Source: TEXAS A&M UNIVERSITY submitted to NRP
PLANT VIRUS INTERACTIONS THAT CONTROL ANTIVIRAL RNA SILENCING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005438
Grant No.
2015-67013-22916
Cumulative Award Amt.
$500,000.00
Proposal No.
2014-04546
Multistate No.
(N/A)
Project Start Date
Jan 1, 2015
Project End Date
Dec 31, 2019
Grant Year
2015
Program Code
[A1121]- Plant Health and Production and Plant Products: Understanding Plant-Associated Microorganisms
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Plant Pathology & Microbiology
Non Technical Summary
Plant viruses cause huge yield and food quality losses in crops grown throughout the world, with damages by some estimated to exceed $60 billion annually. To combat viruses, plants can mount a virus-induced RNA silencing response that specifically recognizes and degrades viral RNA. A detailed understanding of this antiviral RNA silencing in plants will benefit future development of plant disease control strategies, and the use of plants as biotechnological platforms for virus gene-vector mediated protein expression. Our ongoing program focuses on host-dependent molecular plant-virus interactions using Tomato bushy stunt virus (TBSV) that causes diseases worldwide in vegetables, including tomato decline in the U.S. Because of its premier ability to induce and suppress silencing, we also adapted TBSV to study these processes not only in Nicotiana benthamiana, but also in tomato and other vegetables. In addition, we conduct experiments with economically important tobamoviruses and potexviruses. Using these systems we recently revealed: i) antiviral roles for specific ARGONAUTES (AGOs), ii) a connection between antiviral silencing and age-related resistance, iii) that the point of virus entry (roots versus leaves) influences the effectiveness of silencing, and iv) that TBSV-based virus vectors are useful in a variety of crop species. Important questions are: 1) Do one more AGOs function as slicer units of the viral RNA-induced silencing complex (vRISC) for different viruses, 2) How do components of the silencing pathway respond to virus infection, 3) What is the molecular basis for the differential age- and point-of-entry dependent antiviral response, and 4) Can virus-vector mediated protein expression be improved by manipulation of the silencing pathway? Results should lead to a better comprehension of the contribution, dynamics, and regulation of silencing components, also to benefit biotechnological application; all of interest to various areas in life sciences.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121999110175%
2121460110125%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato; 1999 - Tobacco, general/other;

Field Of Science
1101 - Virology;
Goals / Objectives
Even though Arabidopsis Argonaute 1 (AGO1) is thought to be the most important RNA-induced silencing complex (RISC)-associated AGO for miRNA-mediated control and also has been shown to be a key component in silencing against certain viruses in this model plant species, our results in Nicotiana benthamana (Nb) suggest that AGO2 and others also can play a crucial role, indicating a level of flexibility. Furthermore, we have shown that the process responds in an age-dependent manner and that the point-of-entry of a virus may affect its success because of organ-dependent variation in silencing effectiveness. The preliminary experimental analyses also showed that virus-based gene vectors are useful in a variety of crop species. All these results point to a process that appears far from static. Instead the findings lead to the hypothesis that antiviral RNA silencing in plants is dynamic, developmentally coordinated, and can be manipulated to suit biotechnological applications.Objectives. The hypothesis will be tested by examining:Which AGO(s) serve as the slicer unit of vRISC in virus-infected plantsHow the silencing pathway responds to virus infectionThe molecular basis for the differential age- and tissue-dependent antiviral responseHow manipulation of silencing can improve the performance of virus vectorsCompletion of these objectives each will form a major milestone of achievement.
Project Methods
The proposed studies are a logical extension of our long-term program on discovering the basis for resistance of plants against viruses, using Tomato bushy stunt virus (TBSV) as a model. In the past this involved a focus on the role of P19 in the infection process, and we now know that in absence of this protein, TBSV is an effective inducer of RNA silencing that allowed for the first biochemical characterization of a discrete vRISC from plants. Combined with the recent functional genomics data we are now in position to more precisely determine how antiviral silencing is controlled in plants other than Arabidopsis, to serve as guidance for other virus-host systems.Objective 1: We adapted chromatography procedures (gel filtration, ion-exchange, and hydroxyapatite) to isolate an in vitro active virus sequence-specific ribonuclease from infected plants with hallmarks (high MW, siRNA content, requirement for divalent cations) indicative of a bona fide RISC. We showed that such a vRISC can be isolated from Nicotianana benthamiana (Nb) plants infected with either TdP19 (TBSV defective for P19 expression) or from other virus-host systems. A key property that is not biochemically known for any virus-host system is which AGO protein is used to assemble the vRISC. Because we identified a key role of NbAGO2 in silencing against TBSV, in this objective chromatography and related techniques will be used to test the hypothesis that NbAGO2 associated with siRNAs forms the core unit of vRISC either as an unusual sequence-specific catalytic 'slicer' or as a necessary co-factor. However, based on recent findings, other NbAGOs are also considered candidates, and the tomato analogs will now be tested for an antiviral role.Objective 2: From preliminary experiments it appears that not all AGOs respond similarly to TBSV infection and that the response is virus-dependent. This serves to illustrate that the silencing components respond in a dynamic manner to virus infection. To obtain a more complete idea on these dynamics we will monitor the response of key RNA silencing genes to TMV and TBSV infection using transcriptomic analyses.Objective 3: The experiments for this objective are designed to determine how plant age and point of virus entry affect antiviral silencing. The experiments are based on our observations that plant age effects are primarily noted for TBSV as opposed to other viruses, that NbAGO2 is required in older Nb plants for silencing TBSV, and that silencing is strong in Nb and tomato roots. This led to our hypothesis that the expression or activity of NbAGO2 is developmentally controlled. To test this we will compare NbAGO2 expression in differently aged Nb and tomato plants and roots, examine effect of age and tissue on other NbAGOs and silencing factors, compare the effect for roots of tomato and Nb plants at different developmental stages for different viruses, investigate the role of P19, and examine the localization of TBSV and TdP19 (TBSV lacking