Progress 09/15/02 to 09/14/07
Outputs
We participated in the Annual meeting of the American Phytopathological Society in San Digeo, CA in August of 2007. We made presentations to attendees, showing our progress and published the abstracts cited below.
Impacts
Our efforts were focused on gaining a better understanding of Lettuce infectious yellows virus (LIYV) and its interactions with its plant hosts. Part of our effort was focused on two LIYV-encoded proteins, P34 and P26, and to better understand the roles of these proteins in LIYV infection and cytopathology. Localization of P26 and P34 were assessed using GFP-fused or native proteins expressed from a heterologous Tobacco mosaic virus (TMV) vector. Cellular effects of native of fusion proteins were tested by fluorescence microscopy or transmission electron microscopy (TEM). P34:GFP expressed from TMV exhibited perinuclear localization in infected tobacco cells. Perinuclear aggregation was also observed for transgenic ER-targeted GFP in RNA 1-infected tobacco cells, indicating that ER rearrangement, as reported for many other RNA viruses, is also induced by LIYV RNA 1. ER rearranging capability is retained by a mutant RNA 1 where a double stop codon was inserted at the 5'
end of the P34 ORF. The functional significance of perinuclear localization of P34:GFP localization remains to be elucidated, but P34 may colocalize with or contribute to formation of ER-derived replication complexes. C- and N-terminal fusions of P26 to GFP localized differentially, each exhibiting partial localization compared to unfused protein. The GFP:P26 fusion localized to the periphery of infected cells, the P26:GFP fusion aggregated within cells, and the native P26 protein assessed by immunolocalization aggregated at the cell periphery. The work described here adds to our knowledge of the function and roles in cytopathology of two LIYV proteins, P34 and P26. These data indicate that P34 is not essential for most of the membrane rearrangement that occurs in protoplasts during LIYV infections, and also indicates that P26 alone is sufficient to induce formation of the plasmalemma deposits it has previously been associated with. In order to further study LIYV replication
determinants, we generated binary plasmid 35S-driven constructs and attempted to inoculate plants via agro-infiltration. No LIYV infection was observed from agro-infiltration using the two original 35S-driven constructs. Further attempts including co-infiltrations with different gene silencing suppressors and modifying the predicted RNA 1 5' nucleotide sequence were done, however no whole plant infections were obtained. We also used two different ribozymes predicted to give different RNA 1 3' termini in order to see if this might affect RNA 1 replication ability. The Hepatitis delta virus (HDV) ribozyme proved to be effective for yielding infectious LIYV RNA 1 transcripts in protoplasts, as judged by GFP expression of a LIYV RNA 2 defective RNA when it was co-inoculated with RNA 1. We are now constructing additional 35S-driven RNA 1 clones that contain the HDV ribozyme in attempts to develop a direct whole plant infection method.
Publications
- Wang, J., Turina, M., Stewart, L. R., and Falk, B. W. 2007. Optimizing Lettuce infectious yellows virus cDNA inoculation systems for protoplast and plant infection. American Phytopathological Society Annual Meeting, San Diego. S119
- Stewart, L. R., Medina, V., Sudarshana, M., and Falk, B. W. 2007. Functional and cytopathological analysis of two unique Lettuce infectious yellows virus (LIYV)-encoded proteins: P34 and P26. American Phytopathological Society Annual Meeting, San Diego. S155.
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Progress 01/01/06 to 12/31/06
Outputs
Lettuce infectious yellows virus is transmitted from plant to plant by the sweet potato whitefly, Bemisia tabaci. We used recombinant DNA technology to generate specific Lettuce infectious yellows virus genotypes and we found that specific Lettuce infectious yellows virus genotypes are, or are not transmissible by B. tabaci. We showed that the genotypes could be identified in infected plants, and for most genotypes we found no differences in transmission efficiency. We are much closer to understanding the Lettuce infectious yellows virus encoded protein determinants that mediate its transmission by B. tabaci.
