Progress 08/01/99 to 07/31/04
Outputs
Satellite panicum mosaic virus (SPMV) is an 824 nucleotide (nt) RNA that is completely dependent on Panicum mosaic virus (PMV) for replication in millet plants. We have determined that the 17.5 kDa SPMV capsid protein (CP) contributes to and/or coordinates long distance movement in foxtail millet. In parallel, we have determined that SPMV CP greatly reduces in vivo recombination events that delete non-viral (foreign) RNA. We have defined 37 nt cis-acting RNA element that is a determinant for host-specific movement in foxtail millet, but not proso millet. From this work we have subsequently identified a novel RNA that accumulates as a result of this infection--a defective interfering satellite virus RNA, or D-RNA. This SPMV-derived D-RNA completely abolishes SPMV accumulation, yet retains an ability to enhance the accumulation and spread of PMV. This has greatly strengthened our observations concerning the synergism and reveal that the SPMV CP is necessary for the
stability of the SPMV RNA. In addition, the C-terminal portion of the CP is sufficient to induce severe chlorosis/bleaching in millet. Subcellular fractionation has revealed that the SPMV CP is primarily concentrated in cell wall-enriched fractions, a hallmark of plant virus movement proteins. Moreover, we have found that the C-terminal region of the SPMV CP is specifically associated with this cell wall localization. The capsid protein (CP) of SPMV has been implicated as a pathogenicity factor, inducing severe chlorosis on millet plants co-infected with PMV+SPMV, but not PMV alone. SPMV CP expressed from a Potato virus X (PVX) gene vector elicited necrotic lesions on Nicotiana benthamiana, which is not a host for PMV+SPMV infections. Since pathogenicity factors often have the additional feature of acting as suppressors of gene silencing, we devised several strategies to determine if SPMV CP could act in such a capacity. We found that SPMV CP failed to act as a suppressor of
post-transcriptional gene silencing in transgenic N. benthamiana plants silenced for GFP expression, using agroinfiltration or plant virus vectors. However, when the SPMV CP was expressed from the PVX gene vector it did interfere with the known suppressor activity of the PVX p25 protein. This interference was documented by a rebound in the level of GFP expression along the vascular tissues and the veins on upper non-inoculated leaves. Therefore, the roles of the SPMV CP now includes encapsidation of the SPMV RNA, activity as a pathogenicity factor in both host and non-host plants, and the intriguing feature of interfering with suppression of gene silencing. This grant was used to support three graduate students, two of whom have graduated (Ph.D. and M.S.), and 3 postdoctoral fellows, on of whom is now an Assistant Professor. The relevant publications are cited.
Impacts
Our finding that the SPMV CP (Scp) has many of the physicochemcial and biological features of plant virus movement proteins, provides us with several new areas to explore in terms of the biology of host:virus interactions in monocots. Our ongoing dissection of the 824-nt SPMV RNA has shown that although it must have a helper virus replicase provided in trans, there is increasing evidence that SPMV essentially is self contained with the ability to move in the plant in a host-specific manner and has the necessary features for encapsidation. This suggests that this satellite virus may prove amenable to further modifications, such as epitope display, that may have broad impact for biotechnology applications.
Publications
- Batten, J. S., and Scholthof, K.-B. G. 2004. Panicum mosaic virus satellite RNAs. In: "Virus diseases of Poaceae" (H. Lapierre and P. Signoret, eds.). INRA, France.
- Batten, J. S., and Scholthof, K.-B. G. 2004. Satellite panicum mosaic virus. In: "Virus diseases of Poaceae" (H. Lapierre and P. Signoret, eds.). INRA, France.
- Batten, J. S., and Scholthof, K.-B. G. 2004. Panicum mosaic virus satellite RNAs. In: "Virus diseases of Poaceae" (H. Lapierre and P. Signoret, eds.). INRA, France.
- Qiu, W. and Scholthof, K.-B. G.. 2004. Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing. Mol. Plant-Microbe Interact. 17:263-271.
- Desvoyes, B. and Scholthof, K.-B. G. 2000. RNA:protein interactions associated with satellites of panicum mosaic virus. FEBS Lett. 485:25-28.
- Qiu, W. P. and Scholthof, K.-B. G. 2001. Satellite panicum mosaic virus capsid protein induces symptoms in a non-host plant. Phytopathology 91:S74.
- Scholthof, K.-B. G., Mirkov, T. E., and Scholthof, H. B. 2002. Plant virus gene vectors: Biotechnology applications in agriculture and medicine. Genetic Engineering, Principles, and Methods 24:67-86. Plenum Press, NY.
