Progress 08/01/06 to 07/31/11
Outputs
OUTPUTS: ACTIVITIES (listed per objective): The overall hypothesis of this project was that short-interfering RNA (siRNA) binding sites of the Tomato bushy stunt virus (TBSV) P19 suppressor of silencing are essential for maintenance of viral RNA in infected plants by blocking siRNA transfer to the RNA-induced silencing complex (RISC), while different host backgrounds and the P19-interactive host protein Hin19, provide ancillary effects. The project entailed four objectives and the activities for each will be listed separately with an emphasis on those that contributed to major findings over the lifetime of the project summarized in the Outcomes. Objective 1: Test the effect of P19 on formation and activities of Nicotiana benthamiana-derived effector complexes in vitro. We isolated RISC from different plant species infected with TBSV or other viruses and analyzed this for sequence-specific nuclease activity. We developed techniques to dissociate siRNAs from the isolated RISC and then reprogram this with siRNA of a heterologous virus. We also silenced N.benthamiana for expression of different Argonaute (AGO) proteins to determine which ones contribute to silencing against TBSV. Objective II: Infect N. benthamiana with TBSV-P19 mutants specifically defective for siRNA binding to test effects on viral RNA maintenance and the siRNA binding and recovery. We generated thirteen site-specific TBSV mutants encoding P19 with siRNA contact sites altered, and assayed these by testing siRNA binding, siRNA accumulation, and pathogenicity in N. benthamiana. We also used our P19 mutant transgenic N. benthamiana plants. Objective III: Isolate effector complexes from different hosts and analyze the relevance of siRNA binding for the contribution of P19 to systemic invasion of several plant species. We inoculated P19 mutants onto different TBSV hosts and measured virus spread and pathogenicity and accumulation of virus material. We used newly developed GFP-expressing TBSV constructs either competent or defective for expressing P19 and inoculated leaves or roots, or N. tabacum and N. benthamiana suspension cell cultures. Objective IV: Use plants that are either transiently or transgenically compromised for normal Hin19 expression. Hin19 silencing was recalcitrant to initially implemented methods, but we used a newly adapted virus-induced gene silencing (VIGS) approach, which allowed for the co-introduction of several Hin19-silencing inducers at the same time. EVENTS, SERVICES AND PRODUCTS: This reporting year, we presented our findings at the 2011 annual Plant Virology retreat at the Noble Foundation. The PI was an invited speaker at Texas Tech University. DISSEMINATION: Information gathered during the project was incorporated in lectures and courses the PI teaches for undergraduate and graduate students. Information was also dispersed by presentations at meetings, as listed above, and publications listed below. The PI also guest lectured on the findings in the Department of Biological Sciences, at the L.N. Gumilev Eurasian National University, Astanana, Kazakhstan. PARTICIPANTS: Individuals directly involved in the project for this reporting year were the principal investigator Dr. Herman Scholthof, and four scientists in the project were an Assistant Research Scientist Dr. Veria Alvarado, a postdoctoral associate Dr. Shuga Manabayeva, and two graduate students: Denis Odokonyero and Juanita McLachlan. Two undergraduate students have also assisted. TARGET AUDIENCES: During the reporting year, the project involved the participation of a female assistant research scientist, a female postdoctoral associate, and a female technician. The graduate students who participated, one US female and one male from Uganda, were obtaining valuable training to be competitive for employment and advanced training in other research laboratories. The techniques and research topics were incorporated in the PI's undergraduate and graduate courses that also enlisted the participation of the scientists in the laboratory. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES FOR THIS REPORTING YEAR. Outcomes for this year will be listed along with how these contributed to overall key findings for the whole lifetime of the project. For Objective 1, findings contributed to the major outcome that the several biochemical separation techniques we developed to isolate and analyze an in vitro active RISC from N. benthamiana plants infected with TBSV, also works for isolating an antiviral RISC from N. benthamiana plants infected with Tobacco rattle virus. Another novel finding was that siRNAs can be dissociated to inactivate RISC that can then be reconstituted by exogenous addition of siRNAs from a heterologous virus demonstrating that the isolated complex represents