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

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

Quantitative real-time PCR analysis of 18 candidate genes.

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

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 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

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

Non-Uniform Distribution Pattern for Differentially Expressed Genes of Transgenic Rice Huahui 1 at Different Developmental Stages and Environments

<div><p>DNA microarray analysis is an effective method to detect unintended effects by detecting differentially expressed genes (DEG) in safety assessment of genetically modified (GM) crops. With the aim to reveal the distribution of DEG of GM crops under different conditions, we performed DNA microarray analysis using transgenic rice Huahui 1 (HH1) and its non-transgenic parent Minghui 63 (MH63) at different developmental stages and environmental conditions. Considerable DEG were selected in each group of HH1 under different conditions. For each group of HH1, the number of DEG was different; however, considerable common DEG were shared between different groups of HH1. These findings suggested that both DEG and common DEG were adequate for investigation of unintended effects. Furthermore, a number of significantly changed pathways were found in all groups of HH1, indicating genetic modification caused everlasting changes to plants. To our knowledge, our study for the first time provided the non-uniformly distributed pattern for DEG of GM crops at different developmental stages and environments. Our result also suggested that DEG selected in GM plants at specific developmental stage and environment could act as useful clues for further evaluation of unintended effects of GM plants.</p> </div

Changes in electrolyte leakage during Alternaria disease development promoted by chilling stress pre-treatment.

<p>a: the control cotton plants sustained growing at optimal temperature of 28/20°C. b,c,d: Cotton plants performed with chilling stress pre-treatments at 20/16°C, 16/12°C, 12/8°C for 3 days respectively, then inoculated with 1.2×10⁴ conidial/mL inoculum suspension of <i>A. alternata</i> isolate A1, and returned to grow at 28/20°C. All collected data (mean ± standard deviation SD with 6 replicates) were presented as relative values of membrane electrolyte leakage. The chilling stress pre-treatment period was indicated by time points from −3 to 0, and the period after inoculation was indicated by time points from 0 to 15.</p

Numbers of DEP and DEG in HH1 compared with MH63 at different developmental stages and environments.

<p>Numbers of DEP and DEG in HH1 compared with MH63 at different developmental stages and environments.</p