Schematic representation of gDNA (5029 bp) containing <i>ScBx1</i>.

<p>Indicated are: GC-islands and TATA-box motifs, start codon, exons, introns and the two gDNA fragments cloned to the VIGS-BSMV vector for transcriptional gene silencing (<i>pScBx1-fragment I</i> and <i>pScBx1-fragment II</i>). The sites recognized by methylation-sensitive restriction enzymes–<i>Mlu</i>I, <i>Hha</i>I and <i>Hpy</i>CH4IV—located within the cloned fragments of the <i>pScBx1</i> promoter region are indicated. The intron-exon structure of the <i>ScBx1</i> gene is based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171506#pone.0171506.ref023" target="_blank">23</a>].</p

Timetable of VIGS experiment design.

<p>Inoculation of the first (I) leaf of 5 days old seedlings 0 dpi, collection of third (III) and fourth (IV) leaves with virus infection symptoms 14 dpi, collection of young leaves with virus infection symptoms 21 and 99 dpi.</p

Number of rye plants inoculated with VIGS-BSMV control vectors (BSMV:00) and vectors designed for TGS containing promoter fragments of <i>ScBx1</i> (BSMV:<i>pScBx1</i>).

<p>Number of rye plants inoculated with VIGS-BSMV control vectors (BSMV:00) and vectors designed for TGS containing promoter fragments of <i>ScBx1</i> (BSMV:<i>pScBx1</i>).</p

Annotation and profiling of barley <i>GLYCOGEN SYNTHASE3/Shaggy</i>-like genes indicated shift in organ-preferential expression

<div><p>GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 <i>Arabidopsis thaliana GSK</i> genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active <i>GSK</i> genes in barley. We re-evaluated annotation of the <i>GSK</i> genes in the current barley genome (Hv_IBSC_PGSB_v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another <i>GSK</i>, predicted in Hv_IBSC_PGSB_v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in <i>Brachypodium distachyon</i>. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified <i>HvGSK</i> genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of <i>GSKs</i> from the indicated groups showed a shift in organ-preferential expression in <i>A</i>. <i>thaliana</i> and barley illustrating diversification of biological roles of individual <i>HvGSKs</i> in different plant species.</p></div

Identification and VIGS-based characterization of <i>Bx1</i> ortholog in rye (<i>Secale cereale</i> L.)

<div><p>The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, <i>Bx1</i>, <i>TSA</i> and <i>Igl</i>. A gene highly homologous to maize and wheat <i>Bx1</i> has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the <i>Bx1</i> gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. <i>Barley stripe mosaic virus</i> (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the <i>Bx1</i> gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of <i>ScBx1</i> indicates that the function of the <i>ScBx1</i> in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the <i>Igl</i>, which still remains to be identified in rye.</p></div

Relative expression of <i>ScBx1</i> and content of benzoxazinoids in control plants used for PTGS experiments.

<p>Relative expression of <i>ScBx1</i> (<b>A</b>) and content of benzoxazinoids (<b>B</b>) in non-inoculated leaves and in leaves inoculated with the control vector BSMV:α,β_(-),γ_(<i>PDS</i>) (BSMV:<i>PDS</i>). NI—non-inoculated seedlings, SC—leaves abraded with silicon carbide, SC+FES—leaves abraded with silicon carbide in FES buffer. BSMV:<i>PDS</i>—leaves abraded with silicon carbide and inoculated with BSMV:α,β_(-),γ_(<i>PDS</i>) vector suspended in FES. The results represent the mean value and standard deviation from three biological replicates. *–statistical significance (Student’s <i>t</i>-test) p < 0.05.</p

Rye seedlings used for inoculation and symptoms of <i>PDS</i> silencing.

<p>Rye seedlings 5 days after imbibition used for inoculation with the BSMV-derived vectors (<b>A</b>). Rye seedlings 14 days post inoculation (<b>B</b>) and 21 dpi (<b>C</b>). Control plant leaf (<b>D</b>), symptoms of BSMV infection (<b>E</b>), symptoms of <i>PDS</i> silencing (<b>F</b>-<b>J</b>).</p

Normalized expression of <i>ScBx1</i> and normalized content of benzoxazinoids in TGS experiments.

<p>Normalized expression of <i>ScBx1</i> (<b>A</b>) and normalized content of benzoxazinoids (BXs) (<b>B</b>) in control plants (BSMV:00) compared to expression in experimental plants inoculated with BSMV:α,β_{(<i>pScBx1-fragment I</i>)},γ_{(<i>pScBx1-fragment II</i>)} (BSMV:<i>pScBx1</i>). Results are shown on a logarithmic scale. BSMV:00 –the mean value of <i>ScBx1</i> relative expression in plants inoculated with BSMV:α,β_(-),γ_(-) vector assumed as 1.00. BSMV:<i>pScBx1</i>—the mean value of <i>ScBx1</i> normalized expression in leaves inoculated with silencing vector. #17, #22, #29 –the <i>ScBx1</i> normalized expression in plants inoculated with silencing vector. Statistical significance (Student’s <i>t</i>-test): ** p < 0.005, * 0.005 < p < 0.05.</p

Parameters of mass spectrometer analysis.

<p>Parameters of mass spectrometer analysis.</p

Relative level of <i>ScBx1</i> transcript and amount of benzoxazinoids in rye.

<p>Relative level of <i>ScBx1</i> transcript (<b>A</b>) and total amount of DIBOA/DIBOA-Glc and total amount of tested benzoxazinoids (<b>B</b>) in hypocotyls/leaves of rye cv. Stach F1 collected 1, 2, 3, 4, 7, 14, 21 and 99 days after imbibition. The results represent the mean values and the standard deviations from three biological replicates. *—total amount of: HBOA, DIBOA, DIBOA-Glc, DIMBOA, DIMBOA-Glc and MBOA.</p