a bona fide RISC. Lastly, we showed that AGO2 in N. benthamiana is a key factor for silencing against TBSV. Results for Objective 2 contributed to an overall major finding that siRNA binding by P19 is important for its function to prevent viral RNA degradation and concomitant recovery of N. benthamiana plants from infection. Especially, one particular P19 mutant exhibited an unexpected behavior in seemingly accelerating the onset of silencing. Using new GFP-expressing TBSV constructs we showed that in presence of P19 GFP expression is maintained independent of plant age but in absence of P19 silencing occurs in a temperature and plant age-dependent manner. An important development was that we generated and used p19-mutant transgenic plants that allow for elevated levels of foreign gene expression without exhibiting the severe symptomatic effects normally associated with P19 expression in transgenic plants. For objective 3 we extended the use of Agrobacterium-mediated delivery and expression of TBSV-GFP constructs in different hosts and in suspension cultures of N. tabacum and N. benthamiana. We also verified that infection of leaves or roots requires coat protein and P19 in a tissue-dependent and host differential manner. The results suggest that silencing at the site of virus entry is more effective in roots than in leaves. This contributes to the overall major finding that siRNA capture by P19 is crucial for its role in promoting virus spread in different hosts and tissues while other P19-realted responses do not strictly correlate with siRNA binding. For Objective 4, we are finding that silencing of Hin19 is not occurring even with improved virus-induced gene silencing systems, for reasons we will continue to investigate beyond the auspices of this award. Training outcomes: The two new graduate students have become familiar with our studies on TBSV-mediated host responses, especially this year with regards to the contribution of different AGOs to antiviral silencing. Two postdocs were involved, one evaluated the differential requirements for root versus leaf inoculations of different plants, and the role of silencing in suspensions cells of different hosts. The other postdoc (Assistant Research Scientist) is continuing the antiviral RISC analysis and is studying the molecular basis for the plant age-dependent virus induced silencing.
Publications
- Scholthof, H.B., Alvarado, V.Y., Vega-Arreguin, J.C., Ciomperlik, J.J., Odokonyero, D., Brosseau, C., Jaubert, M., Zamora, A., and Moffett, P. (2011). Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana. Plant Physiol., 156:1548-1555.
- Ciomperlik, J., Omarov, R.T., and Scholthof, H.B. (2011). An antiviral RISC isolated from Tobacco rattle virus-infected plants. Virology 412:117-124
- Scholthof, K.-B.G., and Scholthof, H.B. (2011). Induction and suppression of RNA silencing: Insights from plant viral infections - A BARD Workshop Report. Plant Molec. Biol. 75:205-210
- Saxena, P., Hsieh, Y-C., Alvarado, V.Y., Sainsbury, F., Saunders. K., Lomonossoff, G., and Scholthof, H.B. (2011). Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotech. J. 9:703-712.
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Progress 08/01/09 to 07/31/10
Outputs
OUTPUTS: ACTIVITIES (listed per objective): The overall hypothesis of this project is that short-interfering RNA (siRNA) binding sites of the Tomato bushy stunt virus (TBSV) P19 suppressor of silencing are essential for maintenance of viral RNA in infected plants by blocking siRNA transfer to RISC, while different host backgrounds and the P19-interactive host protein Hin19, provide ancillary effects. The project entails four objectives and the activities for each will be listed separately. Objective 1: Test the effect of P19 on formation and activities of Nicotiana benthamiana-derived effector complexes in vitro. We continuously use a variety of separation and chromatography procedures to isolate and analyze RISC from different plant species infected with TBSV either active or inactive for P19 expression. The activity of the complex is tested in vitro by addition of RNA substrate, the siRNA composition is measured, and plant-specific Argonaute antisera are used for immunoblotting experiments. We also isolated RISC from plants infected with viruses other than TBSV to establish if our findings with TBSV reflect a general phenomenon. Objective II: Infect N. benthamiana with TBSV-P19 mutants specifically defective for siRNA binding to test effects on viral RNA maintenance and the activity of DICER (siRNA generation) and RISC (recovery) in those plants. We generated thirteen site-specific TBSV mutants encoding P19 with siRNA contact sites altered, and assayed these by testing siRNA binding, siRNA accumulation, and pathogenecity and movement in different plant species. We