Laccaria bicolor pectin methylesterases are involved in ectomycorrhiza development with Populus tremula x Populus tremuloides

The development of ectomycorrhizal (ECM) symbioses between soil fungi and tree roots requires modification of root cell walls. The pectin-mediated adhesion between adjacent root cells loosens to accommodate fungal hyphae in the Hartig net, facilitating nutrient exchange between partners. We investigated the role of fungal pectin modifying enzymes in Laccaria bicolor for ECM formation with Populus tremula x Populus tremuloides. We combine transcriptomics of cell-wall-related enzymes in both partners during ECM formation, immunolocalisation of pectin (Homogalacturonan, HG) epitopes in different methylesterification states, pectin methylesterase (PME) activity assays and functional analyses of transgenic L. bicolor to uncover pectin modification mechanisms and the requirement of fungal pectin methylesterases (LbPMEs) for ECM formation. Immunolocalisation identified remodelling of pectin towards de-esterified HG during ECM formation, which was accompanied by increased LbPME1 expression and PME activity. Overexpression or RNAi of the ECM-induced LbPME1 in transgenic L. bicolor lines led to reduced ECM formation. Hartig Nets formed with LbPME1 RNAi lines were shallower, whereas those formed with LbPME1 overexpressors were deeper. This suggests that LbPME1 plays a role in ECM formation potentially through HG de-esterification, which initiates loosening of adjacent root cells to facilitate Hartig net formation

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery

Laccaria bicolor pectin methylesterases are involved in ectomycorrhiza development with Populus tremula × Populus tremuloides

The development of ectomycorrhizal (ECM) symbioses between soil fungi and tree roots requires modification of root cell walls. The pectin-mediated adhesion between adjacent root cells loosens to accommodate fungal hyphae in the Hartig net, facilitating nutrient exchange between partners. We investigated the role of fungal pectin modifying enzymes in Laccaria bicolor for ECM formation with Populus tremula × Populus tremuloides. We combine transcriptomics of cell-wall-related enzymes in both partners during ECM formation, immunolocalisation of pectin (Homogalacturonan, HG) epitopes in different methylesterification states, pectin methylesterase (PME) activity assays and functional analyses of transgenic L. bicolor to uncover pectin modification mechanisms and the requirement of fungal pectin methylesterases (LbPMEs) for ECM formation. Immunolocalisation identified remodelling of pectin towards de-esterified HG during ECM formation, which was accompanied by increased LbPME1 expression and PME activity. Overexpression or RNAi of the ECM-induced LbPME1 in transgenic L. bicolor lines led to reduced ECM formation. Hartig Nets formed with LbPME1 RNAi lines were shallower, whereas those formed with LbPME1 overexpressors were deeper. This suggests that LbPME1 plays a role in ECM formation potentially through HG de-esterification, which initiates loosening of adjacent root cells to facilitate Hartig net formation

JEV NS2B(H)-NS3 Kinetic Parameters for Fluorogenic Substrates.

<p>JEV NS2B(H)-NS3 Kinetic Parameters for Fluorogenic Substrates.</p

Effect of pH and ionic strength on the proteolytic activity of NS2B(H)-NS3(pro) from JEV.

<p>(<b>A</b>) Shown are activities as assayed by using 150 µM Ac-nKRR-amc substrate at 37°C at pH ranging from 6.5–11.0 by using different buffers (50 mM MES, MOPS, Tris, CAPS). (<b>B</b>) Ionic strength dependence of JEV NS2B(H)–NS3(pro) enzyme activity. Assay was performed at 37°C using 100 µM Pyr-RTKR-amc, 0.5 µM enzyme and 50 mM Tris-HCl pH 9.5 buffer containing 20% glycerol (v/v) in the presence of increasing salt concentrations (0–200 mM).</p

Purification profile of JEV NS2B(H)-NS3(pro) by Ni²⁺ affinity column.

