De Novo Foliar Transcriptome of <em>Chenopodium amaranticolor</em> and Analysis of Its Gene Expression During Virus-Induced Hypersensitive Response

<div><h3>Background</h3><p>The hypersensitive response (HR) system of <em>Chenopodium</em> spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for <em>Chenopodium</em>, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of <em>C. amaranticolor</em>, a <em>Chenopodium</em> species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance.</p> <h3>Methodology and Principal Findings</h3><p>Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons). BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10⁻⁵. Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs) and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by <em>Tobacco mosaic virus</em> and <em>Cucumber mosaic virus</em> by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized.</p> <h3>Conclusions</h3><p>To our knowledge, this study is the first description of the genetic makeup of <em>C. amaranticolor</em>, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further functional characterization to dissect the molecular mechanisms and regulatory pathways of the HR-type of virus resistance in <em>Chenopodium</em>.</p> </div

GO categories of biological process (BP), cellular component (CC) and molecular function (MF) for the early water-stress responding genes (P-6h <i>vs</i>. P-0h) in <i>A</i>. <i>sparsifolia</i> primary roots.

<p>(A) the early water-stress inducible genes; (B) the early water-stress repressed genes. The right y-axis shows the number of genes in a category, and the left y-axis indicates the percentage of a specific category of genes in that main category.</p

Summary of unigene annotations against public databases.

<p>Abbreviations: NR, non-redundant protein sequence; NT, non-redundant nucleotide; Pfam, Protein Family Database; KEGG, Kyoto Encyclopedia of Genes and Genomes database; KOG, euKaryotic Ortholog Groups; GO, Gene Ontology.</p><p>Summary of unigene annotations against public databases.</p

Effect of water stress induced by PEG-6000 on development of the <i>Alhagi sparsifolia</i> primary roots.

<p>(A) Seeds of <i>A</i>. <i>sparsifolia</i> (Scale bar = 2.0 cm). (B) Typical phenotype of the <i>A</i>. <i>sparsifolia</i> seedlings at 7 days post treatment with PEG-6000 (Scale bar = 1.0 cm). The <i>A</i>. <i>sparsifolia</i> seeds were first treated with concentrated sulfuric acid (98%) for 20 min, and then put on fully wetted filter paper at 25°C in the dark for 24 h. The germinated seeds were selected and transferred to petri dishes containing filter paper saturated with the different percentages of PEG-6000 solution (0, 90, 150, 220, 270 and 320 g/L), and were remained at 25°C in the dark for 3 days followed by 4 additional days but under 16-h light/8-h dark. At least 15 seedlings were included in each different treatment, and three independent experiments were preformed. (C) Average length of the primary roots of <i>A</i>. <i>sparsifolia</i> treated with PEG-6000 at 7 days. Error bars represent SE. P values determined by Student t test (** p < 0.01).</p

Characteristics of homology search of unigenes against the NR database.

<p>(A) E-value distribution of the top BLAST hits for each unigene with a cut-off E-value of 1.0E-5. (B) Similarity distribution of the best BLAST hits for each unigene. (C) Species distribution is shown as the percentage of the total homologous sequences with an E-value of at least 1.0E-5. We used all plant proteins in the NCBI NR database for homology search and extracted the first hit of each sequence for analysis.</p

GO categories of biological process, cellular component and molecular function for the transcriptome of the <i>A</i>. <i>sparsifolia</i> primary roots.

<p>The right y-axis shows the number of genes in a category, and the left y-axis indicates the percentage of a specific category of genes in that main category.</p

Transcriptomic Analysis of the Primary Roots of <i>Alhagi sparsifolia</i> in Response to Water Stress

<div><p>Background</p><p><i>Alhagi sparsifolia</i> is a typical desert phreatophyte and has evolved to withstand extreme dry, cold and hot weather. While <i>A</i>. <i>sparsifolia</i> represents an ideal model to study the molecular mechanism of plant adaption to abiotic stress, no research has been done in this aspect to date. Here we took advantage of Illumina platform to survey transcriptome in primary roots of <i>A</i>. <i>sparsifolia</i> under water stress conditions in aim to facilitate the exploration of its genetic basis for drought tolerance.</p><p>Methodology and Principal Findings</p><p>We sequenced four primary roots samples individually collected at 0, 6, 24 and 30h from the <i>A</i>. <i>sparsifolia</i> seedlings in the course of 24h of water stress following 6h of rehydration. The resulting 38,763,230, 67,511,150, 49,259,804 and 54,744,906 clean reads were pooled and assembled into 33,255 unigenes with an average length of 1,057 bp. All-unigenes were subjected to functional annotation by searching against the public databases. Based on the established transcriptome database, we further evaluated the gene expression profiles in the four different primary roots samples, and identified numbers of differently expressed genes (DEGs) reflecting the early response to water stress (6h <i>vs</i>. 0h), the late response to water stress (24h <i>vs</i>. 0h) and the response to post water stress rehydration (30h <i>vs</i>. 24h). Moreover, the DEGs specifically regulated at 6, 24 and 30h were captured in order to depict the dynamic changes of gene expression during water stress and subsequent rehydration. Functional categorization of the DEGs indicated the activation of oxidoreductase system, and particularly emphasized the significance of the ‘Glutathione metabolism pathway’ in response to water stress.</p><p>Conclusions</p><p>This is the first description of the genetic makeup of <i>A</i>. <i>sparsifolia</i>, thus providing a substantial contribution to the sequence resources for this species. The identified DEGs offer a deep insight into the molecular mechanism of <i>A</i>. <i>sparsifolia</i> in response to water stress, and merit further investigation.</p></div

Statistics of DGE sequencing data from <i>C. amaranticolor</i> infected with distinct viruses.

a<p>The reads were mapped to foliar transcriptom of <i>C. amaranticolor</i> with 112, 452 unigenes.</p>b<p>The conservative degree of mismatch was no more than 2 bp.</p

Changes in gene expression profiles of the TMV- and CMV-inoculated <i>C. amaranticolor</i> leaves at two different time points post-inoculation.

<p>The comparisons were made between inoculated and uninoculated (healthy leaves) at each time point. The numbers of up-regulated and down-regulated genes are summarized.</p

GO categories of biological process, cellular component and molecular function for the foliar transcriptome of <i>C. amaranticolor</i>.

<p>The right y-axis shows the number of genes in a category, while the left y-axis indicates the percentage of a specific category of genes in that main category.</p