GDNF induces cytosolic Ca²⁺ signaling through Tyr1015 of RET.

<p>(<b>A</b>) Schematic representation of GFP-tagged RET constructs. (<b>B</b>) Constructs expressed in HeLa cells. (<b>C–F</b>) Representative single-cell Ca²⁺ recordings of GFP positive HeLa cells loaded with Fura-2/AM and subsequently treated with GDNF (100 ng/ml). (<b>C</b>) Cells expressing the RET^WT construct responded to GDNF with either Ca²⁺ transient (top trace) or Ca²⁺ oscillations (bottom trace). Cells expressing RET¹⁰¹⁵ (<b>D</b>) or RET^1015/1062 (<b>F</b>) failed to trigger a Ca²⁺ response following GDNF exposure. Cells expressing RET¹⁰⁶² (<b>E</b>) responded to GDNF in a similar manner as cells expressing RET^WT.</p

Characteristics of Ca²⁺ responses triggered by GDNF in RET^WT cells treated with various inhibitors.

a<p>Non-responding cells have no Ca²⁺ increase exceeding 1.25 of the baseline.</p>b<p>Transient responding cells have one Ca²⁺ peak exceeding 1.25 of the baseline.</p>c<p>Oscillatory responding cells have at least three Ca²⁺ peaks exceeding 1.25 of the baseline.</p>d<p>[number of cells/number of experiments].</p>e<p>[concentration of extracellular Ca²⁺].</p

GDNF-induced Ca²⁺ signaling phosphorylates ERK1/2 and CaMKII.

<p>(<b>A</b>–<b>D</b>) Western blot of HeLa cells transfected with RET^WT or RET¹⁰¹⁵ treated with GDNF (100 ng/ml). GDNF triggers time dependent phosphorylation of ERK1/2 (pERK1/2) in RET^WT cells that is suppressed by BAPTA (10 µM) (<b>A</b>). Less pERK1/2 is observed in cells transfected with RET¹⁰¹⁵ than RET^WT (<b>B</b>). GDNF-induced phosphorylation of CaMKII (pCaMKII) or pERK1/2 is suppressed when blocking PLC with U73122 (5 µM) (<b>C</b>) or knocking down PLCγ with siRNA (PLCγ-siRNA) (<b>D</b>). Treating RET^WT cells with the U73122 analogue U73343 (5 µM) had no effect on GDNF-activated pCaMKII or pERK1/2 (<b>C</b>). Increased Caspase-3 cleavage was not detected in cells treated with the inhibitors BAPTA or U73122 (<b>C</b>).</p

RET Tyr1015 mediates GDNF-stimulated migration <i>in vivo</i>.

<p>(<b>A</b>) Cartoon illustrating mouse embryo electroporation and GDNF-bead stimulated migration. (<b>B</b>–<b>D</b>) Migration of cortical progenitors in organotypic brain slices from embryos electroporated with RET^WT (<b>C</b>) or RET¹⁰¹⁵ (<b>D</b>) treated without beads (Control) or with beads (indicated with circles) soaked in PBS (Vehicle) or GDNF (500 ng/ml) placed in the cortical plate (CP). GFP positive RET^WT expressing progenitors (green) stimulated with GDNF beads (<b>B</b>, <b>C</b>) show significantly enhanced migration from the ventricular zone (VZ) towards the CP, as compared to Control, Vehicle, or inhibition of PLC with U73122 (5 µM). In RET¹⁰¹⁵ expressing progenitors GDNF beads failed to stimulate migration (<b>B</b>, <b>D</b>). Scale bars, 100 µm.</p

A RET/PLCγ/InsP₃R-cascade stimulates GDNF-induced Ca²⁺ release.

<p>(<b>A–H</b>) Representative single-cell Ca²⁺ recordings of GFP positive RET^WT expressing cells loaded with Fura-2/AM and preincubated with inhibitors as indicated, following treatment with GDNF (100 ng/ml). Inhibiting PLC with U73122 (5 µM) (<b>A</b>) or knocking down PLCγ with siRNA (<b>B</b>) blocked the cytosolic Ca²⁺ response induced by GDNF. Cells transfected with the Mock-siRNA retain the Ca²⁺ response (<b>C</b>). Inhibiting InsP₃R with 2-APB (5 µM) abolished the Ca²⁺ response induced by GDNF (<b>D</b>), while inhibiting RyR with ryanodine (a, 20 µM) or dantrolene (b, 10 µM) had no effect (<b>E</b>). Depleting intracellular Ca²⁺ stores with the SERCA Ca²⁺-ATPase inhibitor Thapsigargin (1 µM) blocked the Ca²⁺ response (<b>F</b>). Zero extracellular Ca²⁺ eliminated the GDNF-induced Ca²⁺ response (<b>G</b>), whereas a low extracellular concentration of Ca²⁺ (1 mM) produced a normal Ca²⁺ response (<b>H</b>).</p

Endogenous RET is expressed in the embryonic neocortex.