P19) upon leaf versus root inoculation.Objective 4: Tobacco mosaic virus (TMV)-based vectors are among the best performing in Nb but need to be tested in other hosts, and compared to TBSV, the host range for TMV is less broad. TBSV locally infects over 100 species, many of which we have tested for foreign gene expression. Therefore, this vector system may hold great potential for application in hosts such as several vegetables. Within this context, we also found that unlike in Nb, silencing in tomato and pepper prohibits TdP19 infection even in inoculated leaves. This suggests that these plants have very effective antiviral silencing systems, and applications of virus vectors in such hosts can be expected to benefit substantially from compromising the silencing cascade. As first demonstrated over a decade ago and now globally used, RNA silencing (in general or against virus vectors) can be alleviated by expression of a suppressor but this often causes severe symptoms upon prolonged expression that may impact the eventual applicability of the virus vector. Therefore, options should be explored to generate host platforms in which the virus vector is ineffectively targeted by silencing thereby excluding the need for expression of a suppressor. We have virus vectors for different plant/crop species, and can manipulate the silencing pathway to impede the plants' defenses against virus infections. In this objective we aim to use available and to be assembled gene knock-down constructs to target AGOs (-1,-2,-5,-7) and DCLs (-2,-4) in Nb and tomato, followed by monitoring expression levels by virus vectors. Efforts: The research efforts address a new fundamental niche of study that has high potential to yield novel mechanistic interpretations of immediate relevance for understanding virus silencing in crop species and for biotechnology. Moreover, considering the evolutionary conservation of RNA silencing, efforts towards a better comprehension of the antiviral silencing process in plants is not only of interest to those studying plant-microbe interactions but will very likely have an impact in various areas in life sciences. The efforts to reach target audiences will include guest lectures and presentations at conferences; incorporation of project activities in formal classroom lectures; having postdocs, graduate and undergraduate students participate in the project; and by publishing results in peer-reviewed journals. These information exchanges will effect changes in knowledge by sharing an improved understanding of antiviral RNA silencing, changes in actions by stimulating the development of better virus control and the implementation of new biotechnological approaches, and potentially changes conditions to the benefit of our food supply and more sustainable use of environmental resources.Evaluation: This proposal entails an ambitious effort, especially Objective 1, but we have ample and solid preliminary data, techniques are working, and tools are readily available. We are thus favorably positioned to proceed rapidly and complete the studies in four years. Success will be measured by evaluating the progress for the objectives. For Objectives 1 and 2, the silencing in Nb and tomato, and activity assays can be completed in the first two years, the measuring of NbAGO2 and the siRNA analyses in years 2 through 4. The gene expression analyses (Objective 2) can be initiated right away for optimization to be finalized in year 2, and expression analyses for Objective 3 will be performed in parallel. The remaining experiments for Objective 3; the P19 mutant testing will be performed during years 1 and 2, and testing of developmental effects and localization during year 3-4. The virus vector related experiments will involve gene constructions in year 1, and gene knock-down experiments and vector performance in years 2-4.Milestones: This study can be anticipated to yield new fundamental insight into the processes that control defense-related antiviral silencing in plants. Our recent results provided evidence for the involvement of a new AGO protein (NbAGO2) in antiviral defense. An important milestone for Objective 1 is to acquire direct biochemical evidence whether this AGO is a component of vRISC in Nb, which is currently unknown for virus-host systems, except perhaps for some indirect evidence for AGO1 in Arabidopsis. The milestone for success of Objective 2 is to understand details on the expression regulation of silencing components. The experiments for Objective 3 have great potential to bring new insights into an old concept, by determining how antiviral RNA silencing is related to age-related resistance in plants. Objective 3 is also addressing an intriguing observation how the effectiveness of silencing differs for leaves versus roots. The milestone of success for Objective 4 is to demonstrate the great potential of virus vectors to further harness their potential in biotechnology.

Progress 01/01/15 to 12/31/19

Outputs
Target Audience:The life span of the project involved the participation of two postdocs, three female graduate students, three male graduate students, two female Research Associates, two female undergraduate students, and three male undergraduate students. The undergraduate students obtained valuable training to boost their competitiveness for graduate school as evidenced by their admission to competitive programs. Because of their training under the auspices of this project, the graduate students all obtained a position of their choice (e.g., Ph.D. program, industry, or postdoc). The techniques and research topics were incorporated in my graduate courses in Plant Virology, Molecular Methods, and a graduate level Plant Pathology lab course. These classes continued to incorporate techniques adapted and developed as part of the project. We also had collaborations with scientists in Kazakhstan, with a laboratory at Texas A&M AgriLife in Weslaco TX, and with a group at Colorado Sate University in Fort Collins, CO. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Individuals involved during the life span of the project were the principal investigator, two postdocs, six graduate students, two research associates, and five undergraduate students. Training and professional development outside regular courses and mentoring activities were provided by having each trainee work on sub-objectives of the project. How have the results been disseminated to communities of interest?Information gathered during the project was incorporated in lectures the PI gives in Molecular Methods and Plant Virology, and courses taught in microbial diseases for undergraduate students, and in guest lectures at other institutions. Information was also presented at meetings (abstracts are listed with the products) and through publications, which in reality measure the success of the project. What do you plan to do during the next reporting period to accomplish the goals?The funding for the project has ended and accordingly the outcome of Objectives has mostly been published, or papers have been submitted. The remaining plan is to address the comments that will be made by manuscript reviewers.