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Progress 01/01/03 to 12/31/03
Outputs
During the past year, our emphasis has been on determining the role of the SPMV capsid protein (Scp) in movement. We have developed several new tools to investigate expression and localization of Scp in millet plants infected with PMV+SPMV. The SPMV CP is primarily localized the the cell wall and membrane fractions, as is typical for virus movement proteins. Futhermore, we have used our previously constructed mutants as well a panel of new Scp mutants to determine that this localization is primarily determined by the C-terminal half of the Scp protein. We are currently developing an assay to determine if the Scp is phosphorylated. In addition, we have been very successful in using immunoprecipitation to pull-down Scp from infected millet, and have determined that the polyclonal antibody is specific for the C-terminal end of the protein. We are currently preparing an antibody for the N-terminal portion of the Scp, which will be important as we begin to screen our
mutants for encapsidation. We have also constructed five Scp insertion mutants that are intended to 'display' 11 amino acids on the exposed portion of the SPMV virions. These are currently being evaluated for infectivity, symptoms on millet, and their ability to encapsidate SPMV RNA.
Impacts
Our finding that the SPMV CP (Scp) has many of the physicochemcial and biological features of plant virus movement proteins, provides us with several new areas to explore in terms of the biology of host:virus interactions in monocots. Our ongoing dissection of the 824-nt SPMV RNA has shown that although it must have a helper virus replicase provided in trans, there is increasing evidence that SPMV essentially is self contained with the ability to move in the plant in a host-specific manner and has the necessary features for encapsidation. This suggests that this satellite virus may prove amenable to further modifications, such as epitope display, that may have broad impact for biotechnology applications.
Publications
- R. Omarov, W. Qiu, and K.-B. G. Scholthof. 2003. A small 5'-untranslated region of satellite panicum mosaic virus determines host-specific movement in millet plants. Phytopathology 93:S68.
- K.-B. G. Scholthof. 2003. One foot in the furrow: linkages between agriculture, plant pathology, and public health. Annu. Rev. Public Health. 24:153-74.
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Progress 01/01/02 to 12/31/02
Outputs
During the past year we have derived several new mutations on the hairpin cis-element at the 5' end of the SPMV genome. These mutants have been made on the wildtype cDNA, as there is still some question of the role of the CP in the movement process. These experiments should resolve if the host specific block of movement of is solely dependent on the lack of the element or if the SPMV CP and/or packaging contributes to host specificity for movement. The mutants include a BamHI site at the apex of the hairpin, a fill-in at this site in to produce a ClaI site mutant, and deletion of the 5' half of the hairpin. A fortuitous `flip-clone' of the 5' half of the hairpin, was characterized which will allow for investigation of the spacer effect on movement and translation of the SPMV CP. To date, all of the clones are infectious on proso and pearl millet plants. Packaging and time course experiments are in progress. To determine the essential components associated with disease
development and packaging, we have initiated a plant transformation program. Since the D-RNA packaging experiments were not successful, this provides an alternative strategy. To date, we have successfully regenerated both millet host plants from embryo and stem cultures and are in the first stages of transforming plants with cDNAs of the 17 kDa wildtype SPMV CP, the 10-kDa C-terminal portion, and a minus-sense clone.
Impacts
These studies will allow for the further investigation of the role of SPMV RNA and it capsid in the development of disease and the requirements for host specific movement. It will be valuable for developing new tools for biotechnology and to elucidate the packaging strategies of these unusual small infectious RNAs.
Publications
- Scholthof, K.-B. G., Mirkov, T. E., and Scholthof, H. B. 2002. Plant viral genomes in genetic engineering. In: "Genetic Engineering, Principles, and Methods" (J. K. Setlow, ed.). Volume 24. Plenum Press, New York.
- Jeff Batten, Ph.D. Dissertation. "The Replication Strategy of Panicum Mosaic Virus". Texas A&M University. August 2002.
