UNDERSTANDING THE MECHANISMS OF VIRUS REPLICATION IN PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001898
• Project Start Date: Nov 22, 2013
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Biochemistry

Non Technical Summary
Applied and basic research on many plant plus-strand RNA viruses have provided valuable insight into a variety of cellular processes and provided tools for novel technologies that benefit agriculture. Studies with potato virus X (PVX) and other plant viruses have resulted in development of vectors for myriad agricultural technologies, including antibody and vaccine production. Studies on replication complexes of the plant virus PVX will generate valuable details on how viruses multiply their genomes, and will provide further opportunities for development of new viral-based technologies and anti-viral strategies. Knowledge obtained from these studies will be utilized to educate students in the Agricultural Sciences and provide them with up-to-date information on basic and practical aspects of plant pathogens and their effects on agriculture.

Animal Health Component

10%

Research Effort Categories

Basic

80%

Applied

10%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	2499	1000	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

plant pathogens

plant virus

plant virus replication

plant rna virus

potato virus x

rna folding

Goals / Objectives
RNA-directed RNA synthesis is essential for survival of numerous prokaryotic and eukaryotic RNA viruses. Despite diverse hosts, many plus-strand RNA viruses exhibit similar mechanisms for various aspects of genome expression. Potato virus X (PVX) is an excellent model system for both genetic and biochemical analyses of RNA synthesis. This virus contains a relatively small, single genomic RNA that is functionally monocistronic and is available as an infectious clone. A protoplast replication system has been developed for quantification of plus-and minus-strand PVX RNA accumulation, which is a powerful system for genetic analyses. Replication of PVX to high levels in tobacco plants has enabled the development of soluble extracts that support template-dependent PVX RNA synthesis and utilization of infected plants for revertant studies. Extensive studies in our laboratory have found that terminal and internal conserved elements and long-distance interactions among these elements are required for synthesis of both minus-and plus-strand RNA in vivo and in vitro. A novel aspect of these interactions is that that both termini share the same subset of internal elements. The hypothesis that interactions between terminal and internal conserved elements form dynamic RNA configurations that facilitate transcription will be studied, as outlined in the three goals listed below. 1. Determine minimal requirements for different cis-acting element combinations for Potato Virus X (PVX) RNA synthesis in vivo and in vitro. 2. Compare the relative contributions of the internal elements during PVX plus-strand RNA synthesis in vivo and in vitro. 3. Evaluate the use different methods to tag the viral RNA and test their potential utility for isolation of viral replication complexes. 4. Utilize information derived from objectives above to develop materials for classroom instruction and mentoring of students interested in agricultural research.

Project Methods
Methods for this project are outlined below according to the specific objectives. 1. Methods to determine the minimal requirements for different cis-acting elements in PVX RNA synthesis include molecular analyses of products isolated from infected plant cells. Mutant and wild-type viral RNA samples will be used to infect cells in order to determine which changes to regulatory elements have the greatest effect on virus replication. 2. Experimental approaches described above, along with comparison of quantitative data will be used to determine the relative contributions of the internal elements during PVX plus-strand RNA synthesis. 3. In order to evaluate different methods to tag the viral RNA, computer-simulated folding of different RNA tags in the context of the viral RNA will first be conducted. Once potential configurations are determined, specific tags will be cloned into the PVX RNA vectors and used to generate tagged viral RNA. These product RNAs will be further tested for their potential to enhance isolation of viral replication complexes from plant cells or in vitro transcription reactions. 4. Data obtained from methods described above will be evaluated in terms of relevance to the field of RNA biochemistry. As new information is obtained, it will be incorporated into lecture content (slides, videos, online materials) for students enrolled in classes within the College of Agriculture at NCSU and potentially off-campus.

Progress 11/22/13 to 09/30/18

Outputs
Target Audience:p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} The target audiences for this project include scientists in the field of virology who benefit from new insights derived from the outcomes of this project. Knowledge gained from our mechanistic studies will help to provide insights important for development of agricultural biotechnology, potentially benefiting farmers in making agricultural improvements. Students represent another large target audience in that the research from this project provides useful new data that can be relayed to students in the classroom, giving them insight into current research trends and real world examples in the context of an experimental framework. Educating students about plant virus research benefits the State of North Carolina by providing a highly qualified workforce and an educated public aware of the importance of scientific research to technology development in our state and the country. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Hemenway has received considerable training and opportunities for professional development as part of the DELTA IDEA grant initiative. How have the results been disseminated to communities of interest?The educational materials developed are utilized in both classroom and online courses every year. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Progress towards understanding goals #1-3 was limited by lack of research funds or personnel support. Goal #4: Considerable progress has been made on development of new classroom and online instructional materials. Dr.Hemenway developedInstructional materials, including videos, taped lectures and learning tools that are used for both classroom instruction and for online courses every year.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audiences for this project include scientists in the field of virology who benefit from new insights derived from the outcomes of this project. Knowledge gained from our mechanistic studies will help to provide insights important for development of agricultural biotechnology, potentially benefiting farmers in making agricultural improvements. Students represent another large target audience in that the research from this project provides useful new data that can be relayed to students in the classroom, giving them insight into current research trends and real world examples in the context of an experimental framework. Educating students about plant virus research benefits the State of North Carolina by providing a highly qualified workforce and an educated public aware of the importance of scientific research to technology development in our state and the country. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided development opportunities for teaching assistants. How have the results been disseminated to communities of interest?p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} Instructional resourceshave been made available to students enrolled in the classroom at NCSU, and also to online students at NCSU and across the country. What do you plan to do during the next reporting period to accomplish the goals?Further refinement of content and optimization of methods to foster' students critical-thinking skills.

