Activity of Fusion Prophenoloxidase-GFP and Its Potential Applications for Innate Immunity Study

<div><p>Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three <i>Drosophila melanogaster</i> PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu²⁺ was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by <i>Drosophila</i> serine protease, and rPPO1-GFP binds to <i>Micrococcus luteus</i> and <i>Beauveria bassiana</i> spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action <i>in vitro</i>. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed <i>in vivo</i>.</p></div

Resistance Risk and Resistance-Related Point Mutations in Target Protein Cyt b of the Quinone Inside Inhibitor Amisulbrom in Phytophthora litchii

Amisulbrom is a novel quinone inside inhibitor, which exhibits excellent inhibitory activity against phytopathogenic oomycetes. However, the resistance risk and mechanism of amisulbrom in Phytophthora litchii are rarely reported. In this study, the sensitivity of 147 P. litchii isolates to amisulbrom was determined, with an average EC50 of 0.24 ± 0.11 μg/mL. The fitness of resistant mutants, obtained by fungicide adaption, was significantly lower than that of the parental isolates in vitro. Cross-resistance was detected between amisulbrom and cyazofamid. Amisulbrom could not inhibit the cytochrome bc1 complex activity with H15Y and G30E + F220L point mutations in cytochrome b (Cyt b) in vitro. Molecular docking indicated that the H15Y or G30E point mutation can decrease the binding energy between amisulbrom and P. litchii Cyt b. In conclusion, P. litchii might have a medium resistance risk to amisulbrom, and a novel point mutation H15Y or G30E in Cyt b could cause high amisulbrom resistance in P. litchii

Presentation_1_Spatial Patterns and Drivers of Microbial Taxa in a Karst Broadleaf Forest.PDF

<p>Spatial patterns and drivers of soil microbial communities have not yet been well documented. Here, we used geostatistical modeling and Illumina sequencing of 16S rRNA genes to explore how the main microbial taxa at the phyla level are spatially distributed in a 25-ha karst broadleaf forest in southwest China. Proteobacteria, dominated by Alpha- and Deltaproteobacteria, was the most abundant phylum (34.51%) in the karst forest soils. Other dominating phyla were Actinobacteria (30.73%), and Acidobacteria (12.24%). Soil microbial taxa showed spatial dependence with an autocorrelation range of 44.4–883.0 m, most of them within the scope of the study plots (500 m). An increasing trend was observed for Alphaproteobacteria, Deltaproteobacteria, and Chloroflexi from north to south in the study area, but an opposite trend for Actinobacteria, Acidobacteira, and Firmicutes was observed. Thaumarchaeota, Bacteroidetes, Gemmatimonadetes, and Verrucomicrobia had patchy patterns, Nitrospirae had a unimodal pattern, and Latescibacteria had an intermittent pattern with low and high value strips. Location, soil total phosphorus, elevation, and plant density were significantly correlated with main soil bacterial taxa in the karst forest. Moreover, the total variation in soil microbial communities better explained by spatial factors than environmental variables. Furthermore, a large part of variation (76.8%) was unexplained in the study. Therefore, our results suggested that dispersal limitation was the primary driver of spatial pattern of soil microbial taxa in broadleaved forest in karst areas, and other environmental variables (i.e., soil porosity and temperature) should be taken into consideration.</p

The effect of combined and independent components of neural induction reagent on FAK and Src activity in HMSCs.

<p>(A) The ECFP/FRET ratio images of the Src/FAK biosensor in representative HMSCs before and after treatment. (B) The time courses of normalized Src/FAK ECFP/FRET ratio (mean ± SEM) in different experiments shown in panel (A). (C) The increase of Src/FAK ECFP/FRET ratio after treatment (mean ± SEM). (*) indicates significant difference between groups with p-value<0.05 by t-test; (**) indicates a group is significantly different from other groups in the same cluster, p-value<0.05 by t-test.</p

FAK/Src inter-regulation in HMSCs treated with combined neural induction reagent.

<p>(<b>A</b>) The emission ratio images of the Src and FAK biosensors in HMSCs cross-inhibited by FAK and Src inhibitors respectively with treatment of combined neural induction reagent. (<b>B</b>) The time courses of the normalized emission ratio of the Src/FAK biosensor (mean ± SEM) in HMSCs shown in panel (A). (<b>C</b>) The change of the Src/FAK ECFP/FRET ratio (mean ± SEM) in HMSCs shown in panel (A). (**) indicates a group is significantly different from other groups in the same cluster, p-value<0.05 by t-test.</p

FAK/Src activation mechanism upon differentiation induction.

<p>The schematics diagram showing different activation patterns of FAK/Src with distinct regulation mechanisms upon the treatment of different induction medium on HMSCs.</p

Unveiling Vacuolar H⁺‑ATPase Subunit a as the Primary Target of the Pyridinylmethyl-Benzamide Fungicide, Fluopicolide

An estimated 240 fungicides are presently in use, but the direct targets for the majority remain elusive, constraining fungicide development and efficient resistance monitoring. In this study, we found that Pcα-actinin knockout did not influence the sensitivity of Phytophthora capsici to fluopicolide, which is a notable oomycete inhibitor. Using a combination of Bulk Segregant Analysis Sequencing and Drug Affinity Responsive Target Stability (DARTS) assays, the vacuolar H+-ATPase subunit a (PcVHA-a) was pinpointed as the target protein of fluopicolide. We also confirmed four distinct point mutations in PcVHA-a responsible for fluopicolide resistance in P. capsici through site-directed mutagenesis. Molecular docking, ATPase activity assays, and a DARTS assay suggested a fluopicolide-PcVHA-a interaction. Sequence analysis and further molecular docking validated the specificity of fluopicolide for oomycetes or fish. These findings support the claim that PcVHA-a is the target of fluopicolide, proposing vacuolar H+-ATPase as a promising target for novel fungicide development

The effect of combined and independent components of muscle induction reagent on FAK and Src activity in HMSCs.

<p>(A) The ECFP/FRET ratio images of the Src/FAK biosensor in representative HMSCs before and after treatment. (B) The time courses of normalized Src/FAK ECFP/FRET ratio (mean ± SEM) in different experiments shown in panel (A). (C) The increase of Src/FAK ECFP/FRET ratio after treatment (mean ± SEM). (*) indicates significant difference between groups with p-value<0.05 by t-test.</p

FAK/Src inter-regulation in HMSCs treated with combined osteogenic reagent.

<p>(<b>A</b>) The emission ratio images of the Src and FAK biosensors in HMSCs cross-inhibited by FAK and Src inhibitors with treatment of combined osteogenic reagent, respectively. (<b>B</b>) The time courses of the normalized emission ratio of the Src/FAK biosensor (mean ± SEM) in HMSCs shown in panel (A). (<b>C</b>) The change of the Src/FAK ECFP/FRET ratio (mean ± SEM) in HMSCs shown in panel (A). (**) indicates a group is significantly different from other groups in the same cluster, p-value<0.05 by t-test.</p

The effect of combined and independent components of osteogenic reagent on KRas-Src activity in HMSCs.

<p>(<b>A</b>) The ECFP/FRET ratio images of KRas-Src biosensors in representative HMSCs before and after treatment. (<b>B</b>) The time courses of normalized KRas-Src ECFP/FRET ratio (mean ± SEM) in different experiments shown in panel (A). (<b>C</b>) The increase of ECFP/FRET ratio after treatment (mean ± SEM). (*) indicates significant difference between groups with p-value<0.05 by t-test.</p