Impacts
What was accomplished under these goals? For Objective 1, we tested the contribution of different ARGONAUTES in Nicotiana benthamiana (NbAGOs) to the defense against silencing-sensitive GFP-expressing constructs based on Tomato bushy stunt virus (TBSV). We published that a sequence-specific antiviral RISC can be isolated from plants and a new finding was that this RISC seemed to have a higher affinity for longer viral RNAs during in vitro RNA cleavage assays. As published, we determined the contribution of different ARGONAUTES in Nicotiana benthamiana (NbAGOs) to the defense against silencing-sensitive GFP-expressing constructs based on Tomato bushy stunt virus (TBSV) (Tombusvirus), Sunn-hemp mosaic virus (Tobamovirus), and Foxtail mosaic virus (Potexvirus). Upon Tobacco rattle virus (TRV)-mediated down-regulation of NbAGO1, -4, -5, or -6, no effects were noted on susceptibility to any virus construct, whereas knockdown of NbAGO2 specifically prevented silencing of P19-defective TBSV (TGdP19). Down-regulation of a new gene referred to as NbAGO5L showed some reduced silencing for TGdP19 but not for the other two virus constructs, whereas silencing of NbAGO7 gave rise to a subtle increase in susceptibility to all three viruses. This publication also revealed a dominant role for NbAGO1 in silencing to the detriment of the effect exerted by other NbAGOs. Using our newly developed and published method of virus-mediated delivery of gene editing components, we were able to either confirm or newly demonstrate the contribution of NbAGO2, DCL2, and HEN1 as anti-TBSV components. For Objective 2, we published on the generation and utility of hairpin transgenic plants in which NbAGO2 expression is specifically knocked-down. We showed that this has little effect on plant development and the trait is passed on to the next generation. Using a variety of viruses we showed that the transgenic plants are generally more susceptible to infection, which manifests itself at the local or systemic level in a virus-dependent manner. We have also confirmed that NbAGO1 and particularly NbAGO2 expression is highly elevated upon infection of plants with TMV or TBSV, as one would expect for crucial RISC-associated components. As we published, we also showed that co-infiltrating different TRV-NbAGO constructs simultaneously did not enhance virus susceptibility. However, an unexpected finding was that whenever the TRV-NbAGO1 construct was present, this compromised silencing of genes targeted by co-infiltrated constructs, as shown upon co-infiltration of TRV-NbAGO1 with either TRV-NbAGO2 or TRV-Sul (targeting Magnesium chelatase I). Only after a prolonged period (~2 months) did TRV-Sul mediated systemic bleaching occur in these co-infected plants, suggesting that TRV-NbAGO1 hinders the silencing ability of other TRV-NbAGO constructs. In conclusion, this study revealed dominant effects of TRV-NbAGO1 For Objective 3, we found that NbAGO2 seemed consistently relatively higher expressed in roots, which agrees with our publication that antiviral silencing is stronger in roots than in leaves. We also have completed the monitoring of the expression of silencing components in plants of different ages. These experiments showed that NbAGO2 expression is elevated in plants of all ages upon TBSV infection and that NbAGO2 expression is very prominent in young leaves. This leads us to conclude in a publication that is forthcoming that it is not the lack of NbAGO2 expression that prevents our observed lack of antiviral silencing in young plants but that something else is contributing. An intriguing finding, also soon to be published, is the observation of a similar age-related effect whereby in younger plants virus-mediated CRISPR/Cas governed gene editing is more efficient because it is less effectively targeted by silencing than in older plants. For Objective 4. Plant viral vectors enable the expression of valuable proteins at high levels in a relatively short time. For many purposes it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector, often due to silencing-related effects. However, we found that such a co-expression could be achieved by deploying a strategy for simultaneous delivery by two compatible and non-competitive viruses that each express a separate protein. As published we developed two agro-launchable virus vector systems based on Tomato bushy stunt virus (TBSV) (expressing the strong silencing suppressor P19) and Tobacco mosaic virus (TMV). Both vectors were used to express green fluorescent protein (GFP), but of different sizes to distinguish them during molecular detection on immunoblots. In addition, experiments were conducted by co-infections of TBSV-GFP with TMV-RFP expressing red fluorescent protein. The results in Nicotiana benthamiana and tomato demonstrated that the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a biotechnological platform for fast and high level expression of potentially valuable protein complexes, for instance those that need to form oligomers for activity. We also published the development of viruses as delivery tools of single guide RNAs (sgRNAs) in an effort to optimize gene editing by the CRISPR/Cas system. As either published or soon to be published, upon carefully conducted biochemical and molecular studies we have documented that a novel 5' sgRNA processing step occurs in plants much to the benefit of the editing efficiency, and this has not been reported before. Furthermore, as soon to be published we successfully expressed both the sgRNA as well as the Cas9 protein from a single virus vector backbone as a one-component delivery system, but this performed only at notable levels when P19 was co-expressed to counteract the negative effects of silencing. In addition, because N. benthamiana is genetically poorly defined we have also used experiments with the diploids N. attenuata and N. otophora and verified that these can also be used for virus-mediated gene editing. We have now also been able to use the TBSV backbone to express sgRNAs, and in general observed that the presence of a guide RNA positively affects the ability of the virus to cause a systemic infection. The finding that viruses can be used to co-express foreign genes in the same plants, tissues, and cells opens up avenues of having higher order oligomeric complexes formed. This could lead to a change in action in future application of innovative gene expression approaches in plants. This could lead to changes in condition with regards to custom production of value-added products in plants. A change in knowledge can be anticipated based on our discovery that NbAGO2 has dominant effects and contributes at various levels to the antiviral defense, and that NbAGOs other than NbAGO2 contribute at various levels to the antiviral defense. Particular impact can also be anticipated from our demonstration that virus-vector systems can be used to deliver guide RNAs for gene editing. This could lead to changes in action and conditions in future application of innovative transient protein expression and gene editing. Significant impact can also be anticipated upon our recent finding that TBSV can be used to genetically explore which components contribute to antiviral silencing by using CRISP/Cas gene editing in conjunction with virus infection. The implementation of virus-mediated gene editing could lead to a change in action in future application of innovative transient gene editing approaches in plants, and lead to changes in condition with regards to functional genomics and biotechnological applications in plants. A change in knowledge can be anticipated based on our finding that an endogenous enzyme machinery in plants properly processes progenitor sgRNAs into the correct template for programming Cas9.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Odokonyero, D., Mendoza, M.R., Alvarado, V.Y., Zhang, J., Wang, X., and Scholthof, H.B. (2015). Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses. Virology 486:209-218.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Mendoza, M. (2016). Argonaute 2 and antiviral silencing in plants. M.S. Thesis, Texas A&M University.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Mendoza, M., Payne, A., Castillo, S., Crocker, M., Shaw, B.D. and Scholthof, H.B. (2017). Expression of separate proteins in the same plant leaves and cells using two independent virus-based gene vectors. In: Update on Plant Virus Infection - a Cell Biology Perspective: Frontiers in Plant Science. DOI: 10.3389/fpls.2017.01808.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Odokonyero, D., Mendoza, M., Moffett, P., and Scholthof, H.B. (2017). Tobacco rattle virus (TRV) mediated silencing of Nicotiana benthamiana ARGONAUTES (NbAGOs) reveals new antiviral candidates and dominant effects of TRV-NbAGO1. Phytopathology 107:1-11.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cody, W., Scholthof, H.B., (co-senior author) and Mirkov, T.E. (co-senior author) (2017). Multiplexed gene editing and protein delivery using a Tobacco mosaic virus viral vector. Plant Physiology 174:113.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhumabek, A.T., Abeuova, L.S., Mukhametzhanov, N.S., Scholthof, H.B., Ramanculov, E.M., and Manabayeva, S.A. (2018). Transient expression of the bovine leukemia virus envelope glycoprotein gp51 in plants by a recombinant TBSV vector. J. Virol. Meth. 255:1-7.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Cody, Will B. (2018). A viral-based toolbox for efficient gene editing in Nicotiana species. Ph.D. Dissertation, Texas A&M University.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Chiong, K. (2018). Tobacco mosaic virus as a gene editing platform. M.S. Thesis, Texas A&M University.