Impacts
This research has led to a greater understanding of transmission of Lettuce infectious yellows virus by its whitefly vector, Bemisia tabaci. Lettuce infectious virus is representative of a newly recognized group of plant viruses, many of which are very economically important. The information from our work will directly apply to those viruses as well.
Publications
- Ng, C.K.J., and B.W. Falk. 2006. Virus-vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annu. Rev. Phytopathol. 44:183-212.
- Ng, C.K.J., and B.W. Falk. 2006. Bemisia tabaci transmission of specific Lettuce infectious yellows virus derived from in vitro synthesized transcript-inoculated protoplasts. Virology 352: 209-215.
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Progress 01/01/05 to 12/31/05
Outputs
We have made substantial progress this year. Cytological, immunological, and mutagenesis approaches were used to identify the viral factors associated with the formation of plasmalemma deposits (PLDs) in whole plants and protoplasts infected by Lettuce infectious yellows virus (LIYV). Transmission electron microscopy and immunogold labeling using polyclonal antibodies to four of the five LIYV RNA 2-encoded large proteins, capsid protein (CP), minor capsid protein (CPm), HSP70 homolog (HSP70h), and P59, showed specific labeling of LIYV virions or virion aggregates around the vesiculated membranous inclusions, but not PLDs in LIYV infected Nicotiana benthamiana, N. clevelandii, Lactuca sativa, and Chenopodium murale plants, and N. tabacum protoplasts. In contrast, antibodies to the RNA 2 encoded P26 showed specific labeling of PLDs but not virions in both LIYV infected plants and protoplasts. Virion like particles (VLPs) were seen in protoplasts infected by all LIYV RNA
2 mutants except for the CP (major capsid protein) mutant. PLDs were more difficult to find in protoplasts, but were seen in protoplasts infected by the CP and CPm mutants, but not in protoplasts infected by the P26, HSP70h or P59 mutants. Interestingly, although the CPm mutant showed VLPs and PLDs, the PLDs did not show associated virions virion like particles as was always observed for PLDs seen in protoplasts infected by wild type LIYV. Immunoblot analyses performed on purified LIYV virions showed that P26 was not detected with purified virions, but was detected in the cell wall, 1000g and 30,000g pellet fractions of LIYV infected plants. These data suggest that P26 is associated with the LIYV induced PLDs, and in contrast to the other RNA 2-encoded large proteins, P26 is not a virion protein.
Impacts
Our work has identified specific Lettuce infectious yellows virus proteins associated with virion formation, cell biology and replication. These findings are yielding important fundamental information, but also may provide new opportunities to control virus diseases of plants.
Publications
- Ng, C. K. J., and Falk, B. W. 2006. Virus vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annual Review of Phytopathology. In Press.
- V. Medina, M. R. Sudarshana, T. Tian, K. S. Ralston, H.H. Yeh, and B. W. Falk. 2005. The Lettuce infectious yellows virus (LIYV) encoded P26 is associated with plasmalemma deposits within LIYV infected cells. Virology 333: 367 373.
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Progress 01/01/04 to 12/31/04
Outputs
We have made substantial progress this year. Nicotiana benthamiana plants, transgenic for the LIYV-encoded P34 and P26 were generated and have been grown through the T1 generation. Plants were analyzed by PCR and shown to contain the expected transgene. T2 generation plants will be grown and analyzed for protein production and ability to complement specific LIYV mutants, also generated during the past year. Full-length cDNA sequences of the LIYV genomic RNAs 1 and 2 have been cloned into Ti plasmids. These are designed to be transcribed via the 35S promoter after Agrobacterium tumefaciens infiltration into plant leaves. So far we have no evidence for infectivity via agroinfiltraton, but experiments are continuing. Virus-encoded silencing suppressors are important proteins that affect a virus ability to replicate within susceptible plants. We have screened the LIYV genome via a co-infiltration silencing suppression assay to determine if any of the LIYV-encoded proteins
exhibit typical silencing suppressor activity. None gave obvious silencing suppression activity in this assay. We have attempted to construct a genetically marked LIYV in order to assess replication and movement within plants. Our first attempts used an RNA2 defective RNA engineered to express the green fluorescent protein. This was successful but did not exhibit obvious visible GFP in plants. Therefore we also engineered a LIYV RNA2-encoded P26:GFP fusion. As this is in the full-length RNA2 we hope this will successfully allow us to monitor LIYV replication and expression in whole plants.