<p>This figure shows a chromatogram from the purification of JEV NS2B(H)-NS3(pro) by Ni²⁺ affinity chromatography The column was washed with 0.1 M Tris-HCl, pH 7.5, 0.3 M NaCl, 30 mM imidazole (peak A) and NS2B(H)-NS3pro was eluted with elution buffer (0.1 M Tris-HCl, pH 7.5, 0.3 M NaCl, 0.3 M imidazole (peak B).</p

JEV NS2B(H)-NS3(pro) catalysed substrate hydrolysis rates at different substrate concentrations.

<p>The graph shows reaction velocities of JEV NS2B(H)-NS3(pro) for hydrolysis of Boc-GRR-amc (A); Boc-LRR-amc (B); Ac-nKRR-amc (C), Bz-nKRR-amc (D), Pyr-RTKR-amc (E), and Abz-(R)₄SAGnY-amide (F). Assays were performed at 37°C in 50 mM Tris-HCl, pH 9.5, 30% v/v glycerol. Kinetic parameters were determined by non-linear fitting of untransformed data to the Michaelis-Menten equation. Data are reported as the mean of three experiments ± standard error (SEM).</p

Structures of JEV and WNV proteases bound to Bz-nKRR-H.

<p>(<b>A</b>) Superimposition of the WNV X-ray structure (PDB ID 2FP7) on the JEV homology model. The WNV structure is shown in blue (NS2B) and cyan (NS3); the JEV structure in orange (NS2B) and green (NS3). The Bz-nKRR-H inhibitor in the WNV structure is shown in stick representation in yellow. (<b>B</b>) Detailed interactions of Bz-nKRR-H with WNV (from X-ray structure). (<b>C</b>) Superimposition of the Bz-nKRR-H X-ray structure (PDB ID 2FP7) on the JEV homology model. Potential hydrogen bonds indicated by dashed lines. Putative interacting amino acid residues of WNV and JEV are represented in cyan and green, respectively, and Bz-nKRR-H in yellow.</p

SDS-PAGE and Western blot analysis of NS2B(H)-NS3(pro).

<p>(<b>A</b>) Samples from the IMAC column were loaded onto a 15% SDS-PAGE gel and electrophoresis was performed in Tris-glycine buffer. The gel was stained with Coomassie-Blue. Lane M, broad range protein marker; lane 2, IMAC flow-through; lane 3, 30 mM imidazole wash peak; lane M imidazole elution peak. (<b>B</b>) Western blot profile using N-terminal anti-His antibody on 15% SDS-PAGE. Lane 1, 30 mM imidazole peak fraction; lane 2, 0.3 M imidazole peak fraction; lane 3, 0.3 M imidazole after dialysis (desalting); lane 4, 0.3 M imidazole peak fraction after dialysis and concentrating (Centricon, Millipore). The band representing the intact protein shows the highest intensity upon induction of expression.</p

Schematic representation of primer binding sites and physical map of pTrcHisA/NS2B(H)-NS3(pro).

<p>(A) The figure illustrates the NS2B(H)-NS3(pro) fragment and the primer binding positions. The NS2B(H), NS2B C terminal 11 amino acid residues linker and NS3 protease domains are shown. The NS2B-NS3 cleavage site is represented as a triangle. PCR primers are shown in maroon lines representing overlapping sequences. Amino acid positions within NS2B and NS3 are shown as black letters. (B) Shown is the recombinant plasmid, pTrcHisA/NS2B(H)-NS3(pro) of JEV encoding the 31 kDa NS2B(H)-NS3(pro) from JEV. The plasmid backbone contains the <i>trc</i> promoter (pTrc), <i>lac</i> operator (<i>lac</i>O), polyhistidine (His)₆ tag, Xpress™ epitope (Xpress), ampicillin resistance gene (AmpR) and lacI^q repressor genes (lacIq). The plasmid map was generated by the Vector NTI program.</p