are also testing the effect of plant age on the induction of virus-induced gene silencing. Objective III: Isolate effector complexes from different hosts and analyze the relevance of siRNA binding for the contribution of P19 to systemic invasion of several plant species. We inoculated P19 mutants onto different TBSV hosts and measured virus spread and pathogenicity and accumulation of virus material. In addition, biochemical separation techniques are used to measure P19 and siRNA accumulation in different plants infected with TBSV. We are also testing new GFP expressing TBSV constructs in presence of absence of P19 by inoculation of leaves or roots, and by agroinfiltration on leaves and by expressing these in N. tabacum and N. benthamiana suspension cell cultures. Objective IV: Use plants that are either transiently or transgenically compromised for normal Hin19 expression. Hin19 silencing was recalcitrant to initially implemented methods, but we are now using a newly adapted virus-induced gene silencing (VIGS) approach. EVENTS, SERVICES AND PRODUCTS: This reporting year, we have presented our findings at the 2010 annual meetings of the American Phytopathological Society (APS) and the American Society for Virology (ASV). In Spring 2010, the PI was an invited speaker at a BARD workshop in Eilat, Israel. DISSEMINATION: Information gathered during the project is incorporated in lectures and courses the PI teaches for undergraduate and graduate students. Information is also dispersed by presentations at meetings, as listed above, and publications listed below. PARTICIPANTS: Individuals involved in the project for this reporting year were the principal investigator Dr. Herman Scholthof, and four scientists in the project were an Assistant Research Scientist Dr. Veria Alvarado, a postdoctoral associate Dr. Shuga Manabayeva, and two graduate students: Denis Odokonyero and Juanita McLachlan. Two undergraduate students have also assisted. We also hosted an international scholar Dr. Emily Ayo-John (Fall 2009) who was a Borlaug USDA Faculty Exchange Fellow from Nigeria. TARGET AUDIENCES: During the reporting year, the project involved the participation of a female assistant research scientist, a female postdoctoral associate, and a female visiting international scientist who temporarily joined the group to obtain training in molecular plant virology and biotechnology. The graduate students who participated, one US female and one male from Uganda, are obtaining valuable training to be competitive for employment and advanced training in other research laboratories. The techniques and research topics are incorporated in my undergraduate and graduate courses that also enlisted the participation of the scientists in my laboratory. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES FOR THIS REPORTING YEAR. For Objective 1 we showed that the several biochemical separation techniques to isolate and analyze an in vitro active RISC from plants infected with TBSV, also works for isolating an antiviral RISC from plants infected with Tobacco rattle virus. We showed that siRNAs can be dissociated to inactivate RISC that can then be reconstituted by exogenous addition of siRNAs from a heterologous virus. We also raised a very specific N. benthamiana argonaute antiserum. For objective 2 we tested newly optimized TBSV constructs expressing GFP, on plants using standard rub-inoculation and agroinfiltration. The results showed that in presence of P19 GFP expression is maintained independent of plant age but in absence of P19 silencing occurs in a plant age-dependent manner. We also used p19 mutant transgenic plants that allow for elevated levels of foreign gene expression. For objective 3 we extended the work with different hosts by using suspension cultures of N. tabacum and establishing de novo suspension cultures of N. benthamiana. We showed that in presence of P19 high levels of virus replication is achieved in suspension cells. We also showed that infection of leaves or roots requires coat protein and P19 in a tissue-dependent and host differential manner. For Objective 3, we are now using a virus-induced gene silencing system to silence Hin19 and are verifying silencing with our Hin19-specific antiserum. Initial results suggest that Hin19 might influence the systemic distribution of TBSV-GFP expression.
Publications
- Saxena, P., Hsieh, Y-C., (both share first co-authorship), Alvarado, V.Y., Sainsbury,, F., Saunders. K., Lomonossoff, G., and Scholthof, H.B. (last two, co-corresponding authors) (2010).. Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotech. J. (in press).
- Everett, A, Scholthof, H.B., and Scholthof, K.-B. G. (2010) Satellite panicum mosaic virus coat protein enhances the performance of plant virus gene vectors. Virology 396:37-46. Epub 2009 Nov 10.