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Progress 01/01/01 to 12/31/01
Outputs
A defective-RNA derived from SPMV that is fully competent for replication and spread has been confirmed to be the first defective-interfering satellite virus RNA (DI). The SPMV DIs strongly interfere with the accumulation of the parental SPMV, but not with the unrelated 400 nucleotide PMV satellite RNA. These DIs do have an ability to slightly enhance the rate of spread and titer of PMV. The SPMV-derived DIs provide a new tool to investigate fundamental biological questions including the evolution and interactions of infectious RNAs. On the 5'-end of the SPMV RNA we have determined that the 37 nt cis-acting RNA is associated with host-dependent movement. Preliminary mutagenesis experiments show that the insertion of four nucleotides at the apex of the hairpin have no effect on SPMV replication or spread in proso or pearl millet plants. However, dissection of either the 5'- or 3'-half of this 37-nt element abolish movement in foxtail millet plants, confirming the
impression that this element is critical for SPMV fitness in a host-dependent manner, and providing an excellent model system to screen for host-associated proteins that are interacting with SPMV RNA for spread in proso millet vs. foxtail millet. We expressed SPMV capsid protein from two plant virus vectors, Tomato bushy stunt virus and Potato virus X. However, we have not been successful in developing this system to investigate the specific RNA:protein interactions for encapsidation of SPMV.
Impacts
Continued characterization of the SPMV hairpin will be used to identify specific host- and PMV-encoded proteins interactions. This will be used important to confirm the role of the host protein(s) in supporting SPMV movement. This may lead new strategies for plant-disease control by exploiting available and emerging genomic and proteomic technologies. The data will certainly contribute to the fascinating, growing literature on the RNA-based superhighway in plants, yeast, and animals.
Publications
- Qiu, W. P. and Scholthof, K.-B. G. 2001. Capsid protein of satellite panicum mosaic virus (SPMV) and its role as a symptom determinant. Molec. Plant-Microbe Interact. 14:21-30.
- Qiu, W. P. and Scholthof, K.-B. G. 2001. Defective interfering RNAs of a satellite virus. J. Virol. 75:5429-5432.
- Scholthof, K.-B. G. 2001. Panicoviruses. In: "The Encyclopedia of Plant Pathology" (O. C. Maloy and T. D. Murray, eds.). John Wiley & Sons, New York.
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Progress 01/01/00 to 12/31/00
Outputs
Deletion analyses of the satellite panicum mosaic virus (SPMV) genome has resulted in the discovery of a unique RNA, a defective satellite virus RNA. This RNA accumulates in a manner reminiscent of classic defective interfering RNAs in that passage experiments in millet plants revealed its emergence, particularly from capsid protein derivatives. This defective RNA is independent of SPMV, requiring only panicum mosaic virus for its replication and spread. Infectious cDNA clones of these defective RNAs were derived from millet plants. The SPMV defective RNAs are ca. 400 nucleotides in length (vs. 824 nucleotides for SPMV). The symptom phenotypes associated with the defective RNAs are intermediate, between the mild PMV symptoms and the severe phenotype associated with wild-type SPMV.
Impacts
The discovery of a defective RNA produced by a satellite virus provides a unique opportunity to dissect the minimum features (sequence and structure) that are associated with virus replication in grasses. In addition, this defective RNA provides an opportunity to investigate the underlying mechanisms related to the origin of satellite RNAs and satellite viruses. Further dissection of this defective RNA may allow it to be engineered as a virus vector for the expression of foreign RNAs or peptides in plants.
Publications
- Qiu, W. P. and Scholthof, K.-B. G. 2000. In vitro and in vivo generated defective RNAs of satellite panicum mosaic virus define cis-acting RNA elements required for replication and movement. J. Virol. 74:2247-2254.
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Progress 01/01/99 to 12/31/99
Outputs
We have completed a survey of the incidence of Panicum mosaic virus and its satellites in two locations in south Texas (Corpus Christi and College Station). We found that all symptomatic St. Augustinegrass plants are infected with PMV and most are also infected with satellite panicum mosaic virus (SPMV) and/or PMV satellite RNA. The preliminary investigation of the cis-acting elements of SPMV has determined that the capsid protein (CP) is not required for replication or movement in co-infections with PMV. We have defined that the extreme 5'-end of SPMV is necessary for replication as well as the entire 3' untranslated trailer. In addition, the small ca. 400 nucleotide RNAs that accumulated in association with the SPMV CP derivatives is a defective-RNA. The cDNA clone of this RNA is infectious and only requires PMV for replication and movement.
Impacts
The survey was the first of its kind and provides a working definition of 'St. Augustine decline' an economically important disease of St. Augustine grass (Stenotaphrum secundatum) in the Gulf Coast States. The D-RNA will allow us to refine the mapping of the cis-elements on the SPMV genome that are required for replication and movement and may provide us with an improved understanding of the origins of satellites.
Publications
- Cabrera, O. and Scholthof, K.-B. G. 1999. The complex viral etiology of St. Augustine decline. Plant Disease 83:902-904.
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