Quantitative real-time PCR analysis of 18 candidate genes.

<p>Quantitative real-time PCR analysis of 18 candidate genes.</p

Silencing of phenylpropanoid pathway-related genes in Zhongzhimian KV3 infected with <i>V</i>. <i>dahliae</i> strain V991.

<p>4A, a, GhCYP71D-silenced Zhongzhimian KV3 plant; b, GhCYP736-silenced Zhongzhimian KV3 plant; c, GhHCT-silenced Zhongzhimian KV3 plant.</p> <p>4B, The disease index (DI) after silencing different genes.</p> <p>Asterisk represents the data point that was statistically different from wild-type and CLCrV-00 Zhongzhimian KV3 plants (p<0.05) analyzed by one-way ANOVA, using the SAS 8.1.</p> <p>The vertical bars indicate standard Deviation.</p> <p>4C, Relative expression levels of candidate genes in the silenced and non-silenced cotton plants at 20 dpi were determined through qRT-PCR.</p> <p>Asterisk represents the data point that was statistically different from the non-silenced (p<0.05) analyzed by one-way ANOVA, using the SAS 8.1.</p> <p>The vertical bars indicate standard Deviation.</p

The quantitative real-time PCR analysis of 18 differentially expressed genes (DEGs) between resistant upland variety Zhongzhimian KV3 and susceptible upland variety 86–1 at 24 h post inpculation (hpi) with <i>V</i>. <i>dahliae</i>.

<p>The relative gene expression levels of cotton genes induced by <i>V</i>. <i>dahliae</i> were normalized against Ct values for cotton ubiquitin gene, which were calculated by the 2^−ΔΔCt method (Livak & Schmittgen, 2001). The data are mean values and standard deviation (bar) of three independent qRT-PCR experiments. Asterisk represents the data point of Zhongzhimian KV3 that was statistically different from the data point of 86–1 (p<0.05) analysed by one-way ANOVA, using the SAS 8.1. The vertical bars indicate standard Deviation.</p

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes (DEGs).

<p>KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes (DEGs).</p

Large-scale identification of <i>Gossypium hirsutum</i> genes associated with <i>Verticillium dahliae</i> by comparative transcriptomic and reverse genetics analysis

<div><p>Verticillium wilt is a devastating disease of cotton, which is caused by the soil-borne fungus <i>Verticillium dahliae</i> (<i>V</i>. <i>dahliae</i>). Although previous studies have identified some genes or biological processes involved in the interaction between cotton and <i>V</i>. <i>dahliae</i>, its underlying molecular mechanism remains unclear, especially in <i>G</i>. <i>hirsutum</i>. In the present study, we obtained an overview of transcriptome characteristics of resistant upland cotton (<i>G</i>. <i>hirsutum</i>) after <i>V</i>. <i>dahliae</i> infection at 24 h post-inoculation (hpi) via a high-throughput RNA-sequencing technique. A total of 4,794 differentially expressed genes (DEGs) were identified, including 820 up-regulated genes and 3,974 down-regulated genes. The enrichment analysis showed that several important processes were induced upon <i>V</i>. <i>dahliae</i> infection, such as plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid-related and ubiquitin-mediated signals. Moreover, we investigated some key regulatory gene families involved in the defense response, such as receptor-like protein kinases (RLKs), WRKY transcription factors and cytochrome P450 (CYPs), via virus-induced gene silencing (VIGS). GhSKIP35, a partner of SKP1 protein, was involved in ubiquitin-mediated signal. Over-expression of GhSKIP35 in <i>Arabidopsis</i> improved its tolerance to Verticillium wilt in transgenic plants. Collectively, global transcriptome analysis and functional gene characterization provided significant insights into the molecular mechanisms of <i>G</i>. <i>hirsutum</i>-<i>V</i>. <i>dahliae</i> interaction and offered a number of candidate genes as potential sources for breeding wilt-tolerance in cotton.</p></div

Functional classifications of differentially expressed genes (DEGs) were determined by GO terms.

<p>The GO categories are presented as follows: A, biological process, B, molecular function, C, cellular component. Pie charts represent the functional distribution, which is expressed as a percentage of the amount of genes.</p

The RLKs and WRKYs are required for resistance against <i>V</i>. <i>dahliae</i> in cotton via VIGS analysis.

<p>A: Silencing of CLA1 gene in <i>G</i>. <i>hirsutum</i> variety Zhongzhimian KV3. Left, plant infiltrated with CLCrV-based empty vector (CLCrV-00); right, plant infiltrated with CLCrV-CLA1.</p> <p>B: Relative expression levels of candidate gene in the silenced and non-silenced cotton plants at 20 dpi were determined through qRT-PCR. Asterisk represents the data point that was statistically different from the non-silenced (p<0.05) analyzed by one-way ANOVA, using the SAS 8.1.</p> <p>The vertical bars indicate standard Deviation.</p> <p>C: a, Negative control-silenced upland cotton (Zhongzhimian KV3, CLCrV-00); b, GhGsSRK-silenced Zhongzhimian KV3 plant; c, GhWRKY2-silenced Zhongzhimian KV3 plant; d, GhWRKY29-silenced Zhongzhimian KV3 plant; e, GhFLS2-silenced Zhongzhimian KV3 plant; f, Negative control-silenced upland cotton (86–1, CLCrV-00); g, GhWRKY13-silenced 86-1plant.</p> <p>D: The disease index (DI) after silencing different genes. The results are presented as mean ±standard deviation (SD) from three replicates with at least 20 plants per replicate. Black represents candidate gene silenced in Zhongzhimian KV3, and blue represents candidate gene silenced in 86–1.</p> <p>Asterisk represents the data point that was statistically different from wild-type and CLCrV-00 plants (p<0.05) analyzed by one-way ANOVA, using the SAS 8.1.</p> <p>The vertical bars indicate standard Deviation.</p