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: DeMell, A. (2019). Developing viruses for gene editing to study virus-specific molecular interactions in Nicotiana species. M.S. Thesis, Texas A&M University.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Cody, W.B., and Scholthof, H.B. (2019) Plant virus vectors 3.0: Transitioning into synthetic genomics. Ann. Rev. Phytopath. 57: 211-230.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Cody, W.B., and Scholthof, H.B. Native eukaryotic RNA degradation pathways process progenitor guide RNAs to create catalytically active Cas9/sgRNA complexes. (submitted).
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Chiong, K., Cody, W.B., and Scholthof, H.B. Tobacco mosaic virus as a gene editing platform (to be submitted).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Cody, W., Scholthof, H.B., and Mirkov, T.E. (2016). Application of a transient viral vector for efficient genome editing in plants. Annual meeting ASPB, Austin TX.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Cody, W., Scholthof, H.B., and Mirkov, T.E. (2016). Application of a transient viral vector for efficient genome editing in plants. Synthetic Biology: Engineering, Evolution & Design (SEED), Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cody, W., Scholthof, H.B., (co-senior author) and Mirkov, T.E. (2017). Viral delivery of a gene editing tool for transient screening of gene function. Annual meeting APS, San Antonio, TX. Phytopathology 107:S5.1.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chiong, K., Cody, W., Mirkov, T.E., and Scholthof, H.B. (2018). A TMV-based viral vector for delivering gene editing tools. Phytopatyhology 108;10S
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Scholthof, H.B. (2015). Tomato bushy stunt virus as a model system to study antiviral RNA silencing. Phytopathology 105 S4.161
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mendoza, M., and Scholthof, H.B. (2016). Co-expression of proteins by two virus vectors in the same cells of infected plants. Annual meeting APS, Miami FL. Phytopathology 106 S4.146
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mendoza, M., B.D. Shaw, and Scholthof, H.B. (2017). Two RNA viruses as tools for the co-expression of proteins in the same cells of infiltrated plants. Annual meeting APS, San Antonio, TX. Phytopathology 107:S5.1.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: DeMell, A., Cody, W., and Scholthof, H.B. (2019). Developing viruses as genome modification platforms to study virus-specific molecular interactions in Nicotiana species. Annual meeting of the American Society for Virology.


Progress 01/01/18 to 12/31/18

Outputs
Target Audience:During 2018 the project involved the participation of three graduate students, three undergraduate students, and a female Research Associate. The undergraduate students obtained valuable training to boost their competitiveness for graduate school as evidenced by their admission to competitive programs. The techniques and research topics were incorporated in my graduate courses in Plant Virology Spring 2018, Molecular Methods Fall 2018, and a graduate level Plant Pathology lab course in Fall 2018. These classes continue to incorporate techniques adapted and developed as part of the project. We also continue collaborations with scientists in Kazakhstan, with a laboratory at Texas A&M AgriLife in Weslaco TX, and with a group at Colorado Sate University in Fort Collins, CO. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Individuals involved in the project were the principal investigator, three graduate students, a research associate, and three undergraduate students. Training and professional development outside regular courses and mentoring activities were provided by having each trainee work on sub-objectives of the project. How have the results been disseminated to communities of interest?Information gathered during the project was incorporated in lectures the PI gives in Molecular Methods and Plant Virology, and courses taught in microbial diseases for undergraduate students, and in guest lectures at other institutions. Information was also presented at meetings (abstracts are listed with the products) and through publications, which in reality measure the success of the project. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Finalize experiments and prepare publication on the TBSV-mediated editing of essential components of the antiviral RNA silencing pathway. Also, continue exploring the basis for the aggressive photype associated with viral constructs expressing sgRNAs, even in absence of Cas9. Objective 2. In conjunction with Objective 1, the contribution of different silencing components will be tested with regards to susceptibility to virus infection. Objective 3. We will prepare a publication and also further examine the correlation between lack of antiviral silencing in young N. benthamiana plants and the observation that gene editing is more efficient in younger plants. Objective 4. We will finalize experiments and publish on a novel processing of virus-mediated delivered guide RNA for CRISPR gene-editing technology. We also aim to finalize a study on comparing the utility and editing in different Nicotiana species.

