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The whitefly, Bemisia tabaci, is one of the most destructive insect pests of agricultural crops in tropical and subtropical areas. They can transmit a huge numbers of plant pathogenic viruses that result in serious crop losses worldwide annually . This B. tabaci cryptic species complex can colonize over thousands of host plant species , and able to transmit over 300 different viruses . Bemisia tabaci is a cryptic species complex, previously known as biotypes that differ from one another in host range, reproductive compatibility, insecticide resistance, endosymbiont composition, and virus transmissibility [4–12].
There are about 40 cryptic species of B. tabaci has been recognized . Among them, B. tabaci MED (Q biotype) is well-known for its rapid invasion ability and special transmission ability of tomato yellow leaf curl virus (TYLCV) . As a consequence, controlling vector is the only practical and effective strategy for viral disease prevention. Over the past few decades, many researchers have investigated the interactions between plant viruses and insect vectors because of its importance in viral epidemiology and disease management [15, 16, 17].
As we know that, the journey of Begomovirus and B. tabaci MED is a physiological puzzle. There are about 192 approved species are in the genus Begomovirus but B. tabaci is a vector for only half of them [18,19]. The reason of not transmitting virus within the same family like honeysuckle yellow vein virus (HYVV) , could be a good target for whitefly-transmitted Begomovirus control. Therefore, a detailed understanding of the genetic and molecular basis of insect-virus interaction will lead the discovery of novel and specific molecular targets for whitefly and whitefly-transmitted Begomovirus control. In this study, I am going to compare the transcriptome profile of B. tabaci MED after feeding TYLCV and HYVV. Methods Whitefly treatments and sample collection To prepare viruliferous and nonviruliferous whiteflies, approximately 1000 newly emerged (1 d) adult whiteflies will be collected and released onto healthy tomato plants in different cages for 48 h. Then the whiteflies will be transferred onto virus-infected and uninfected tomato plants for another 48 h, respectively. After that, they will be separately transferred onto another tomato (Solanum lycopersicom), which will be a TYLCV resistant variety, to eliminate effects of host differences on whiteflies. RNA isolation and cDNA library preparation Total RNA will be extracted from whole body of adult whiteflies (n=100) by following lab protocol.
Transcriptome sequencing and assembly The cDNA libraries will be sequenced for 150 bp paired-end reads by following lab instructions. Analysis of differential gene expression The clean reads from viruliferous and nonviruliferous whiteflies will be separately mapped back to the assembled unigenes. For gene expression analysis, reads per kilobase million Mapped Reads (RPKM) will be calculated to estimate the expression level of genes in each sample. RPKM could eliminate the effect of sequencing depth and gene length on gene expression levels, which will facilitates the comparison of the number of transcript levels generated between samples. DEGseq (v1.18.0) will be used to identify differentially expressed genes (DEGs) between the viruliferous and nonviruliferous whiteflies. Genes with q ≤ 0.05 (adjusted p-value) and log2 ratio ≥ 1 will be considered differentially expressed. GO and KEGG pathway analysis To obtain an overview and for further understanding of the biological functions of genes, all DEGs will be subjected to GO functional annotation by using Blast2GO and mapping terms of KEGG pathway database by using KOBAS.
Enrichment analysis will be performed to identify the GO terms and significantly regulated KEGG pathways. After that, a corrected q ≤ 0.05 as the threshold will be selected to determine significant enrichment of the gene sets. qRT-PCR analysis To confirm the result of the DEG analyses, I will measure the expression of selected genes using comparative CT (ΔΔCT) Real-time Quantitative PCR with β-actin and α-tubulin as the internal control gene by following lab protocol. Three biological replicates will be done at the same time.
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