Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in <i>Nicotiana benthamiana</i>

<div><p>The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in <i>Nicotiana benthamiana</i>. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, <i>NbSKη-RNAi</i>, <i>35S</i>::<i>NbCycD1;1</i> transgenic <i>N</i>. <i>benthamiana</i> plants have the similar phenotype as <i>35S</i>::<i>C4</i> transgenic <i>N</i>. <i>benthamiana</i> plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants.</p></div

NbSKη interacts with and phosphorylates NbCycD1;1.

<p><b>(A</b>-<b>C)</b> Interaction between NbCycD1;1 and NbSKη was validated in Y2H (A), Co-IP (B) and BiFC (C) assays. <b>**</b> indicates NbSKη-Flag. <b>*</b> represents the substrate of NbSKη. <b>(D)</b> NbSKη phosphorylates NbCycD1;1 <i>in vitro</i>. The upper panel shows autoradiography and the bottom panel shows coomassie blue staining. <b>**</b> indicates His-NbSKη. <b>*</b> represents the substrate of NbSKη. <b>(E)</b> NbSKη phosphorylates NbCycD1;1 <i>in vivo</i>. <b>**</b> indicates phosphorylated form of GFP-CycD1;1. <b>*</b> represents the non-phosphorylated form of GFP-CycD1;1. <b>(F)</b> Identification of the phosphorylation of NbCycD1;1 mediated by NbSKη using λ-PPase. Plant tissues expressing GFP-NbCycD1;1 with or without Flag-NbSKη were extracted at 60 hpi. Two aliquots of samples were treated in the reaction system in the absence or presence of λ-PPase for only 10 min at 30°C. <b>**</b> indicates phosphorylated form of GFP-CycD1;1. <b>*</b> represents the non-phosphorylated form of GFP-CycD1;1. Relative values represent the ratio of phosphorylated form and non-phosphorylated form of NbCycD1;1 (top panel) and the accumulation level of NbSKη-Flag (middle panel), respectively.</p

The proposed model to show how TLCYnV C4 interferes with the cell cycle in mature leaves through changing the nuclear-localization of NbSKη.

<p>TLCYnV C4 directs NbSKη to the cyto-membrane and reduces the accumulation of nucleus-localized NbSKη. The redistribution of NbSKη mediated by C4 interrupts the phosphorylation and proteasomal degradation of nucleus-located NbCycD1;1, and then increases the accumulation level of NbCycD1;1. Increased accumulation of NbCycD1;1 induces the callus-like tissues formation and creates an environment suitable for DNA virus replication in mature and differentiated leaves.</p

<i>NbSKη-</i>silenced <i>N</i>. <i>benthamiana</i> plants display the phenotype similar to <i>N</i>. <i>benthamiana</i> plants infected by PVX-C4.

<p><b>(A</b> and <b>B)</b> qRT-PCR (A) and western blot (B) assays of <i>NbSKη</i> gene expression in wild-type (WT) and <i>NbSKη</i>-silenced plants. Relative accumulation level of <i>NbSKη</i> transcripts is normalized against the amount of <i>actin</i> transcript. Error bar denotes the standard deviation of three biological replicates. <b>(C)</b> Growth of the wild-type (WT), mock (TRV-GFP) and <i>NbSKη</i>-silenced (TRV-<i>NbSKη</i>) <i>N</i>. <i>benthamiana</i> plants at 35 dpi. <b>(D)</b> The leaf petioles of <i>NbSKη-</i>silenced <i>N</i>. <i>benthamiana</i> leave mimics that of <i>N</i>. <i>benthamiana</i> leave inoculated with PVX-C4. The phenotype of 1st-5th leaves of plants under different treatments was shown. <b>(E)</b> The petiole length of the 3rd leaf of plants under different treatments. Each treatment had fifteen plants at the same developmental stage. ** represent significant difference (P value <0.01) between treatments tested by <i>t</i>-test. <b>(F)</b> The phenotype of WT, <i>35S</i>::<i>C4</i> or <i>NbSKη</i>-RNAi transgenic <i>N</i>. <i>benthamiana</i> plants (left) and qRT-PCR analysis of <i>NbSKη</i> gene expression in WT and <i>NbSKη</i>-RNAi transgenic plants (right). Relative accumulation level of <i>NbSKη</i> transcripts is normalized against the amount of <i>actin</i> transcript. Error bar denotes the standard deviation of three biological replicates. <b>(G)</b> The petiole length of the 2nd (right column) and 3rd (left column) leaves of WT, <i>35S</i>::<i>C4</i> transgenic or <i>NbSKη</i>-RNAi transgenic <i>N</i>. <i>benthamiana</i> plants. <b>(H)</b> Images of Col-0, <i>pBZR1</i>::<i>BZR-CFP</i> (BZR1 overexpression <i>A</i>. <i>thaliana</i>) or <i>35S</i>::<i>TLCYnV C4</i> transgenic <i>A</i>. <i>thaliana</i>. Arrowheads indicate the downward leaf curling of the leaves. <b>(I)</b> Western blot analysis of BZR1-CFP phosphorylation level. The presence of TLCYnV C4 could decrease the level of phosphorylated BZR1-CFP. BZR1-CFP was detected using a monoclonal antibody against GFP. C4-Myc was detected using a monoclonal antibody against Myc. BZR1-CFP and phosphorylated BZR1-CFP were designated as BZR1 and ^PBZR1, respectively. <b>(J)</b> Quantitative PCR analysis of relative expression levels of BZR1-targeted genes (<i>CPD</i> and <i>DWF4</i>) in Col-0, <i>35S</i>::<i>C4</i> transgenic or <i>pBZR1</i>::<i>BZR1-CFP</i> transgenic <i>A</i>. <i>thaliana</i>.</p

Immuno-cytochemistry and immunoblot analysis of the nuclear-localized accumulation of NbSKη under different treatments.

