probed with polyclonal anti-RTA (1∶5000). The blot was reprobed with the ER membrane marker Dpm1p as a loading control.</p

The trafficking of wild type preRTA-EGFP, preG212E-EGFP and preP95L/E145K-EGFP in yeast.

<p>Localization of the precursor (A) and mature forms (B) of wild type RTA-EGFP, G212E-EGFP and P95L/E145K-EGFP in yeast (W303) was analyzed at 2, 4, 6 and 24 hpi with an Olympus BX41 epifluorescence microscope. Yeast cells (W303) were treated with FM4-64 to stain the vacuole. Merged images show localization of each protein relative to the vacuole.</p

Toxicity, depurination and trafficking of RTA variants.

<p>Toxicity, depurination and trafficking of RTA variants.</p

Expression of wild type RTA and RTA variants in vacuole, membrane and cytosol fractions.

<p>The vacuole (V), membrane (M) and cytosol (C) fractions were isolated from yeast (W303) at 6 hpi. The proteins (5 µg) were separated on a 10% SDS-polyacrylamide gel and probed with monoclonal anti-RTA (1∶5000). The blot was reprobed with the vacuole membrane marker, Vph1p, the ER membrane marker, Dpm1p and the cytosol marker, Pgk1p.</p

Analysis of preRTA and preG83D in <i>png1Δ</i>.

<p>(A) The viability of <i>png1Δ</i> and isogenic wild type (BY4743) expressing preRTA or preG83D. A series of 10-fold dilutions were spotted either on a glucose plate at 0 and 24 h after galactose induction or on a galactose plate after overnight growth in glucose media. The CFU/ml was calculated based on the analysis of at least three different transformants. Differences in CFU/mL for BY4743 and png1Δ were analyzed using two sided, two-sample t tests (*P<0.05). (B) Immunoblot analysis of membrane and cytosol fractions of <i>png1</i>Δ and BY4743 expressing preRTA or preG83D. The proteins (5 µg) were treated with (+) or without (-) Endo H. (C) Ribosome depurination by preRTA or preG83D was analyzed by qRT-PCR after isolation at 2, 4, 6 hpi. Differences in mean depurination by preRTA or preG83D in BY4743 and png1Δ were analyzed using two sided, two-sample t tests (*P<0.05, **P<0.01, ***P<0.001).</p