2011 Annual Report
1a.Objectives (from AD-416)
Insect transmission of plant viruses is a major problem in agriculture that requires proactive research to limit its occurrence. The objective of the proposed cooperative research is to better understand the genes expressed in Bemisia tabaci salivary gland and midgut tissues in whiteflies harboring the begomovirus, Squash leaf curl virus. Next-generation massively-parallel DNA sequencing of Bemisia tabaci expressed sequence tag (EST) libraries will provide a more comprehensive representation of genes that are expressed in these tissues than is currently available and how vectoring this begomovirus affects gene expression. These ESTs could represent novel targets for disrupting normal homeostasis of the insect pest and/or viral disease transmission. As many plant disease causing begomoviruses must cross the epithelial barrier within salivary glands or digestive tracts of their arthropod vector, these tissues represent logical targets for understanding the processes involved in virus transmission. How whiteflies respond to viral infection will yield important new information about how begomoviruses enter their insect hosts and are transmitted to plants.
1b.Approach (from AD-416)
Next-generation sequencing will be used to provide a view of gene expression (e.g. transcriptome) in tissues of Bemisia tabaci vectoring Squash leaf curl virus. The approach follows standard procedures adapted for next-generation sequencing of transcriptomes, including tissue dissection from B. tabaci (naïve and infected with Squash leaf curl virus), RNA extraction, cDNA library construction, massively parallel DNA sequencing of ESTs, annotation, and organization in a PAVE database for mining. Post-sequencing analysis includes verification of changes in gene expression and functional analysis of proteins encoded by transcripts.
The whitefly, Bemisia tabaci, is a major pest of cotton and other crops and vectors many economically important plant viruses. This specific cooperative agreement contributes directly to the goals of CRIS project 5347-22620-021-00D “Sustainable Pest Management Strategies for Arid-Land Crops” and specifically relates to Objective 1 – Develop knowledge and control tactics based on the physiology, biochemistry, genetics and vector-pathogen interactions of insect pests – by expanding our knowledge of the B. tabaci transcriptome, which is extremely lacking based on the global significance of this pest. This project intends to better understand the genes expressed in B. tabaci salivary glands and midgut. Because many plant disease causing begomoviruses must cross the cellular barriers in salivary glands or digestive tracts of their arthropod vector, these tissues represent logical targets for understanding the processes involved in virus transmission. By sequencing the genes expressed in these tissues, we will identify novel targets for disrupting normal homeostasis of the insect pest and/or viral disease transmission. In the first year, 1000 guts and 1000 primary salivary glands were extirpated from adult B. tabaci. Two hundred intact adult whiteflies were collected from the same colony. Sufficient genetic material was obtained from guts for DNA library construction, but additional salivary glands were needed. A paired-end Illumina library was made from the gut preparation and subjected to DNA sequencing and assembly. The assemblies were moved into a sequence management system and annotated. The gut library yielded 229,881 expressed sequences. A library similarly constructed from whole adult whitefly was also subjected to Illumina sequencing yielded 542,617 expressed sequences. Data mining was carried out and genes present uniquely in the gut (whole whitefly was also searched) were selected based on a predicted involvement in stress-related responses. Multiple genes were selected and validated for gene expression, indicating that all processes in the pipeline are functioning optimally, paving the way for additional library constructions. Library preparation is underway for a second sampling of 1000 guts and 2000 salivary glands (extirpatation completed June 2011) for sequencing, assembly, and annotation. The goal is to obtain the complete transcriptome for the whitefly gut and primary salivary glands, respectively, with the next round of sequencing. This information will not only provide needed information to better understand the biology and epidemiology of this pest, but genes could also be targeted for direct control of this pest and/or reduce virus transmission. Progress of the project is maintained via emails, phone meetings, and occasional visits between collaborators.