<p><b>(A)</b> Analysis of nuclear-localized NbSKη accumulation through immuno-cytochemistry and electron microscopy. Thin sections were prepared from <i>N</i>. <i>benthamiana</i> leaf tissues under different treatments. All the sections were probed with anti-GSK3β polyclonal antibody followed by a protein A-gold conjugate. Scale bar = 0.2 μm. <b>(B)</b> Average number of gold particles per 0.25 um² in three randomly selected nuclei in sections from various treatments. ** represents significant difference (P value <0.01) between treatments tested by <i>t-</i>test. <b>(C)</b> Nuclear-cytoplasmic fractionation analysis of the accumulation of the nuclear-localized NbSKη in plant tissues under different treatments. Western blot analysis was conducted with the antibodies specific to the indicated proteins.</p

TLCYnV directs NbSKη to cytoplasmic membrane.

<p><b>(A)</b> Subcellular localization of C4-GFP or GFP-NbSKη fusion protein in <i>N</i>. <i>benthamiana</i> epidermal cells. GFP-tagged proteins were expressed in the transgenic <i>N</i>. <i>benthamiana</i> plants expressing the nucleus marker H2B-RFP (2–4 rows), or in the <i>N</i>. <i>benthamiana</i> plants expressing the cytoplasmic membrane marker (first row). Scale bar = 50 μm. <b>(B)</b> Subcellular fractionation of C4-GFP, GFP, and PIP2A-DsRed. Plant tissues expressing C4-GFP, GFP, or PIP2A-DsRed were fractionated into soluble (S) and membrane-enriched (P) fractions. GFP and PIP2A-DsRed acted as the markers of soluble fraction and membrane-enriched fraction, respectively. 3K, the extracts following centrifugation at 3,000 <i>g</i>; 30K, the extracts following centrifugation at 30,000 <i>g</i>. <b>(C)</b> Membrane flotation assays of GFP-NbSKη, GFP, and PIP2A-DsRed. GFP-NbSKη and PIP2A-DsRed were detected using monoclonal antibodies specifically against GFP and RFP, respectively. <b>(D)</b> BiFC analysis of C4/NbSKη interaction position in epidermal cells of H2B-RFP transgenic <i>N</i>. <i>benthamiana</i> plants. Scale bar = 50 μm. <b>(E)</b> Distribution patterns of GFP-NbSKη in the presence of CFP or C4-CFP expressed in the H2B-RFP transgenic <i>N</i>. <i>benthamiana</i> epidermal cells. Scale bar = 50 μm. <b>(F)</b> Membrane flotation assays of plant extracts expressing CFP alone or co-expressing GFP-NbSKη with CFP or C4-CFP. CFP, C4-CFP, and GFP-NbSKη were detected using a rabbit monoclonal antibody raised against GFP. <b>(G)</b> Subcellular fractionation of plant tissues prepared from <i>N</i>. <i>benthamiana</i> plants expressing GFP-NbSKη with CFP or C4-CFP.</p

Phosphorylation of NbCycD1;1 mediated by NbSKη promotes NbCycD1;1 degradation via 26S proteasome.

<p><b>(A)</b> Detection of the stability of GFP or GFP-NbCycD1;1 protein by semi-<i>in vivo</i> assays. GFP or GFP-NbCycD1;1 protein was analyzed with anti-GFP antibody at different time points after CHX treatment in the presence or absence of ATP. <b>(B)</b> Detection of the stability of GST or GST-NbCycD1;1 protein by <i>in vitro</i> assays. Purified GST or GST-NbCycD1;1 was mixed with additive volumes of fresh plant extract and then diminishing volumes of elution buffer were added for the identical concentration of fusion proteins at 25°C for 2h. GST or GST-NbCycD1;1 protein level was analyzed with anti-GST antibody. <b>(C</b> and <b>D)</b> Tris SDS-PAGE (C) or Tricine SDS-PAGE (D) analysis of the influence of NbCycD1;1 phosphorylation mediated by NbSKη on NbCycD1;1 stability by semi-<i>in vivo</i> assays. <b>(E)</b> Detection of the influence of NbCycD1;1 phosphorylation mediated by NbSKη on NbCycD1;1 stability by <i>in vitro</i> assays. <b>(F)</b> The phosphorylation level of NbCycD1;1 or NbCycD1;1 (T328A) by NbSKη <i>in vitro</i>. <b>**</b> indicates His-NbSKη. <b>*</b> represents the substrate of NbSKη. <b>(G)</b> Western blot analysis of stability of GFP-NbCycD1;1, GFP-NbCycD1;1 (T328A), or GFP by semi-<i>in vivo</i> assays. <b>(H)</b> Western blot analysis of the protein level of NbSKη in wild-type (WT) or <i>NbSKη-</i>RNAi plants. <b>(I)</b> Western blot analysis of the stability of GFP-NbCycD1;1 in WT or <i>NbSKη-</i>RNAi <i>N</i>. <i>benthamiana</i> plants by semi-<i>in vivo</i> assays. <b>(J)</b> Effect of MG132 on the stability of GFP-NbCycD1;1 protein by semi-<i>in vivo</i> assays. GFP-NbCycD1;1 protein levels were analyzed with anti-GFP antibody at different time points after 100 μM CHX and 20 mM ATP treatments in the presence of 100 μM MG132 or an equal volume of DMSO (control). <b>(K)</b> Effect of BL on the stability of GFP-NbCycD1;1 protein in semi-<i>in vivo</i> assays.</p