Impacts
What was accomplished under these goals? Online and classroom course materials have been refined and adapted to optimize student learning.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences for this project include scientists in the field of virology who benefit from new insights derived from the outcomes of this project. Knowledge gained from our studies will help to provide insights important for development of agricultural biotechnology, potentially benefiting farmers in making agricultural improvements. Students represent another large target audience in that the research from this project provides useful new data that can be relayed to students in the classroom, giving them insight into current research trends and real world examples in the context of an experimental framework. Educating students about plant virus research benefits the State of North Carolina by providing a highly qualified workforce and an educated public aware of the importance of scientific research to technology development in our state and the country. Changes/Problems:Due to lack of support from NCSU or external funding agencies, the experimental aspects of the project have been severely limited. The instructional improvements will continue due to support from Delta and the Department of Molecular and Structural Biochemistry. What opportunities for training and professional development has the project provided?Dr. Hemenway was able to acquire online course development skills through her collaboration with the DELTA team. How have the results been disseminated to communities of interest?Dr. Hemenway teaches a classroom andonline version of BCH453/553 course. The online version services students who work in the Research Triangle Park and students who work and liveremotely from campus, in addition to students on campus.Dr. Hemenway's course is being utlized by DELTA as a template for otherfaculty and staff to utlize when they develop their online courses. What do you plan to do during the next reporting period to accomplish the goals?Dr. Hemenway will utilize the Moodle analytics and course evaluations to modify the content and navigation forthe coure online site to improve the learning format online. All content will be continually evaluated for both onlineand classroom sections.

Impacts
What was accomplished under these goals? Progress towards understanding goals #1-3 was limited by lack of research funds or personnel support. Goal #4: Dr. Hemenway developed new classroom and online instructional materials (included recorded lectures).

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audiences for this project include scientists in the field of virology who benefit from new insights derived from the outcomes of this project. Knowledge gained from our studies will help to provide insights important for development of agricultural biotechnology, potentially benefiting farmers in making agricultural improvements. Students represent another large target audience in that the research from this project provides useful new data that can be relayed to students in the classroom, giving them insight into current research trends and real world examples in the context of an experimental framework. Educating students about plant virus research benefits the State of North Carolina by providing a highly qualified workforce and an educated public aware of the importance of scientific research to technology development in our state and the country. Changes/Problems:Due to lack of support from NCSU or external funding agencies, the experimental aspects of the project have been severely limited. The instructional improvements will continue due to support from Delta. What opportunities for training and professional development has the project provided?Dr. Hemenway received considerable training and opportunities for professional development as part of the DELTA IDEA grant initiative. She was able to acquire online course development skills through her collaboration with the DELTA team. How have the results been disseminated to communities of interest?Dr. Hemenway launched the online BCH453/553 course in fall, 2015, and enable students taught in the classroom to also access the online course content. In addition, Dr. Hemenway's course is being utlized by DELTA as a template for other faculty and staff to utlize when they develop their online courses. What do you plan to do during the next reporting period to accomplish the goals?Because Dr. Hemenway taught both the online only version of BCH453/553 at the same time she taught the course in the classroom (with classroom students having same access to online materials), she will utilize the Moodle analytics interface to compare student performance under the two conditions. She will also analyze which materials were most frequently utilized by all students. In addiiton, further modifications to the online materials will be ongoing in order to improve the learning format online and in the classroom.

Impacts
What was accomplished under these goals? Progress towards understanding goals #1-3 was limited by lack of research funds or personnel support. Goal #4: Dr. Hemenway developed new classroom and online instructional materials (included recorded lectures). She launched the online version of BCH453/553 in fall of 2015 and also provided the same materials to students taking the course in the classroom.

Publications

Progress 11/22/13 to 09/30/14

Outputs
Target Audience: The target audiences for this project include scientists in the field of virology who benefit from new insights derived from the outcomes of this project. Knowledge gained from our mechanistic studies will help to provide insights important for development of agricultural biotechnology, potentially benefiting farmers in making agricultural improvements. Students represent another large target audience in that the research from this project provides useful new data that can be relayed to students in the classroom, giving them insight into current research trends and real world examples in the context of an experimental framework. Educating students about plant virus research benefits the State of North Carolina by providing a highly qualified workforce and an educated public aware of the importance of scientific research to technology development in our state and the country. Changes/Problems: Due to lack of support from NCSU or external funding agencies, the experimental aspects of the project have been severely limited. What opportunities for training and professional development has the project provided? Dr. Hemenway has received considerable training and opportunities for professional development as part of the DELTA IDEA grant initiative. How have the results been disseminated to communities of interest? The results will be disseminated online once the course is launched in Fall, 2015. In the meantime, various materials laready developed have been made available to students enrolled in in-class instruction at NCSU. What do you plan to do during the next reporting period to accomplish the goals? Onine course materials will be completed and assembled into the online delivery system prior to August 1, 2015.

Impacts
What was accomplished under these goals? Progress towards understanding goals #1-3 was limited by lack of research funds or personnel support. Goal #4: Considerable progress has been made on development of new classroom and online instructional methods. Dr. Hemenway was a recipient of a NCSU DELTA IDEA grant, which provided support for development of an online course that will be launched during the Fall of 2015. Instructional materials, including videos, taped lectures and learning tools have been developed for this course.

Publications