- Manabayeva, S.A., and Scholthof, H.B. (2010) Direct delivery of viral vectors into plant suspension cells. Phytopathology 100:S76
- Alvarado, V.Y., Ciomperlik, J., and Scholthof, H.B. (2010). Aging-associated enhancement of RNA silencing against Tomato bushy stunt virus (TBSV) in Nicotiana benthamiana. Annual meeting American Society for Virology W11-7.
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: ACTIVITIES (listed per objective): P19 encoded by Tomato bushy stunt virus (TBSV) is a pathogenicity protein that is a structurally well-characterized suppressor of RNA silencing. The overall hypothesis of this project is that short interfering RNA (siRNA) binding sites of P19 are essential for maintenance of viral RNA in infected plants by blocking siRNA transfer to RISC, while different host backgrounds and the P19-interactive host protein Hin19, provide ancillary effects. The project entails four objectives and the activities for each will be listed separately. Objective 1: Test the effect of P19 on formation and activities of Nicotiana benthamiana-derived effector complexes in vitro. We continuously use a variety of separation and chromatography procedures to isolate and analyze RISC from different plant species infected with TBSV either active or inactive for P19 expression. The activity of the complex is tested in vitro by addition of RNA substrate, the siRNA composition is measured, and a plant-specific Argonaute antiserum is used for immunoblotting experiments. Objective II: Infect N. benthamiana with TBSV-P19 mutants specifically defective for siRNA binding to test effects on viral RNA maintenance and the activity of DICER (siRNA generation) and RISC (recovery) in those plants. We generated thirteen site-specific TBSV mutants encoding P19 that has siRNA contact sites altered, and assayed these by testing siRNA binding, siRNA accumulation, and pathogenecity and movement in different plant species. Objective III: Isolate effector complexes from different hosts and analyze the relevance of siRNA binding for the contribution of P19 to systemic invasion of several plant species. We inoculated P19 mutants onto different TBSV hosts and measured virus spread and pathogenicity and accumulation of virus material. In addition, biochemical separation techniques are used to measure P19 and siRNA accumulation in different plants infected with TBSV. Objective IV: Use plants that are either transiently or transgenically compromised for normal Hin19 expression. Hin19 silencing was recalcitrant to initially implemented methods, but we are now using a novel approach with artificial microRNAs (amiRNAs), and a newly adapted virus-induced gene silencing (VIGS) approach. EVENTS, SERVICES AND PRODUCTS: This reporting year, we have presented our findings at the 2009 Annual meeting of the American Phytopathological Society (APS). In Spring 2009, the PI presented the latest findings at the USDA-NRI-CSREES annual Project Directors meeting in Washington D.C, and he was an invited speaker at a symposium jointly organized by the British Society for General Microbiology and the Association of Applied Biologists, in Harrogate UK, and an invited presenter at the annual CPBR symposium in Washington DC. We routinely send P19-and TBSV-associated materials to various laboratories throughout the world. DISSEMINATION: Information gathered during the project is incorporated in lectures and courses the PI teaches for undergraduate and graduate students. Information is also dispersed by presentations at meetings, as listed above, and publications. PARTICIPANTS: Individuals involved in the project for this reporting year were the principal investigator Dr. Herman Scholthof, and three scientists in the project were Dr. Rustem Omarov, an Associate Research Scientist, a postdoctoral associate Dr. Veria Alvarado, and for five months the recent Ph.D. graduate who finished and stayed on for a short while in a postdoctoral capacity, Dr. Yi-Cheng Hsieh. Training and professional development were also provided to two Research Assistants (Ms. Bonnie Seaberg and Ms. Vanessa Vaughn), an undergraduate student (Ms. Kristina Twiggs), and two international visiting scientists (Dr. Shuga Manabayeva and Dr. Ana Maria da Graca Mondjana). TARGET AUDIENCES: During the reporting year, the project involved the participation of a female postdoctoral associate, two female technical/research assistants, and two female visiting international scientists who temporarily joined the group to obtain training in molecular plant virology and biotechnology. The graduate students who finished at the end of the previous reporting year obtained valuable training to be competitive for employment and advanced training in other research laboratories. In fact, the female M.S. student who left for the University of Wisconsin, had offers from Cornell, Berkeley, and Texas A&M. The Associate Research Scientist who was in the laboratory has now a leading academic function in biotechnology in his home country. The techniques and research topics are incorporated in my undergraduate and graduate courses that also enlisted the participation of the scientists in my laboratory. