Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

<div><p>Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In <i>Arabidopsis</i>, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our <i>in vitro</i> biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).</p></div

Model depicting the impact of CIB1 shRNA on KSHV macropinocytosis and productive <i>de novo</i> infection in HMVEC-d cells.

<p>CIB1-shRNA transduction in HMVEC-d cells resulted in the following consequences which are depicted by the red lines: (i) CIB1 protein level was reduced by >90%; (ii) CIB1 association with EphA2 and the associated KSHV induced Src-PI3-K-c-Cbl signal complex was significantly reduced; (iii) KSHV induced EphA2 activation was not sustained and as a consequence, downstream Src and Erk1/2 signal amplification was significantly abrogated; (iv) EphA2 association with Src-PI3-k-c-Cbl signal complex was substantially reduced; (v) EphA2 association with actin modulating myosin IIA and alpha actinin-4 were almost abolished, (vi) as a consequence, actin cytoskeletal rearrangement and membrane blebbing was inhibited, which (vii) impaired KSHV macropinocytosis, productive trafficking, and establishment of <i>de novo</i> infection.</p

CIB1 association with KSHV productive trafficking.

<p>(A) HMVEC-d cells were incubated with medium containing Alexa 488 conjugated LysoTracker alone (no virus, uninfected) or with DiI-labeled KSHV (40 DNA copies/cell) and Alexa 488 conjugated LysoTracker at 37°C for 10 min. Cells were fixed and processed for confocal immunofluorescence analysis using mouse anti-CIB1 antibodies and co-stained with DAPI. (B) HMVEC-d cells were left uninfected or DiI-labeled KSHV infected (40 DNA copies/cell) at 37°C for 10 min and were processed for confocal immunofluorescence using Rab5 and CIB1 antibodies. (A & B) Boxed areas are enlarged in the right most panels for uninfected cells, and left most panels for infected cells. Representation of line-scan signal intensity plots for triple staining was performed on the enlarged infected cell area (bottom panel).</p

Transmission electron microscopic observation of HMVEC-d cells early during KSHV infection.

<p>HMVEC-d cells (10⁶) were infected with purified KSHV (20 DNA copies/cell) at 37°C for 5 and 10 min. Post-infection, cells were washed, fixed, processed for transmission EM, and embedded in 812 resin. Thin sections were made and visualized under a JEOL 100CXII transmission electron microscope. (A–I) Red arrows indicate HMVEC-d cell membrane protrusions induced by enveloped KSHV virion particles at various stages of wrapping and engulfment of KSHV during the process of endocytosis. Magnification 87,000 X (A–F, H and I); G: Enlarged.</p

Effect of CIB1 knockdown on <i>de novo</i> KSHV infection.

<p>(A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.</p