Location: Floral and Nursery Plants Research Unit
2013 Annual Report
Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized.
Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR.
Under Objective 1a: Additional isolates of Alternanthera mosaic virus (AltMV) were detected in Phlox stolonifera and P. divaricata, and in Celosia hybrids from different sources. Several viruses were detected for the first time in new hosts (Ornithogalum mosaic virus in Ixia and Crocosmia; Triteleia mosaic virus in Brodaiea; Ligustrum necrotic ringspot virus in P. divaricata). Potentially ‘new’ potyviruses were detected in Aconitum and Dichlostemma, and Aconitum latent virus carlavirus was detected in Aconitum (a new report for the U.S.). Parts of this work were carried out in collaboration with scientists at Agdia, Inc.; Michigan Dept. of Agriculture; and the Univ. Maryland.
Under Objective 1b: Additional members of the Alphaflexiviridae and Betaflexiviridae were successfully amplified using generic primers, and sequences obtained.
Under Objective 1c: Additional viruses, including some mixed infections, were successfully detected with the Universal Plant Virus Microarray (UPVM). Methods for normalizing amplification of viral and host RNAs were improved, allowing increased sensitivity of detection of low-titered. Several viruses detected under Objective 1a were further characterized and partially sequenced.
Under Objective 2a: Interactions of several natural and mutant sequence variants of AltMV TGB1 were examined with a number of host proteins using Bimolecular Fluorescence Complementation (BiFC), in collaboration with a scientist at Chungnam National University (Korea) and at Beltsville using laser scanning confocal microscopy (LSCM).
Under Objective 2b: Interactions of AltMV CP and TGB3 with plant host proteins were examined by LSCM, BiFC, and yeast two-hybrid analysis. Monoclonal antibodies were developed to several unique and conserved regions of the predicted movement protein of several pelarspoviruses.
Under Objective 3.1: We obtained 20x draft genomes of four landscape strains of X. fastidiosa and are almost done with a fifth strain. We examined published MLST primers in the sequenced strains of X. fastidiosa.
Under Objective 3.2: By using in silico genome subtraction, we identified 76 non-phage related fragments unique only to the r3b2 select agent strains of R. solanacearum. Similarly, we also identified sequences conserved among strains belonging to Ralstonia species complex, but not in other closely related species, as well as other plant pathogenic bacteria including Xanthomonas campestris and Pseudomonas syringii. With this information we designed and tested primers that are specific to R. solanacearum species complex, and to r3b2, respectively. We also developed a multiplex PCR assay by combining the species-, r3b2-, and plant-specific primers in a single reaction to allow fast and reliable detection and differentiation of r3b2 strains in plant extracts.
Summaries to document research conducted under nine Specific Cooperative Agreements or Reimbursable Agreements between the Floral and Nursery Plants Research Unit and others can be found in those specific annual reports.
Damsteegt, V.D., Stone, A.L., Smith, O.P., Mcdaniel, L., Sherman, D.J., Dardick, C.D., Hammond, J., Jordan, R.L., Schneider, W.L. 2013. A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia. Archives of Virology. 158:1235-1244.
Li, W., Teixeira, D.C., Hartung, J.S., Huang, Q., Chen, J., Lin, H. 2012. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis. Journal of Microbiological Methods. 92:79-89.
Jang, C., Seo, E., Nam, J., Bae, H., Gim, Y., Cho, I., Lee, Z., Bauchan, G.R., Hammond, J., Lim, H. 2013. Insights into Alternanthera mosaic virus TGB3 functions: Interactions with Nicotiana benthamiana PsbO correlate with chloroplast vesiculation and veinal necrosis caused by TGB3 overexpression. Frontiers in Plant Science. http://dx.doi.org/10.3389/fpls.2013.00005.
Lim, H., Lee, M., Jang, C., Nam, J., Bae, H., Ju, H., Lee, M., Yu, Y., Hammond, J., Jackson, A.O. 2013. Interactions with the actin cytoskeleton are required for cell wall localization of barley stripe mosaic virus TGB proteins. Plant Pathology Journal . 29:17-30.
Guan, W., Shao, J.Y., Singh, R., Davis, R.E., Huang, Q. 2012. A TaqMan-based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander. Current Microbiology. 92:108-112.
Solovyev, A.G., Makarova, S.S., Remizowa, M.V., Lim, H., Hammond, J., Owens, R.A., Kopertech, L., Schiemann, J., Morozov, S.Y. 2013. Possible role of the Nt-4/1 protein in macromolecular transport in vascular tissue. Plant Signaling and Behavior. 8(10):e25784.
Guaragna, M.A., Lamborn, J., Hammond, J., Schadewijk, T., Jordan, R.L. 2013. First report of Nerine yellow stripe virus in Amaryllis in the United States. Plant Disease. 97(10):1389.