Cloning and expression analysis of potassium channel gene NKT3 from Nicotiana tabacum

Potassium (K+) is the predominant inorganic ion of plant cells. K+ channels in higher plant cells play an important role in regulating the influx and efflux of K+ from cells, and activity of these channels might be involved in plant stress resistance. A completely new K+ channel gene of Nicotiana tabacum was obtained through homologous cloning strategy. The complete cDNA sequence was submitted to the National Center for Biotechnology Information (NCBI) GenBank, designated as NKT3 and the accession number is FJ230956. The phylogenetic analysis indicated that NKT3 is located at the branch of weak-inwardly rectifying K+ channels and might be a member of the Shaker family. The spatial and temporal expression of the gene was also investigated. NKT3 is expressed abundantly in the roots, while little in the leaves of N. tabacum. It might be involved in the process of K+ acquirement and release in tobacco roots.Keywords: Potassium channel gene, NKT3, RACE, Nicotiana tabacu

PERMUTATION TRINOMIALS OVER FINITE FIELDS WITH EVEN CHARACTERISTIC

Permutation polynomials have been a subject of study for a long time and have applications in many areas of science and engineering. However, only a small number of specific classes of permutation polynomials are described in the literature so far. In this paper we present a number of permutation trinomials over finite fields, which are of different forms

Geminivirus Activates <i>ASYMMETRIC LEAVES 2</i> to Accelerate Cytoplasmic DCP2-Mediated mRNA Turnover and Weakens RNA Silencing in <i>Arabidopsis</i>

<div><p>Aberrant viral RNAs produced in infected plant cells serve as templates for the synthesis of dsRNAs. The derived virus-related small interfering RNAs (siRNA) mediate cleavage of viral RNAs by post-transcriptional gene silencing (PTGS), thus blocking virus multiplication. Here, we identified ASYMMETRIC LEAVES2 (AS2) as a new component of plant P body complex which mediates mRNA decapping and degradation. We found that AS2 promotes DCP2 decapping activity, accelerates mRNA turnover rate, inhibits siRNA accumulation and functions as an endogenous suppressor of PTGS. Consistent with these findings, <i>as2</i> mutant plants are resistant to virus infection whereas AS2 over-expression plants are hypersensitive. The geminivirus nuclear shuttle protein BV1 protein, which shuttles between nuclei and cytoplasm, induces AS2 expression, causes nuclear exit of AS2 to activate DCP2 decapping activity and renders infected plants more sensitive to viruses. These principles of gene induction and shuttling of induced proteins to promote mRNA decapping in the cytosol may be used by viral pathogens to weaken antiviral defenses in host plants.</p></div

Cytosolic AS2 is a functional PTGS suppressor.

<p>(A-G) Suppressor activity of AS2, AS2-NES, AS2-NLS and AS2 I88A mutant. <i>N</i>. <i>benthamiana</i> (line 16c) leaves were co-infiltrated with <i>35S</i>:<i>GFP</i> and <i>35S</i>:<i>AS2</i> or 35S:<i>AS2-NES</i> or <i>35S</i>:<i>AS2-NLS</i> or <i>35S</i>:<i>AS2I88A</i> (local silencing, A-D). E-G show systemic silencing. AS2 can interfere with systemic silencing induced by <i>35S</i>:<i>GFP</i> (compare 5F of <i>35S</i>:<i>AS2</i> co-infiltrated with 5E of mock co-infiltrated with <i>35S</i>:<i>GFP</i> and 5G with AS2 I88A mutant). Bar = 10 mm. Bar = 10 mm. (H) Western blot analysis showing GFP protein levels in <i>N</i>. <i>benthamiana</i> leaf (line 16c) infiltrated with <i>35S</i>:<i>GFP</i> along with <i>35S</i>:<i>AS2</i>, <i>35S</i>:<i>AS2-NES</i>, <i>35S</i>:<i>AS2-NLS</i>, <i>35S</i>:<i>AS2I88A</i>, or mock treatment (upper panel). Coomassie blue-staining of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) is shown as a loading control (lower panel). (I) Western blot analysis showing AS2 and its variants protein levels in <i>N</i>. <i>benthamiana</i> leaf. Coomassie blue-staining of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) is shown as a loading control (lower panel).</p

Decapping machinery antagonizes PTGS.

<p>(A) AS2 promotes DCP2 decapping activity in vitro. Addition of BV1 can further increase the ability of AS2 to enhance the decapping activity of DCP2. (B) <i>In vivo AtEXPL1</i> mRNA stability of <i>as2-1</i> mutant and AS2 complementation lines after cordycepin treatment. In WT (Col-0) plants, the half-life(½t) for <i>AtEXPL1</i>mRNA was 40 min. The level of <i>AtEXPL1</i> mRNA in each time point was normalized against that of solvent (DMSO) treatment. <i>Actin2</i> was used as an internal control. Values are mean ± SD (n = 3 biological replicates). (C) Relative virus titer of AS2 complementation lines infected with CaLCuV. Values are mean ± SD (n = 3 biological replicates).</p

<i>as2</i> mutant is resistant to CaLCuV infection.