Impacts
What was accomplished under these goals? As mentioned in the summary and goals, plant viruses cause huge yield and food quality losses in crops grown throughout the world, with damages by some estimated to exceed $60 billion annually. As mentioned with previous reports, but to briefly summarize, the overall aim in the laboratory and also of the present project is to gain knowledge to ultimately develop sustainable control methods and to use viruses in biotechnology. This project focuses on efforts to examine plant factors that are used to activate antiviral defenses also known as RNA silencing whereby the host specifically degrades viral RNA. We also explore how viruses and virus products can be used as delivery tools in plants to produce either high levels foreign proteins, or guide RNAs for CRISPR/Cas gene editing, or both. The corresponding efforts and achievements are briefly summarized. For Objective 1, we tested the contribution of different ARGONAUTES in Nicotiana benthamiana (NbAGOs) to the defense against silencing-sensitive GFP-expressing constructs based on Tomato bushy stunt virus (TBSV) expressing single guide RNAs (sgRNAs) for CRISPR/Cas9 mediated gene editing. When the TBSV-sgRNA was delivered in combination with a transient or transgenic expression construct for Cas9 we showed that the target NbAGO2 gene was edited by performing PCR and restriction enzyme mediated assays on isolated chromosomal DNA. Because NbAG02 is essential for RNA silencing against TBSV, the virus was able to replicate in the edited tissue as evidenced by green fluorescence. In preliminary tests we obtained similar results when DCL2 or HEN1 were targeted by gene editing. This illustrates that TBSV-mediated CRISPR/Cas9 gene editing allows us to carefully dissect the silencing pathway to demonstrate which components are required for silencing the virus. A serendipitous finding in these experiments was that, even in absence of Cas9, certain viral constructs expressing sgRNAs display a more aggressive phenotype with more severe disease symptoms. For Objective 2, we previously used VIGS or experimented with the hairpin transgenic plants in which NbAGO2 expression is specifically knocked-down. Using a variety of viruses we again showed that the RISC-programming disruption makes these plants more susceptible to infection. We have also confirmed that NbAGO1 and particularly NbAGO2 expression is highly elevated upon infection of plants with TMV or TBSV, as one would expect for crucial RISC-associated components. In conjunction with Objective 1, we now have now developed a clean gene editing tool to functional genetically explore the antiviral silencing pathway. For Objective 3, we completed tests on the differential expression of silencing components in leaves versus roots. NbAGO2 is consistently relatively higher expressed in roots. We also have completed the monitoring of the expression of silencing components in plants of different ages. These experiments showed that NbAGO2 expression is elevated in plants of all ages upon TBSV infection and that NbAGO2 expression is very prominent in young leaves. This leads us to conclude in a publication that is forthcoming that it is not the lack of NbAGO2 expression that prevents our observed lack of antiviral silencing in young plants but that something else is contributing. An intriguing finding mentioned the previous year, and as now confirmed, is the observation of a similar age-related whereby in younger plants virus-mediated CRISPR/Cas governed gene editing is more efficient because it is less effectively targeted by silencing than in older plants. For Objective 4, we further developed the use of viruses as delivery tools of sgRNAs in an effort to optimize gene editing by the CRISPR/Cas system. Upon carefully conducted biochemical and molecular studies we have documented (to be published) that a novel 5' sgRNA processing step occurs in plants much to the benefit of the editing efficiency, and this has not been reported before. Furthermore, we successfully expressed both the sgRNA as well as the Cas9 protein from a single virus vector backbone as a one-component delivery system. In addition, because N. benthamiana is genetically poorly defined we have also used experiments with the diploids N. attenuata and N. otophora and verified that these can also be used for virus-mediated gene editing. Anticipated impacts as well as changes in knowledge, action and condition are provided in the Non-Technical Summary. Here, these are briefly addressed specifically with respect to the findings during this reporting period. Substantial impact can be anticipated upon our recent finding that TBSV can be used to genetically explore which components contribute to antiviral silencing by using CRISP/Cas gene editing in conjunction with virus infection. The implementation of virus-mediated gene editing could lead to a change in action in future application of innovative transient gene editing approaches in plants, and lead to changes in condition with regards to functional genomics and biotechnological applications in plants. A change in knowledge can be anticipated based on our finding that an endogenous enzyme machinery in plants properly processes progenitor sgRNAs into the correct template for programming Cas9. As indicated previously, it is likely that our reports and publications will influence research in other laboratories, and we continue to supply laboratories worldwide with research materials.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhumabek, A.T., Abeuova, L.S., Mukhametzhanov, N.S., Scholthof, H.B., Ramanculov, E.M., and Manabayeva, S.A. (2018). Transient expression of the bovine leukemia virus envelope glycoprotein gp51 in plants by a recombinant TBSV vector. J. Virol. Meth. 255:1-7.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Cody, Will B. (2018). A viral-based toolbox for efficient gene editing in Nicotiana species. Ph.D. Dissertation, Texas A&M University.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Chiong, K. (2018). Tobacco mosaic virus as a gene editing platform. M.S. Thesis, Texas A&M University.
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Cody, W.B., and Scholthof, H.B. Plant virus vectors 3.0: Transitioning into synthetic genomics. Ann. Rev. Phytopath. (submitted).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chiong, K., Cody, W., Mirkov, T.E., and Scholthof, H.B. (2018). A TMV-based viral vector for delivering gene editing tools. Phytopatyhology 108;10S


Progress 01/01/17 to 12/31/17

Outputs
Target Audience:This year the project involved the participation of three graduate students, two undergraduate students, and female Research Associate. The undergraduate students obtained valuable training to further their competitiveness for graduate school admission. The techniques and research topics were incorporated in my graduate courses in Plant Virology Spring 2017, Molecular Methods Fall 2017, and a graduate level Plant Pathology lab course in Fall 2017. These classes continue to incorporate techniques adapted and developed as part of the project. We also continue collaborations with scientists in Kazakhstan, with a laboratory at Texas A&M AgriLife in Weslaco TX, and with a group at Colorado Sate University in Fort Collins, CO. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Individuals involved in the project were the principal investigator, three graduate students, a research associate, and two undergraduate students. Training and professional development outside regular courses and mentoring activities were provided by having each trainee work on sub-objectives of the project. How have the results been disseminated to communities of interest?Information gathered during the project was incorporated in lectures the PI gives in Molecular Methods and Plant Virology, and courses taught in microbial diseases for undergraduate students, and in guest lectures at other institutions. Information was also presentated at meetings (abstracts are listed with the products) and through publications, which in reality measure the success of the project. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will aim to target different components of the antiviral silencing pathway by using our virus-mediated gene editing, and then monitor the effect on virus infection. We also will further explore our finding that guide RNA expression stimulates systemic infection. Objective 2. In conjunction with Objective 1, the contribution of different silencing components will be tested with regards to susceptibility to virus infection. Objective 3. We will further examine the correlation between lack of antiviral silencing in young N. benthamiana plants and the observation that gene editing is more efficient in younger plants. Objective 4. We will compare the performance of different gene vectors across a platform of many different Nicotiana species. We will publish our data on novel processing of virus-mediated delivered guide RNA for CRISPR gene-editing technology. We also aim to optimize gene editing by providing a suppressor of gene silencing, to express guide RNA and Cas9 from the same viral vector, and to target specific genes for biotechnology purposes.