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES FOR THIS REPORTING YEAR. For Objective 1 we continued to use several biochemical separation techniques to isolate and analyze an in vitro active RISC from plants infected with TBSV. We confirmed that this antiviral RISC is associated with TBSV-specific siRNAs, it preferentially and endonucleolytically cleaves TBSV RNA, and the complex is enriched for proteins recognized by the Argonaute antiserum. As a method test for Objective IV, we initiated a screen to silence individual Argonautes in N. benthamiana to determine which Argonaute is the slicer component of the RISC. An exciting finding was that the siRNAs can be dissociated to inactivate RISC that can then be reconstituted by exogenous addition of siRNAs. For objective 2 we generated multiple site-specific TBSV mutants to encode P19 that has siRNA contact sites altered. The selected ones indeed no longer sequestered siRNAs but not all mutations equally affected host range or symptomatology. We did observe a correlation between the ability of P19 to bind siRNAs and systemic invasion of spinach and pepper plants. For objective 3 we isolated P19-siRNA complexes from plant species in addition to N. benthamiana. Results suggest that only in N. benthamiana is the siRNA accumulation prolific. For Objective IV, we continued our species screening with our Hin19-specific antiserum and confirmed that Hin19 is expressed constitutively across a wide range of plant species. We are screening plants silenced with the amiRNA strategy and we have five segments cloned in an intermediate plasmid, ready to be used for VIGS. For this purpose the PI has obtained training and materials for implementing this strategy. Training outcomes: A graduate student who finished his Ph.D. stayed on as a postdoctoral associate for five months and now has a postdoctoral positon at the University of Maryland. Two M.S. students finished last year, one stayed on as a Research Assistant for a year, and she has now a job of her interest elsewhere; the other student continued for Ph.D. studies at UW Madison. The Associate Research Assistant since two months is Head of a Biotechnology Laboratory in Kazakhstan.
Publications
- Manabayeva, S.A., Shamekova, M., Hsieh, Y, Omarov, R.T., and Scholthof, H.B. (2009). Tomato bushy stunt virus inoculation of roots versus leaves reveals differential effects by the coat protein and the P19 silencing suppressor. Phytopathology 99:S7.
- Hsieh, Y.-C., Omarov, R.T., and Scholthof, H.B. (2008). Diverse and newly recognized effects associated with siRNA binding site modifications on the Tomato bushy stunt virus P19 silencing suppressor. J. Virol. 83:2188-200.
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: ACTIVITIES (listed per objective): Tomato bushy stunt virus (TBSV) encodes a pathogenicity protein termed P19 that is a structurally well-characterized suppressor of RNA silencing. The overall hypothesis of this project is that the short interfering RNA (siRNA) binding sites of P19 are essential for maintenance of viral RNA in infected plants by blocking siRNA transfer from DICER to RISC, while different host backgrounds and the P19-interactive host protein Hin19, can provide ancillary effects. The project entails four objectives and the activities for each will be listed separately. Objective 1: Test the effect of P19 on the formation and activities of Nicotiana benthamiana-derived effector complexes in vitro. We have continued to use gel filtration, ion exchange and hydroxyapatite chromatography to isolate an in vitro active ribonuclease complex from different plant species infected with TBSV either active or inactive for P19 expression. The activity of the complex is tested in vitro by addition of RNA substrate. We also selected a plant-specific Argonaut peptide to raise antiserum. Objective II: Infect N. benthamiana with TBSV-P19 mutants specifically defective for siRNA binding to test effects on viral RNA maintenance and the activity of DICER and RISC isolated from those plants. We have analyzed ten site-specific TBSV mutants (that were generated in the previous reporting year) to encode P19 that has siRNA contact sites altered, by testing siRNA binding, siRNA accumulation, and pathogenecity and movement in different plant species. Objective III: Isolate effector complexes from different hosts and analyze the relevance of siRNA binding for