Impacts
As our work proceeds I anticipate that our findings will impact our overall understanding of virus:plant cell interactions and determinants of RNA replication. These findings will yield important fundamental information, but also may provide new opportunities to control virus diseases of plants.
Publications
- Falk, B. W. and Hull, R. 2003. Plant RNA virus diseases. In Encyclopedia of Plant and Crop Science, Marcel Dekker Inc. pp. 1023-1025.
- Ng, C. K. J., Tian, T. and Falk, B. W. 2004. Quantitative parameters determining whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus (LIYV) and an engineered defective RNA. Journal of General Virology 85:2697-2707.
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Progress 01/01/03 to 12/31/03
Outputs
We have initiated experiments to gain a detailed understanding of the asynchronous replication of the Lettuce infectious yellows virus (LIYV) genomic RNAs. Transmission electron microscopy and LIYV-specific antibodies were used to immunologically identify LIYV-encoded proteins within LIYV-infected plant cells and to compare these results to cytopathological effects induced by related viruses of the genus Crinivirus. All viruses were found to induce formation of the double-membrane bound vesicles where we believe that LIYV replication occurs. We also used immunological analyses and cell fractionation to show that the LIYV RNA 2-encoded P26 is not associated with LYV virions, or with the vesicles. In contrast P26 is associated with plasmalemma deposits in LIYV-infected cells, and cell fractionation shows P26 to be primarily associated with the cell wall fraction. We also made antibodies to the RNA 1 protein, P34. P34 was previously reported by us as P32, but recent
analyses by our lab revealed a previous sequencing error thereby increasing the size of the ORF encoding this protein, giving a product of 34,000 MW instead of the previously reported 32,000 MW. Antibodies to P34 were used with cell fractionation studies and show P34 to be in the soluble fraction from LIYV-infected plant cells. To further understand cytopathology and replication, transgenic N. benthamiana plants were generated for these two LIYV-encoded genes, those encoding P34, and for P26. Successful transformants have been obtained and the plants are currently being analyzed.
Impacts
As our work proceeds I anticipate that our findings will impact our overall understanding of virus:plant cell interactions and determinants of RNA replication. These findings will yield important fundamental information, but also may provide new opportunities to control virus diseases of plants.
Publications
- Medina, V., Rodrigo, G., Tian, T., Juarez, M., Dolja, V. V., Achon, M. A. and Falk, B. W. 2003. Comparative cytopathology of Crinivirus infections in different plant hosts. Annals of Applied Biology 143:99-110.
- Medina, V., Sudarshana, M. R., Tian, T., Ralston, K. and Falk, B. W. 2003. The Lettuce infectious yellows virus (LIYV)-encoded P26 protein is in the cell wall fraction of infected plants. ASV abstract P40-8, p. 264.
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Progress 01/01/02 to 12/31/02
Outputs
We have initiated experiments to gain a detailed understanding of the asynchronous replication of the Lettuce infectious yellows virus (LIYV) genomic RNAs. Two LIYV-encoded proteins, P32 and P26 have been subcloned and expressed in E. coli, and are being used in cell fractionation and RNA binding assays. Both also will be used for antibody production to facilitate immuno-localization of the respective proteins within LIYV-infected cells. Several specific LIYV mutants have been generated and these will be used for protoplast-based replication studies.
Impacts
As our work proceeds I anticipate that our findings will impact our overall understanding of virus:plant cell interactions and determinants of RNA replication. These findings will yield important fundamental information, but also may provide new opportunities to control virus diseases of plants
Publications
- No publications reported this period
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