Virus-induced gene silencing database for phenomics and functional genomics in Nicotiana benthamiana

Abstract

Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.

Keywords: Nicotiana benthamiana; functional genomics; gene silencing; tomato; virus‐induced gene silencing.

Figure 1

Representative phenotypes presented in the VIGS database and distribution of silenced plants in phenotypic categories. Silencing of NbME and NbTI library genes by VIGS resulted in various phenotypes in Nicotiana benthamiana. Representative photographs of some of the most common phenotypes are presented. Plants were photographed approximately 3 weeks after inoculation with the TRV‐VIGS vector (a). Approximately 1,300 plants were individually scored for the visual phenotypes under the mentioned categories. The chart represents the distribution of silencing phenotypes of genes from the libraries (b). Phenotype descriptions: NbTI06E09 silencing shows severely stunted plants, crinkled leaves, reduced apical growth, and severe cell death on top leaves; NbME14A8 silencing shows stunted, bushy plants, and albino green leaves; NbME12B6 silencing shows stunted, bushy plants, green‐white mottled, crinkled leaves, and spotted cell death on leaves; NbME12B7 silencing shows moderately stunted plants and yellow leaves; NbME12B10 silencing shows severely stunted plants, thick, and mosaic leaves; NbTI02D02 silencing shows cell death. Four replicates were carried out for each experiment, and two independent experiments were performed

Figure 2

Functional categories of ESTs in the database. Nucleotide sequences in the VIGS database were annotated, and the gene function(s) was identified by BLAST analysis. The pie chart shows the representation of the sequences under different functional categories

Figure 3

Summary of the gene ontology analysis for the Nicotiana benthamiana ESTs. The distribution of gene ontology (GO) terms in three GO categories (biological process, cellular component, and molecular function) is shown in the pie chart. The number of GO terms is counted according to the heterologous BLAST search from Arabidopsis. The charts presented are based on information from tomato. All 4,117 EST sequences were used for the GO analysis

Figure 4

Pipeline of the VIGS database construction. The framework for the VIGS database along with its content organization is presented. The database was constructed based on three inputs namely two EST library sequences, photographs of visual phenotypes, and phenotype descriptions. These inputs were processed as indicated in the middle panel. Specifically, the nucleotide sequences were annotated and a BLAST search was constructed. Phenotype information was organized and presented in a searchable format. Users are able to perform the functions as depicted in the right‐most panel

Figure 5

Screenshot of few tabs from the database and description of contents. This screenshot is taken from the VPGD web site. It has four tabs namely, Home, VIGS Database, Material Request, and BLAST. The Home tab contains the basic description of the database including the background and relevant literature. The VIGS Database page displays all information related to the clone upon clicking the clone name or NCBI ID or keyword search. The display includes silencing phenotype description, gene name annotation, GO terms, EST sequence, and photograph of silencing phenotype. The Material Request tab provides details of construct availability and biosafety information. The BLAST tab has a built‐in BLAST page. Query sequence can be used to search for homologs in the database, and this will show all the information available for the desired clone. The red arrows indicate key information provided in the database, and this is to guide the readers to notice the description in the legend