the contribution of P19 to systemic invasion of several plant species. We inoculated P19 mutants onto different TBSV hosts and measured virus spread and pathogenicity and accumulation of virus material. Objective IV: Use plants that are either transiently or transgenically compromised for normal Hin19 expression. Hin19 silencing was recalcitrant to virus induced gene silencing and therefore we are attempting a novel approach with artificial microRNAs (amiRNAs) specifically designed to target Hin19 mRNA. EVENTS, SERVICES AND PRODUCTS: This reporting year, we have presented our findings at the 2008 Annual Plant Virology Retreat at the Noble Foundation in Ardmore Oklahoma, at the 2008 annual meeting of the American Society of Virology (ASV), and at the annual meeting of the American Phytopathological Society (APS). The PI was an invited speaker at the USDA-NRI-CSREES annual Project Directors meeting in Washington D.C. We routinely send p19-associated materials to various laboratories throughout the world. DISSEMINATION: Information gathered during the project is incorporated in lectures I give and courses I teach for undergraduate and graduate students. Information is also dispersed by presentations at meetings, as listed above, and publications. PARTICIPANTS: As last year, individuals involved in the project are the principal investigator Dr. Herman Scholthof, and a leading scientist in the project is Dr. Rustem Omarov, an Associate Research Scientist. A new postdoctoral associate (Dr. Veria Alvarado) has recently joined the laboratory specifically for Objective 4. During the reporting year, training and professional development were provided to three graduate students (Yi-Cheng Hsieh, Jessica Ciomperlick, and Bonnie Seaberg), a technician (Vanessa Vaughn) and an international visiting scientist (Dr. Shuga Manabayeva). TARGET AUDIENCES: During the reporting year, the project involved the participation of two female graduate students, a new female postdoc, a female technician, and also a female visiting scientist from Kazakhstan who is here to obtain training in molecular plant virology and biotechnology. The graduate students obtain valuable training to be competitive for employment and advanced training in other research laboratories. In fact, one of the female M.S. students has joined a Ph.D. program at the University of Wisconsin, Madison. The international visiting scientist who was in the laboratory the previous year, has now a leading biotechnology function in her home country. The techniques and research topics are incorporated in my undergraduate and graduate courses that also enlist the participation of the Associate Research Scientist and graduate students. This involves the incorporation of laboratory sessions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES FOR THIS REPORTING YEAR Objective 1: The RISC-like ribonuclease complex associated with TBSV-specific siRNAs is only readily detectable in absence of P19. It endonucleolytically cleaves TBSV RNA and a novel outcome is that the complex preferentially targets genome-sized RNA and immunoblot assays with our newly developed antiserum suggest that the active complex is enriched for RISC-associated Argonaute protein(s). Objective 2: Some of the P19 mutations have no or little effect whereas others cause reduced symptom severity and show variable levels of siRNA accumulation. The potential outcome of our p19 mutagenesis work may have a measurable impact for plant biotechnology. Objective 3: We tentatively observed a correlation between the ability of P19 to bind siRNAs and systemic invasion of spinach and pepper plants. A strict correlation between local lesion formation and siRNA binding is not evident. One particular mutant caused accelerated onset of silencing concomitant with the inability to detect viral RNA and proteins, but surprisingly host RNAs and proteins were also susceptible in a host-dependent manner. Objective 4: As part of our work on utilizing viruses as gene vectors, we have tested the Tobacco rattle virus system, to silence Hin19, but so far have not been successful. Subsequently, we have generated T-DNA constructs expressing Hin19-amiRNAs and these are currently tested for their ability to silence Hin19. For this purpose we are using our previously raised Hin19-specific antiserum. Training outcomes: Three graduate students have finished their Ph.D. (1) or M.S. degrees (2). The Ph.D. graduate is interviewing for a postdoc position, one of the M.S. students is enrolled in a Ph.D. program elsewhere (see participants) and one is currently a Research Assistant in the laboratory.
Publications
- Qiu, W, and Scholthof, H.B. 2007. Tomato bushy stunt virus gene vectors. Current Protocols in Microbiology. Curr. Protoc. Microbiol 7:16I.4.1-16I.4.16.