<p>(A) WT (Col-0), <i>as1-1</i> or <i>as2-1</i> plants infected with CaLCuV. Note typical viral symptoms of early-stage flowers (10 dpi) in WT and <i>as1-1</i> inflorescence. Bar = 10mm. (B) WT and mutant plants infected with CaLCuV. Pictures were taken at 28 dpi. Bar = 10mm. (C) Flowers of WT and mutant plants from (B) (28 dpi). Bar = 10mm. (D) Relative CaLCuV DNA levels (DNA-A and DNA-B) in infected (by particle bombardment) WT, <i>as1-1</i>, and <i>as2-1</i> plants (28 dpi). <i>Actin2</i> levels served as an internal plant genomic DNA control. Values are mean ± SD (n = 3 biological replicates). (E) Relative CaLCuV DNA levels (DNA-A and DNA-B) in infected (by particle bombardment) WT, <i>as2-1</i>, and <i>35S</i>:<i>AS2</i> overexpression (<i>35S</i>:<i>AS2</i>) plants (18 dpi). <i>AtActin2</i> levels served as an internal plant genomic DNA control. Values are mean ± SD (n = 3 biological replicates).</p

<i>AS2</i> overexpression blocks PTGS in <i>Arabidopsis</i>.

<p>(A) The <i>Arabidopsis</i> L1 line (Col-0 background) carries a <i>35S</i>:<i>GUS</i> that has been silenced by PTGS [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005196#ppat.1005196.ref024" target="_blank">24</a>]. The L1 line was re-transformed with <i>35S</i>: <i>AS2</i> and <i>35S</i>:<i>AS2-YFP</i>. Note the re-activation of GUS expression by <i>35S</i>: <i>AS2</i> and <i>35S</i>:<i>AS2-YFP</i>. Bar = 10 mm. (B) <i>GUS</i> mRNA and <i>GUS-</i>specific siRNA analysis in L1, <i>35S</i>: <i>AS2</i>/L1 and <i>35S</i>:<i>AS2-YFP</i>/L1 lines.</p

BV1 interacts with AS2 and DCP2

<p>(A) Sub-cellular localization of AS2-EGFP fusion protein expressed from a native <i>AS2</i> promoter in transgenic <i>Arabidopsis</i> root. Propidiumiodide (PI) was used to stain the nucleus and plant cell wall. Note cytoplasmic AS2 bodies. Bar: 10μm. (B) <i>In vitro</i> interaction of BV1 with AS1 and AS2. Around 50% of the input AS2 protein can be pulled down by GST-BV1, whereas less than 10% of the AS1 protein can be pulled down by GST-BV1. (C) <i>In vitro</i> assay of self-association of WT AS2 and its I88A mutant. Equal input amounts of His-AS2 and His-AS2-I88A were used for GST-AS2 pull down assays. (D) <i>In vitro</i> interaction of BV1 or AS2 with various protein subunits of the P-body. Arrowheads and asterisks indicate the bait proteins and the prey proteins, respectively, in each experiment. (E) <i>In vivo</i> interaction between AS2 and DCP2. Nuclear extracts from <i>AS2p</i>:<i>AS2-EGFP</i> plants were immunoprecipitated by GFP-agrose beads (GFPtrack). The immunoprecipitate was detected by anti-DCP2 antibody. (F) <i>In vivo</i> interaction between BV1 and DCP2. Nuclear extracts from <i>35S</i>:<i>BV1-ECFP</i> plants was immunoprecipitated by GFP-agrose beads (GFPtrack). The immunoprecipitate was detected by anti-DCP2 antibody. (G) BiFC assay. cDNAs encoding proteins fused to the N- or the C-terminal portion of YFP were constructed. Aux/IAA-ECFP (IAA-ECFP) was used as a nuclear marker. The co-expressed proteins are indicated. Bar = 50μm. (H) BiFC assay of <i>in vivo</i> interaction of DCP2 with AS2, AS2-I88A, AS2-NES and AS2-NLS. Aux/IAA-ECFP (IAA-ECFP) was used as a nuclear marker. The co-expressed proteins are indicated above the photographs. Bar = 50μm. (I) BV1 expression triggers nuclear exit of AS2-DCP2 to the cytosol. Aux/IAA-ECFP (IAA-ECFP) was used as a nuclear marker and also a negative control for BV1-ECFP. The co-expressed proteins are indicated above the photographs. Bar = 50μm. (J) BV1 expression increases the number of P body marked by DCP2-AS2 complex. Twenty cells were examined in this experiment.</p

Proposed mechanism of BV1 and AS2.

<p>The viral protein BV1 causes nuclear exit of AS2 to the cytosol. Cytosolic AS2 becomes localized in P-bodies to activate decapping thereby inhibiting PTGS and facilitating virus replication.</p