<p>Immunohistochemistry of an E14.5 mouse forebrain coronal slice (<b>A</b>, Scale bars, 250 µm) and cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ) regions (<b>B</b>, Scale bars, 25 µm) for RET and TuJ1. Western blot (<b>C</b>), reverse transcription PCR (35 cycles) (<b>D</b>) and real-time PCR (<b>E</b>) analysis for RET in cortical tissue. Cerebellar tissue and NIH3T3 cells were used as controls. TATA-box binding protein (TBP) was the house keeping gene. (<b>F</b>) Representative single-cell Ca²⁺ recording of a RET^WT expressing primary cortical neuron loaded with Fura-2/AM and subsequently treated with GDNF (100 ng/ml).</p

GDNF-induced Ca^2<b>+</b> signaling stimulates cell motility <i>in vitro</i>.

<p>(<b>A</b>) Cell motility assay in HeLa cells transfected with RET^WT or RET¹⁰¹⁵ and treated with GDNF (100 ng/ml) for 6–8 h. (<b>B</b>) Cell motility was significantly higher in RET^WT transfected cells treated with GDNF, as compared to control cells without GDNF. Buffering cytosolic Ca²⁺ with BAPTA (10 µM) or inhibiting PLC with U73122 (5 µM) abolished the cell motility. GDNF failed to stimulate cell motility in cell transfected with RET¹⁰¹⁵. Bars represent the average number of cells in the scratch. * <i>P</i><0.05 versus control.</p

Characteristics of Ca²⁺ responses induced by GDNF in cells expressing various RET constructs.

a<p>Non-responding cells have no Ca²⁺ increase exceeding 1.25 of the baseline.</p>b<p>Transient responding cells have one Ca²⁺ peak exceeding 1.25 of the baseline.</p>c<p>Oscillatory responding cells have at least three Ca²⁺ peaks exceeding 1.25 of the baseline.</p>d<p>[number of cells/number of experiments].</p

A single-step reverse transcription loop-mediated isothermal amplification assay for rapid and accurate detection of <i>Pepper vein yellows virus</i>

<p>Pepper vein yellows virus (PeVYV) is a member of Polerovirus and infects solanaceous crops worldwide. In this study, a highly efficient and easy-to-use single-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for PeVYV detection. This assay used two outer (F3 and B3), two inner (FIP and BIP) and two loop (LF and LB) primers in a single reaction. The optimized reaction was determined to be at 62°C for 45 min and the whole reaction took less than 1 h. This assay was about 10 times more sensitive than the conventional one-step RT-PCR. This RT-LAMP assay should allow specific detection of PeVYV in plant tissues. We recommend this PeVYV-specific RT-LAMP assay for large scale field studies of PeVYV infection.</p

DataSheet_2_CRISPR/Cas9-mediated gene editing of vacuolar ATPase subunit d mediates phytohormone biosynthesis and virus resistance in rice.doc

Vacuolar ATPases (V-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; OsV-ATPase subunit d (OsV-ATPase d) is part of an integral, membrane-embedded V0 complex in the V-ATPase complex. Whether OsV-ATPase d is involved in phytohormone biosynthesis and resistance in rice remains unknown. The knockout mutants of OsV-ATPase d in rice were generated using the CRISPR/Cas9 system, and mutation of OsV-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed that OsV-ATPase d is probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Compared to wild type, mutation of OsV-ATPase d significantly increased JA and ABA biosynthesis and resistance against Southern rice black-streaked dwarf virus (SRBSDV), but it decreased resistance against Rice stripe virus (RSV) in rice. The data presented in this study reveal that OsV-ATPase d mediates phytohormone biosynthesis and virus resistance in rice and can be selected as a potential target for resistance breeding in rice.</p