Impacts
What was accomplished under these goals? As mentioned in the summary and goals, plant viruses cause huge yield and food quality losses in crops grown throughout the world, with damages by some estimated to exceed $60 billion annually. The overall aim in the laboratory and also of the present project is to gain knowledge to ultimately develop sustainable control methods and to use viruses in biotechnology. This project focuses on efforts to examine plant factors that are used to activate antiviral defenses also known as RNA silencing whereby the host specifically degrades viral RNA. We also explore how viruses and virus products can be used as biotechnology tools to produce high levels of value-added pharmaceutical or bioenergy-optimizing foreign proteins in plants. The corresponding efforts and achievements are briefly summarized. For Objective 1, we finalized testing the contribution of differentARGONAUTESinNicotiana benthamiana(NbAGOs) to the defense against silencing-sensitive GFP-expressing constructs based onTomato bushy stunt virus(TBSV) (Tombusvirus), Sunn-hemp mosaic virus(Tobamovirus), andFoxtail mosaic virus(Potexvirus). As already mentioned last year, and now confirmed, uponTobacco rattle virus(TRV)-mediated down-regulation ofNbAGO1,-4,-5, or-6, no effects were noted on susceptibility to any virus construct, whereas knockdown ofNbAGO2specifically prevented silencing of P19-defective TBSV (TGdP19). Surprisingly, we discovered that a new gene referred to asNbAGO5Lshowed some reduced silencing for TGdP19 but not for the other two virus constructs, whereas silencing ofNbAGO7gave rise to a subtle increase in susceptibility to all three viruses.This study also revealed a dominant role forNbAGO1in silencing to the detriment of the effect exerted by otherNbAGOs. During this reporting year we published the above findings inPhytopathology. For Objective 2, we have continued experiments with the hairpin transgenic plants in which NbAGO2 expression is specifically knocked-down. Using a variety of viruses we again showed that the RISC-programming disruption makes these plants more susceptible to infection. We have also confirmed that NbAGO1 and particularly NbAGO2 expression is highly elevated upon infection of plants with TMV or TBSV, as one would expect for crucial RISC-associated components. We also confirmed that one line of empty vector transformedN. benthamianaplants is specifically resistant to infection withFoxtail mosaic virus(FMV). We have learned that virus GFP-expressing FMV variants not expressing the coat protein are capable of replication and cell-to-cell movement in these plants. This suggests that the restriction for infection is related to coat protein expression. For Objective 3, we are continuing tests on the differential expression of silencing components in leaves versus roots. NbAGO2 seemed consistently relatively higher expressed in roots. We also have carefully monitored the expression of silencing components in plants of different ages. These experiments showed that NbAGO2 expression is elevated in plants of all ages upon TBSV infection and that NbAGO2 expression is very prominent in young leaves. This leads us to conclude that it is not the lack of NbAGO2 expression that prevents our observed lack of antiviral silencing in young plants but that something else is contributing. We have also found a similar age-related effect by showing that in younger plants, virus-mediated CRISPR/Cas governed gene editing is less effectively targeted by silencing than in older plants, and consequently the editing is more efficient in younger plants. For Objective 4, we finalized the comparison between the performance of two virus vector systems, based on eitherTobacco mosaic virus(TMV) orTomato bushy stunt virus(TBSV). Both express active suppressors to minimize the effect of silencing. We showed that both systems perform well inN. benthamianaand tomato while the TBSV-based system has the advantage of being operational in other plants such as lettuce and cowpea. We also showed that they co-express cargo proteins in the same tissues and cells. These findings are published inFrontiers of Plant Science. We also published the development of viruses as delivery tools of single guide RNAs (sgRNAs) in an effort to optimize gene editing by the CRISPR/Cas system (Plant Physiology). Results thus far show that TMV-mediated delivery of sgRNAs leads up to 70% editing. We have now also been able to use the TBSV backbone to express sgRNAs, and in general observed that the presence of a guide RNA positively affects the ability of the virus to cause a systemic infection. We have also found that a novel 5' sgRNA processing step occurs in plants much to the benefit of the editing efficiency, and this has not been reported before. Furthermore, we successfully edited a lipase gene inN. benthamiana, using the virus mediated guide RNA delivery tools, and are investigating the effects on oil production. Anticipated impacts as well as changes in knowledge, action and condition are provided in the Non-Technical Summary. Here, these are briefly addressed specifically with respect to the findings during this reporting period. Substantial impact can be anticipated upon our recent publication that different NbAGOs contribute to antiviral silencing, and from of our novel virus-based systemto deliver guide RNAs for CRISPR/Cas9 mediated gene editing. As mentioned last year, this could lead to a change in action in future application of innovative transient gene editing approaches in plants, and lead to changes in condition with regards to functional genomics and biotechnological applications in plants. A change in knowledge can be anticipated based on our report that two different viruses can co-exist in the same plants, tissues and cells. It is likely that our reports and publications will influence research in other laboratories, and we continue to supply laboratories worldwide with research materials.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Mendoza, M., Payne, A., Castillo, S., Crocker, M., Shaw, B.D. and Scholthof, H.B. (2017). Expression of separate proteins in the same plant leaves and cells using two independent virus-based gene vectors. In: Update on Plant Virus Infection - a Cell Biology Perspective: Frontiers in Plant Science. DOI: 10.3389/fpls.2017.01808.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cody, W., Scholthof, H.B., (co-senior author) and Mirkov, T.E. (co-senior author) (2017). Multiplexed gene editing and protein delivery using a Tobacco mosaic virus viral vector. Plant Physiology 174:113.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Odokonyero, D., Mendoza, M., Moffett, P., and Scholthof, H.B. (2017). Tobacco rattle virus (TRV) mediated silencing of Nicotiana benthamiana ARGONAUTES (NbAGOs) reveals new antiviral candidates and dominant effects of TRV-NbAGO1. Phytopathology 107:1-11.
