2010 Annual Report
1a.Objectives (from AD-416)
To develop and validate a universal plant virus microarray for detection and differentiation of plant viruses. To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts.
1b.Approach (from AD-416)
ARS and the Cooperator will assemble the lists of viral taxa to be represented on the viral detection microarray, and virus-infected samples from which to amplify nucleic acids to validate the microarray. This information and material will be utilized by both ARS and the Cooperator to jointly develop and validate the microarray for detection of target viruses, and to make validation results available to collaborators via a web server. The COOPERATOR will perform analysis of viral sequences to identify suitable sequences for the development of oligonucleotides, supervise production of the microarrays based on the selected oligonucleotides, perform assays to validate the array with as many viruses as possible from the collections of all cooperators, and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples.
This research is part of a larger collaboration including USDA-ARS scientists at Beltsville, MD; the Donald Danforth Plant Science Center, St. Louis, MO; Washington University, St. Louis, MO; the University of Utah, Salt Lake City, UT; Oklahoma State University, Stillwater, OK; and Cornell University, Ithaca, NY. The overall goals are to be able to detect any plant virus in extracts of infected plants, and to identify previously characterized viruses to the species level, or previously uncharacterized viruses to at least the viral family or genus level.
Progress was made towards validating additional viruses on a MiniPlantViroChip microarray as precursor to the Universal Plant Virus Microarray. The ability to control plant viral and viroid diseases requires the ability to correctly detect and identify the virus or viroid, and thus obtain knowledge about the likely vectors or other means of transmission. The ability to assign a previously uncharacterized virus to a known taxonomic group allows inference of many important characters, and facilitates the selection of appropriate methods to obtain further specific sequence information to develop specific detection and identification reagents. The first step in developing a Universal Plant Virus Microarray (UPVM) is to design and select oligonucleotide probes that will collectively provide information allowing identification of isolates of a previously characterized virus to the correct species, and of a previously uncharacterized species to the correct genus or family. A ‘MiniPlantViroChip’ based on oligonucleotides previously designed for a panviral microarray has been further validated using extracts from virus-infected plants received from collaborators in the UPVM project; nucleic acid extracts have been labeled and hybridized to the ‘MiniPlantViroChip’, and results analyzed. Viruses representing genera in the families Geminiviridae (single-stranded DNA genome), Caulimoviridae (double-stranded DNA genome), Bromoviridae, Closteroviridae, Flexiviridae, Potyviridae, and Virgaviridae (all with single-stranded RNA genomes) have been successfully detected and correctly identified. Printing of the initial batch of the full 9,600 probe UPVM will allow validation of a much larger number of viruses representing additional viral families, and the viroids. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.
Communications to monitor progress were carried out by e-mail and conference calls between the various partners, by a group meeting during the Annual meeting of the America Phytopathological Society, and by written and oral reports to the NRI Plant Biosecurity Program.