- Seaberg, L.B, Hsieh, Y, and Scholthof, H.B. 2008. Host impact on foreign gene integrity in a virus vector. Phytopathology 98: S143.
- Ciomperlik, J.J., Omarov, R.T, and Scholthof, H.B. 2008. Characterization of a RNAi associated anti-viral ribonuclease in Nicotiana benthamiana. Phytopathology 98:S39.
- Hsieh, Y., Omarov, R.T., and Scholthof, H.B. 2008. Substitutions of tombusvirus p19 short interfering RNA binding sites perturb virus-host interactions. Annual meeting American Society for Virology W41-4.
- Omarov, R.T., Ciomperlik, J.J. and Scholthof, H.B. 2008. RNA silencing-effector complexes involved in Tombusvirus RNA degradation. Annual meeting American Society for Virology W41-5.
- Scholthof, H.B. 2007. Heterologous expression of viral RNAi suppressors: RISC management. Plant Physiology 145:1110-1117.
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Progress 08/01/06 to 07/31/07
Outputs
Tomato bushy stunt virus (TBSV) encodes a pathogenicity protein termed P19 that is a structurally well-characterized suppressor of RNA silencing. Our extensive biochemical knowledge of P19 in combination with the novel tools and methods recently developed in the laboratory provide us with a unique opportunity to address the overall hypothesis that the siRNA binding sites of P19 are essential for maintenance of viral RNA in infected plants by blocking siRNA transfer from DICER to RISC, while different host backgrounds and the P19-interactive host protein Hin19, can provide ancillary effects. The project entails four objectives that will each be listed together with the progress made for the first year. Objective 1: Test the effect of P19 on the formation and activities of Nicotiana benthamiana-derived effector complexes in vitro. We have used gel filtration, ion exchange and hydroxyapatite chromatography to isolate an in vitro active ribonuclease complex from plants
infected with TBSV inactive for P19 expression. The ribonuclease complex is associated with TBSV-specific siRNAs, it preferentially cleaves TBSV RNA and does so endonucleolytically, and the activity is RNA dosage and divalent cation dependent. Objective II: Infect N. benthamiana with TBSV-P19 mutants specifically defective for siRNA binding to test effects on viral RNA maintenance and the activity of DICER and RISC isolated from those plants. We have generated ten site-specific TBSV mutants to encode P19 that has siRNA contact sites altered. Some of the mutations have no or little effect on infectivity or symptoms. However, other cause reduced symptom severity and show variable levels of siRNA accumulation. Some mutations also affected the overlapping gene that specifies the cell-to-cell movement protein, with one specific mutation abolishing the ability of the virus to move, as shown with GFP imaging. Objective III: Isolate effector complexes from different hosts and analyze the
relevance of siRNA binding for the contribution of P19 to systemic invasion of several plant species. We have inoculated siRNA binding mutants to different hosts and have tentatively observed a correlation between the ability of P19 to bind siRNAs and systemic invasion of spinach and pepper plants. A correlation between local lesion formation and siRNA binding is not yet apparent. We have conducted preliminary isolations of P19-siRNA complexes from plant species in addition to N. benthamiana but the results are not yet conclusive. Objective IV: Use plants that are either transiently or transgenically compromised for normal Hin19 expression. Because of technical problems with Agrobacterium-mediated transformation we have started to use virus induced gene silencing based on the Tobacco rattle virus system, to silence host genes. We have successfully used this on selected host genes and are now incorporating this system for this objective on Hin19. We have used our Hin19-specific
antiserum and demonstrated that Hin19 is expressed constitutively across a wide range of plant species; so far only one out of twenty-six species lacked detectable amounts of Hin19.
Impacts
The studies on the correlation between silencing suppression and infectivity, and to biochemically characterize the in vitro activity of virus-induced effector complexes is important from a fundamental perspective towards understanding how plants defend themselves against pathogen attack. The long-term beneficial impact for sustainable U.S. agriculture is that the information from our studies can potentially be used in plant biotechnology and to develop environmentally safe and effective virus control strategies for crop plants.
Publications
- Omarov, R., Ciomperlik, J., and Scholthof, H.B. 2007. RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex. Proc. Natl. Acad. Sci. USA 104:1714-1719
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