  • Type: Other Status: Published Year Published: 2017 Citation: Mendoza, M., B.D. Shaw, and Scholthof, H.B. (2017). Two RNA viruses as tools for the co-expression of proteins in the same cells of infiltrated plants. Annual meeting APS, San Antonio, TX. Phytopathology 107:S5.1. https://doi.org/10.1094/PHYTO-107-12-S5.1
  • Type: Other Status: Published Year Published: 2017 Citation: Cody, W., Scholthof, H.B., (co-senior author) and Mirkov, T.E. (2017). Viral delivery of a gene editing tool for transient screening of gene function. Annual meeting APS, San Antonio, TX. Phytopathology 107:S5.1. https://doi.org/10.1094/PHYTO-107-12-S5.1


Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Fort this reporting year the project involved the participation of a female and two male graduate students, one female Research Associate, and a female Honors undergraduate student. The undergraduate student obtained valuable training to further her elected career in health sciences. The techniques and research topics were incorporated in my graduate courses in Molecular Methods Fall 2016, a new graduate level Plant Pathology lab course in Fall 2016, and guest lectures at Colorado State University (CSU), Fort Collins in Spring 2016. These classes continue to incorporate techniques adapted and developed as part of the project. We also have collaborated with a laboratory at Texas A&M AgriLife in Weslaco TX, and with a group at CSU. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Individuals involved in the project were the principal investigator, three graduate students, a research associate, and one undergraduate student. Training and professional development outside regular courses and mentoring activities were provided by having each trainee work on sub-objectives of the project. How have the results been disseminated to communities of interest?Information gathered during the project was incorporated in lectures the PI gives in Molecular Methods and Plant Virology, and courses taught in microbial diseases for undergraduate students, and in guest lectures at other institutions. Information was also dispersed by presentations at meetings (abstracts are listed with the products) and through publications, which in reality measure the success of the project What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Further examine the effect of different NbAGOs on susceptibility to virus infection. In conjunction with Objective 4, we are continuing to test if TBSV-based vectors or vectors based on different viral backbones, can be used to deliver guide RNA against NbAGOs to knock-out their function. Serious attempts aim at over-expressing NbAGO2 and with immune-precipitation determine if this NbAGO2 is a component of the antiviral RISC assembly. Objective 2. In conjunction with Objective 1, the contribution of different NbAGOs will be tested with regards to susceptibility to virus infection. Objective 3. As in the present reporting year, we will further examine if young N. benthamiana plants are generally more susceptible to virus infection or if this is somehow specific to TBSV and related viruses. Objective 4. Publish our data on virus-mediated delivery of more than one protein in the same cells of infected plants, and expand the utility to different host platforms. Also, publish our data on virus-mediated guide RNA delivery for CRISPR gene-editing technology to knock-out specific genes in plants, and analyze gene expression.

Impacts
What was accomplished under these goals? As mentioned in the summary and goals, plant viruses cause huge yield and food quality losses in crops grown throughout the world, with damages by some estimated to exceed $60 billion annually. The overall aim in the laboratory and also of the present project is to gain knowledge to ultimately develop sustainable control methods and to use viruses in biotechnology. This project focuses on efforts to examine plant factors that are used to activate antiviral defenses also known as RNA silencing whereby the host specifically degrades viral RNA. We also explore how viruses and virus products can be used as biotechnology tools to produce high levels of value-added pharmaceutical or bioenergy-optimizing foreign proteins in plants. For Objective 1, we determined the contribution of different ARGONAUTES in Nicotiana benthamiana (NbAGOs) to the defense against silencing-sensitive GFP-expressing constructs based on Tomato bushy stunt virus (TBSV) (Tombusvirus), Sunn-hemp mosaic virus (Tobamovirus), and Foxtail mosaic virus (Potexvirus). Upon Tobacco rattle virus (TRV)-mediated down-regulation of NbAGO1, -4, -5, or -6, no effects were noted on susceptibility to any virus construct, whereas knockdown of NbAGO2 specifically prevented silencing of P19-defective TBSV (TGdP19). Down-regulation of a new gene referred to as NbAGO5L showed some reduced silencing for TGdP19 but not for the other two virus constructs, whereas silencing of NbAGO7 gave rise to a subtle increase in susceptibility to all three viruses. We have also initiated experiments to target different NbAGOs using CRISPR/Cas9 gene editing to examine the effect on virus susceptibility. For Objective 2, we showed that co-infiltrating different TRV-NbAGO constructs simultaneously did not enhance virus susceptibility. However, an unexpected finding was that whenever the TRV-NbAGO1 construct was present, this compromised silencing of genes targeted by co-infiltrated constructs, as shown upon co-infiltration of TRV-NbAGO1 with either TRV-NbAGO2 or TRV-Sul (targeting Magnesium chelatase I). Only after a prolonged period (~2 months) did TRV-Sul mediated systemic bleaching occur in these co-infected plants, suggesting that TRV-NbAGO1 hinders the silencing ability of other TRV-NbAGO constructs. In conclusion, this study revealed dominant effects of TRV-NbAGO1 For Objective 3, we have continued our investigations on the differential expression of silencing components in leaves versus roots. NbAGO2 levels were relatively higher expressed in roots. Follow-up experiments also confirmed that NbAGO2 expression is elevated in plants of all ages upon TBSV infection and that NbAGO2 expression is very prominent in young leaves. This leads us to firmly conclude that something other than NbAGO2 is contributing to the age-related effect. Objective 4. Plant viral vectors enable the expression of valuable proteins at high levels in a relatively short time. For many purposes it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that each express a separate protein. Here we report on the use of two agro-launchable virus vector systems based on Tomato bushy stunt virus (TBSV) and Tobacco mosaic virus (TMV). Both vectors were used to express green fluorescent protein (GFP), but of different sizes to distinguish them during molecular detection on immunoblots. In addition, experiments were conducted by co-infections of TBSV-GFP with TMV-RFP expressing red fluorescent protein. The results in Nicotiana benthamiana and tomato demonstrated that the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a biotechnological platform for fast and high level expression of potentially valuable protein complexes, for instance those that need to form oligomers for activity. Anticipated impacts as well as changes in knowledge, action and condition are provided in the Non-Technical Summary. Here, these are briefly addressed specifically with respect to the findings during this reporting period. Particular impact can be anticipated from our virus-vector systems that can be used to over-express proteins in the same cells, but that can also deliver guide RNAs for gene editing. This could lead to changes in action in future application of innovative transient protein expression and gene editing approaches in plants. This could potentially lead to changes in condition with regards to custom production of value-added products in plants. A change in knowledge can be anticipated based on our discovery that NbAGOs other than NbAGO2 contribute at various levels to the antiviral defense. Our reports and publications might influence research in other laboratories, and we continue to supply laboratories worldwide with research materials.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Omarov, R., Ciomperlik, J., and Scholthof, H.B. (2016). An in vitro reprogrammable antiviral RISC with size-preferential ribonuclease activity. Virology 490:41-48 (highlighted paper).
  • Type: Other Status: Other Year Published: 2016 Citation: Mendoza, M., and Scholthof, H.B. (2016). Co-expression of proteins by two virus vectors in the same cells of infected plants. Annual Program Directors meeting USDA-NIFA-AFRI, Washington D.C.
