Unknown,Transcriptomics,Genomics,Proteomics

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Detoxification activity and energy cost is attenuated in the whiteM-oM-,M-^Bies feeding on Tomato yellow leaf curl China virus-infected tobacco plants

ABSTRACT: We compared the transcriptional profiles of female adult whiteflies of B. tabaci Middle East-Asia Minor 1 feeding on TYLCCNV-free and TYLCCNV-infected tobacco plants using the next-generation sequencing technique. Culture of B (MEAM1 cryptic species) whitefly was maintained on cotton plants. One thousand of newly emerged adults of whitefly on cotton were released onto the leaves of healthy and viruliferous tobacco plants. After 72 h of oviposition, all the adult whiteflies were discarded, and the progeny allowed to develop to adults. The cultures of MEAM1 on tobacco plants were maintained in climate chambers at 27 M-BM-1 1M-BM-0C, a photoperiod of 14 h light/10 h darkness and 70 M-BM-1 10% relative humidity. Approximately 1,000 female adult whiteflies newly emerged from mock-inoculated tobacco plants and 1,000 female adult whiteflies newly emerged from virus-infected tobacco plants were collected and stored at -80M-BM-0C. The RNA was extracted and sequenced using Illunima Analyzer II.

ORGANISM(S): Bemisia tabaci

SUBMITTER: Xiaowei Wang

PROVIDER: E-GEOD-30409 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Here we performed a transcriptomic study on complete symptom development process of CMV-infected Nicotiana tabacum using Solexa/Illumina's high-throughput digital gene expression (DGE) system. 12 DGE libraries (from six virus-infected samples and six corresponding mock-inoculated samples) were constructed, and the gene expression variations between the virus-infected sample and the mock-inoculated sample in each symptom stage were compared. Thousands of differentially expressed genes were obtained by the comparison, and KEGG pathway analysis of these genes suggested that many biological processes (such as phtosynthesis, pigment metabolism and plant-pathogen interaction) were related to the symptom development. The authenticity of the DGE data was further confirmed by analyzing real-time RT-PCR using several random-selected genes. We sequenced a cDNA library constructed from mixture of total RNA from six virus-infected samples and six mock-inoculated samples to get gene information for tobacco leaves in different symptom stages, including vein clearing, mosaic, severe chlorosis, partial recovery, total recovery and re-mosaic, and 95,916 Unigenes were obtained Tobacco leaves were inoculated by CMV-infected leaf homogenate and healthy leaf homogenate, respectivley. Six time points with different symptom stage were selected, and one virus-infect sample and one mock-inoculated sample were collected at each time. In order to average out variation of different plants, five leaves from five different plants were mixed to prepare every RNA sample. Twelve individual tag libraries of samples (six infected samples and six mock-inoculated samples) were constructed in parallel. For the gene expression analysis, the twelve samples were grouped into six groups, and each group contained a virus-infected sample and a mock-inoculated sample collected at the same time. In each group, the DGE data of virus-infected sample were compared to that of mock-inoculated sample to obtain the gene expression variations. Illumina sequencing of transcripts from virus-infected and mock-inoculated samples to get gene information for tobacco leaves in different symptom stages. In order to get more gene information, the systemically infected leaves and mock-inoculated leaves were harvested at six time points and five leaves from five different plants were collected at each time point. The RNA-Seq analysis provided gene information for mock-inoculated and virus-infected tobacco leaves in different symptom stages.

Project description:Phloem-feeding pests cause extensive crop damage throughout the world yet little is understood about how plants perceive and defend themselves from these threats. The silverleaf whitefly (SLWF; Bemisia tabaci type B) is a good model for studying phloem-feeding insect-plant interactions as SLWF nymphs cause little wounding and have a long, continuous interaction with the plant. Using the Arabidopsis ATH1 GeneChip, the global responses to Silverleaf Whitefly 2nd instar feeding were examined. Experiment Overall Design: The silverleaf whitefly colony (Bemisia tabaci type B; Bemisia argentifolii Bellows and Perring) was maintained on Brassica napus cv. âFlorida broad leafâ grown under fluorescent and incandescent lights (180 Î¼E m-2 s-1) 27Â°C with 55% relative humidity under long-day (16 hr light: 8 hr dark) conditions in the Insectory and Quarantine Facility at the University of California, Riverside. Adult whiteflies are collected from infested plants by aspiration into 15-ml falcon tubes. Experiment Overall Design: Individual Arabidopsis thaliana ecotype Columbia plants were grown for 21 days in 4-inch diameter, round pots under fluorescent and incandescent lights (180 Î¼E m-2 s-1) with 50% relative humidity, 23Â° C and an 8 hr-light/16-hr dark cycle. One hundred adult whiteflies were collected into each 15-ml falcon tube and the tube was placed upright in each pot. Plants were individually encased with 5- by 10-inch nylon bags that were secured to each pot with a rubber band. The whiteflies were released by unscrewing the falcon tube. After seven days, the adult whiteflies were removed from the plants by aspiration. The infested and non-infested plants were caged for the remainder of the experiment to ensure any adults that escaped aspiration could not reach the plants. Rosette tissue was collected after 28 days, when 2nd and 3rd instars were observed on wild-type Columbia plants. Developmentally matched leaves were harvested from uninfested plants. Infestations were performed in two growth chambers; each chamber contained one replicate experiment, which included 10 control and 10 infested plants. This experiment was repeated for a total of 8 biological replicate experiments. Experiment Overall Design: Total RNA from the eight biological replicates was isolated using the RNAwiz protocol (Ambion Inc., Austin, TX) and purified using a RNAeasy column (Qiagen, Valencia, CA). RNA from the two biological replicates performed in each growth chamber were pooled to eliminate variance due to different environmental factors. This yielded the infested and control RNA pools used in the microarrays (Exp1 and Exp2) and RT-PCRs (Exp3 and Exp4). The quality of the RNA was determined by A260/A280 absorbance readings. RNA integrity (1 Âµg) was verified by fractionation on a 1% formaldehyde gel. Experiment Overall Design: Hybridization Experiment Overall Design: Biotin-labeled cRNAs were synthesized from infested and control RNAs for Exp1 and Exp2 at the UC Irvine Microarray Facility using the Affymetrix Eukaryotic One-Cycle Target Labeling Assay protocol (Affymetrix GeneChip Expression, Analysis Technical Manual, Affymetrix, Santa Clara, CA). The labeled cRNA was hybridized to Affymetrix Arabidopsis genome ATH1 Chip arrays, washed, and scanned using a Hewlett Packard Genearray scanner (Hewlett-Packard, Palo Alto, CA). Experiment Overall Design: MAS 5.0 was performed using the standard parameters (Affymetrix GeneChip Expression, Analysis Technical Manual, Affymetrix, Santa Clara, CA.) Genes with âabsentâ calls in replicate experiments were removed from further analysis.

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Detoxification activity and energy cost is attenuated in the whiteM-oM-,M-^Bies feeding on Tomato yellow leaf curl China virus-infected tobacco plants

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