Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor.     

Genome-Wide Survey and Expression Analysis of Chlamydomonas reinhardtii U-box E3 Ubiquitin Ligases (CrPUBs) Reveal a Functional Lipid Metabolism Module

E3 ubiquitin ligases determine the substrate specificity of ubiquitination. Plant U-box (PUB) E3 ligases, with a typical 70-amino acid U-box domain, participate in plant developmental processes and environmental responses. Thus far, 64 PUB proteins have been identified in Arabidopsis and 77 PUB proteins have been identified in Oryza. However, detailed studies on U-box genes in the model microalgae Chlamydomonas reinhardtii are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in C. reinhardtii. Following BLASTP analysis, 30 full-length U-box genes were identified in the C. reinhardtii genome sequence. Bioinformatics analyses of CrPUB genes were performed to characterize the phylogenetic relationships, chromosomal locations and gene structures of each member. The 30 identified CrPUB proteins are clustered into 3 distinct subfamilies, and the genes for these proteins are unevenly distributed among 14 chromosomes. Furthermore, the quantitative real-time RT-PCR or semi-quantitative RT-PCR analysis of 30 CrPUB mRNA abundances under nitrogen starvation showed that 18 CrPUB genes were induced by N starvation and that 7 genes were repressed in the N-poor environment. We selected five CrPUB genes exhibiting marked changes in expression under N-free conditions for further analysis in RNAi experiments and examined the oil content of these gene-silenced transgenic strains. The silencing of CrPUB5 and CrPUB14, which are typically down-regulated under N starvation, induced 9.8%-45.0% and 14.4%-61.8% lipid accumulation, respectively. In contrast, the silencing of CrPUB11, CrPUB23 and CrPUB28, which are markedly up-regulated under N-free conditions, decreased the lipid content by 5.5%-27.8%, 8.1%-27.3% and 6.6%-27.9%, respectively. These results provide a useful reference for the identification and functional analysis of this gene family and fundamental information for microalgae lipid metabolism research.     

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.     

Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses.     

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.     

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.)

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.     

RNA interference mediated knockdown of juvenile hormone esterase gene in Bemisia tabaci (Gennadius): Effects on adults and their progeny

Juvenile hormone is responsible for regulating metamorphosis and reproduction in insects. Analysis of key elements of juvenile hormone regulation would enhance the understanding of this complex mechanism. Juvenile hormone esterase plays an important role in maintaining juvenile hormone titres in insects. In this study, effects of knockdown of juvenile hormone esterase gene (jhe) in Bemisia tabaci were studied using RNA interference (RNAi) technique. dsRNA corresponding to two conserved regions of jhe gene, substrate binding pocket site (jhe1), catalytic triad site (jhe2), green fluorescent protein gene (gfp) as control were synthesized. dsRNAs incorporated in artificial diet (20% sucrose solution) @ 2.5, 1.0, 0.5 and 0.1 μg/μl were fed to adult whiteflies for 48 h, followed by shifting whiteflies to live plants for next generation biology study. Based on qRT-PCR analyses, reduced jhe gene expression was observed in adult whiteflies after dsRNA feeding @ 2.5 and 1.0 μg/μl. jhe gene knockdown affects the survival and reproduction of whiteflies adversely in a dose-dependent manner. Moreover, oral feeding of dsRNA to adult whiteflies @ 2.5 and 1.0 μg/μl showed adverse effects on next generation of whitefly viz., lower egg hatchability and shortened egg incubation period. Minimum number of viable eggs (1.04 and 1.80 eggs/female) were observed when whiteflies were fed with highest concentration of dsjhe1 and dsjhe2 as compared to control (16.58 eggs/female). These data suggest that jhe gene acts as a major biological player in whitefly and its progeny and further indicate to be potential target for managing whitefly population.     

Evaluation of stability and validation of reference genes for RT-qPCR expression studies in rice plants under water deficit

Many studies use strategies that allow for the identification of a large number of genes expressed in response to different stress conditions to which the plant is subjected throughout its cycle. In order to obtain accurate and reliable results in gene expression studies, it is necessary to use reference genes, which must have uniform expression in the majority of cells in the organism studied. RNA isolation of leaves and expression analysis in real-time quantitative polymerase chain reaction (RT-qPCR) were carried out. In this study, nine candidate reference genes were tested, actin 11 (ACT11), ubiquitin conjugated to E2 enzyme (UBC-E2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta tubulin (β-tubulin), eukaryotic initiation factor 4α (eIF-4α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin TIP41 (TIP41-Like) and cyclophilin, in two genotypes of rice, AN Cambará and BRS Querência, with different levels of soil moisture (20%, 10% and recovery) in the vegetative (V5) and reproductive stages (period preceding flowering). Currently, there are different softwares that perform stability analyses and define the most suitable reference genes for a particular study. In this study, we used five different methods: geNorm, BestKeeper, ΔCt method, NormFinder and RefFinder. The results indicate that UBC-E2 and UBQ5 can be used as reference genes in all samples and softwares evaluated. The genes β-tubulin and eIF-4α, traditionally used as reference genes, along with GAPDH, presented lower stability values. The gene expression of basic leucine zipper (bZIP23 and bZIP72) was used to validate the selected reference genes, demonstrating that the use of an inappropriate reference can induce erroneous results.