  • Type: Other Status: Published Year Published: 2016 Citation: Mendoza, M., and Scholthof, H.B. (2016). Co-expression of proteins by two virus vectors in the same cells of infected plants. Annual meeting APS, Miami FL. Phytopathology 106 S4.146


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:The project involved the participation of a female and a male graduate student, one female Honors undergraduate student, and a male Honors undergraduate student. The undergraduate students obtained valuable training to further their elected career in Public Health or Molecular Entomology. The techniques and research topics were incorporated in my graduate courses in Molecular Methods Fall 2015, Plant Virology Spring 2015, and a new graduate level Plant Pathology lab course in Fall 2015. These classes continue to incorporate techniques adapted and developed as part of the project. We also have collaborated with scientists in Kazakhstan, with a laboratory at Texas A&M AgriLife in Weslaco TX, and with a group at Virginia Tech. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Individuals involved in the project were the principal investigator, two graduate students and two undergraduate students. Training and professional development outside regular courses and mentoring activities were provided by having each student work on sub-objectives of the project, in particular on testing different virus vectors in different hosts. How have the results been disseminated to communities of interest?Information gathered during the project was incorporated in lectures the PI gives in Molecular Methods and Plant Virology, and courses taught in microbial diseases for undergraduate students. Information was also dispersed by presentations at meetings (abstracts are listed with the products) and through publications, which in reality measure the success of the project. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Further examine the effect of NbAGO2-hairpin expression, or control empty vector transgenic plants on susceptibility to virus infection. In conjunction with Objective 4, test if TBSV can be used to deliver guide RNA against NbAGO2 to knock-out its function and thereby allow the TBSV vector (not expressing P19) to maintain virus load. Testing if NbAGO2 pull-down experiments yield an in virto active RISC-like antiviral nuclease. Objective 2. Experiments are also underway to examine the contribution of additional NbAGOs to antiviral silencing and re-examine how these respond to virus infection. Objective 3. Tests are being conducted to investigate if young N. benthamiana plants are generally more susceptible to virus infection or if this is somehow specific to TBSV and related viruses. Objective 4. Produce publishable data on virus-mediated delivery of more than one protein in the same cells of infected plants, and expand the utility to different host platforms. Also, obtain publishable data on guide RNA delivery for CRISPR gene-editing technology to knock-out specific genes in plants, and analyze gene expression.

Impacts
What was accomplished under these goals? As mentioned in the summary and goals, plant viruses cause huge yield and food quality losses in crops grown throughout the world, with damages by some estimated to exceed $60 billion annually. The overall aim in the laboratory and also of the present project is to gain knowledge to ultimately develop sustainable control methods and to use viruses in biotechnology. This project focuses on efforts to examine plant factors that are used to activate antiviral defenses also known as RNA silencing whereby the host specifically degrades viral RNA. We also explore how viruses and virus products can be used as biotechnology tools to produce high levels of value-added pharmaceutical or bioenergy-optimizing foreign proteins in plants. The corresponding efforts and achievements are briefly summarized. For Objective 1, we have now firmly demonstrated that a sequence-specific antiviral RISC can be isolated from plants and a new finding was that this RISC seemed to have a higher affinity for longer viral RNAs during in vitro RNA cleavage assays. We have also obtained an NbAGO2 over-expression vector for agroinfiltration of plants. Upon infection of leaves in which NbAGO2 is over-expresssed, this allows us to perform co-immunoprecipitation experiments and test if the pulled-down complex has RISC-like nuclease activity, using techniques we recently published. For Objective 2, we have published that we can successfully and specifically knock-down NbAGO2 expression using RNA hairpin technology in transgenic N. benthamiana. We showed that this has little effect on plant development and the trait is passed on to the next generation. Using a variety of viruses we showed that the transgenic plants are generally more susceptible to infection, which manifests itself at the local or systemic level in a virus-dependent manner. Subsequent experiments have shown that NbAGO1 and particularly NbAGO2 expression is highly elevated upon infection of plants with TMV or TBSV. For Objective 3, we have further scrutinized the differential expression of silencing components in leaves versus roots. NbAGO2 seemed consistently relatively higher expressed in roots. We also have carefully monitored the expression of silencing components in plants of different ages. These experiments showed that NbAGO2 expression is elevated in plants of all ages upon TBSV infection and that NbAGO2 expression is very prominent in young leaves. This leads us to conclude that it is not the lack of NbAGO2 expression that prevents our observed lack of antiviral silencing in older plants but that something else is contributing. Further experiments are planned. For Objective 4, we have compared the performance of two virus vector systems, based on either Tobacco mosaic virus (TMV) or Tomato bushy stunt virus (TBSV). Both express active suppressors to minimize the effect of silencing. We showed that both systems perform well in N. benthamiana while the TBSV-based system has the advantage of being operational in other plants such as lettuce and cowpea. We also showed that they co-express cargo proteins in the same tissues and cells. Experiments are ongoing to use viruses as delivery tools of small guide RNAs in an effort to optimize gene editing by the CRISPR/Cas system. Furthermore, we will test if the performance can be further enhanced in N. benthamiana plants expressing the NbAGO2-hairpin. Anticipated impacts as well as changes in knowledge, action and condition are provided in the Non-Technical Summary. Here, these are briefly addressed specifically with respect to the findings during this reporting period. Of particular impact is our finding that viruses can be used to co-express foreign genes in the same plants, tissues, and cells. This opens up avenues of having higher order oligomeric complexes formed. This could lead to a change in action in future application of innovative gene expression approaches in plants. This could potentially lead to changes in condition with regards to custom production of value-added products in plants. A change in knowledge can be anticipated based on our discovery that NbAGO2 has effects beyond silencing at the site of infection to contribute at various levels to the antiviral defense. Our reports and publications might influence research in other laboratories, and we continue to supply laboratories worldwide with research materials.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Odokonyero, D., Mendoza, M.R., Alvarado, V.Y., Zhang, J., Wang, X., and Scholthof, H.B. (2015). Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses. Virology 486:209-218.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Scholthof, H.B. (2015). Tomato bushy stunt virus as a model system to study antiviral RNA silencing. Phytopathology 105 S4.161
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Omarov, R ., Ciomperlik, J., and Scholthof, H.B. An in vitro reprogrammable antiviral RISC with size-preferential ribonuclease activity. Virology 490:41-48. All the preparatory work was done in 2015 even though the technical publication year is 2016.