CHARACTERIZATION OF EMERGING VIRUSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1005716
• Project No.: KY012039
• Project Start Date: Jan 1, 2015
• Project End Date: Dec 31, 2019

Project Director
Goodin, M.

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Plant Pathology

Non Technical Summary
"Eight major crops (corn, soybeans, wheat, cotton, rice, barley, oats and canola) account for almost 95% of the total cropland used in the United States (USDA's National Agricultural Statistics Service (NASS)), and five major crops (soybeans, corn, cotton, sugar cane, and rice) account for almost 90% of the total cropland used in Brazil (FAPRI, Brazilian Ministry of Agriculture (CONAB), Brazilian Institute of Geography and Statistics (IBGE), and Institute for International Trade Negotiations (ICONE))." Fundamental principles of plant pathology predict that given the scale and similarity of Brazilian and U.S. farming systems emergence of pathogens in one of these countries poses a threat to agriculture in the other. Through collaborative efforts between these countries effective controls can be deployed. Building from recently established collaborations, this proposal seeks to enlarge research of emerging viruses that threaten agriculture. In addition to studies of pathogens themselves, the proposed research will provide research and teaching opportunities for University of Kentucky students interested in the multiple dimensions of sustainable agriculture.

Animal Health Component

0%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2232	1030	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2232 - Coffee;

Field Of Science
1030 - Cellular biology;

Keywords

coffee

genomics

virus

agriculture

international

Goals / Objectives
This proposal seeks to enlarge research of emerging viruses that threaten agriculture. In addition to studies of pathogens themselves, the proposed research will provide research and teaching opportunities for University of Kentucky students interested in the multiple dimensions of sustainable agriculture.Objective 1. Deep sequencing to determine CoRSV quasispecies cloud sizes and temperature-dependent changes in gene expression of infected Chenopodium quinoa.Objective 2. To compare the effect of gene expression induced by serologically distinguishable strains of Potato yellow dwarf virus.

Project Methods
We will follow upon our current experience gained in determination of the CoRSV and PYDV-C genomes, which are similar to methods used for discovery of other novel viral genomes, including a plant-adapted rhabdovirus. Guided by our time course assay (Fig.3), we will isolate total RNA (Qiagen) from mock- and CoRSV-inoculated plants that have been incubated at 28oC for 0, 10 or 30 days. As was successful for our prior microarray experiments, we will isolate RNA from inoculated and systemic leaves pooled from a minimum of five plants at each time point. We will define "systemic leaves" as the two newest fully expanded leaves on a plant that show veinal chlorosis indicative of CoRSV infections. The corresponding leaves from mock-inoculated leaves will serve as a control. To reduce the cost and size of datasets, we will conduct RNA-Seq analysis on systemic leaves at all time 28 oC points (six samples total). A corresponding set of samples from mock inoculated leaves from plants incubated at 26 oC, will serve as a control for identification of temperature-dependent changes in gene expression and splice variants (three samples). CoRSV-inoculated leaves from plants incubated at 28 oC for 10 and 30 dpi (2 samples total) will control for tissue-specific changes in gene expression. Total RNA will be isolated using the Qiagen RNeasy kit. Construction of cDNA libraries and sequencing will be performed by the Genome Informatics Facility, at Iowa State University (http://gif.biotech.iastate.edu). Sequence data will be generated using an Illumina HiSeq instrument, with is currently not available at the University of Kentucky. Transcriptome assembly will be done using the Trinity assembler. The resulting transcripts will be analyzed for differential gene expression; because the many differential expression packages have their own unique advantages and limitations, we will perform this analysis using several packages, including but not limited to Cuffdiff and DESeq. Data output will include a comparison of differentially expressed genes with regard to the two treatments (temperature and CoRSV inoculation). We will furthermore use software such as Cuffdiff, DEXSeq and MISO to search for and differentially analyze splicing variations, which are hypothesized to play a role in temperature-dependent changes in susceptibility to infection.Data output will include a comparison of differentially expressed genes with regard to the two treatments (temperature and CoRSV inoculation). We will also extract all CoRSV-derived transcripts from the datasets to determine if there is a temperature-dependent variation in the sequences that compose the viral quasispecies. This analysis will also reveal time and temperature dependent changes in viral titer and the relative ratios of viral mRNA transcripts to each other (Note: negative strand RNA viruses such as CoRSV and PYDV encode a RNA-dependent RNA polymerase that synthesizes poly-adenylated transcripts from genomic RNA templates that are similar in structure to host-cell mRNAs).

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Plant pathologists were reached through publication of scientific literature. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two postdoctoral sholars received training in microscopy, virus molecular biology, and preparation of manuscripts for publication. How have the results been disseminated to communities of interest?The research has been disseminated through publication in Plant Disease, a journal read frequently by the target audience of growers, exension agents, plant pathologists, virologists, and soybean seed producers. What do you plan to do during the next reporting period to accomplish the goals?This research has been used as preliminary data in a grant proposal submitted to the National Science Foundation to continue research on this and related viruses. Research grant proposals will be submitted to the USDA as well in the near future.

Impacts
What was accomplished under these goals? Characterization of soybean yellow shoot virus, an emerging potyvirus infecting soybean plants in Brazil A novel virus, capable of inducing severe symptoms in soybean, was first observed in experimental fields in Lavras-MG, Brazil, and named soybean yellow shoot virus. In a collaboration between the Figueira and Goodin laboratories, SoyYSV was completely sequenced and its structural, biological, and molecular properties were determined. Among the most common potyviruses present in Brazil, only SoyYSV induced local necrotic lesions in Carica papaya L. The complete genome of SoyYSV was determined to be 9,052 nucleotides, encoding a single ORF that is subsequently cleaved generating 11 proteins. SoyYSV was transmitted by Myzus persicae and Aphis gossipii, but lacks the HCPro domain required for aphid transmission in Potyvirus genus members. No seed transmission was observed. This report constitutes the first detailed characterization of SoyYSV. The ability of SoyYSV to infect soybean plants with genetic resistance to soybean mosaic virus (SMV) is of substantial concern, as it suggests the potential to cause losses in soybean. Fortunately, among some soybean cultivars genetic resistance to SoyYSV was identified, despite being susceptible to SMV. Studies with the purpose of mapping these resistance genes for a possible resistance pyramiding have already been started. A more detailed characterization of this virus has become important to guide development of control measures in the future.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Geraldino Duarte PS, Figueira AR, van Lent J, Galvino-Costa SBF, Farman M, Goodin M. 2019. Characterization of soybean yellow shoot virus, a new member of the family Potyviridae infecting soybean plants in Brazil. Plant Disease

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

Outputs
Target Audience:Mr. Chanyong Jang, graduate student in the Goodin lab gave an oral presentation at the 2016 South Eastern Regional Virology Conference. Goodin gave invited presentations at the 2016 Annual meeting of the American Phytopathological Society, the Brazilian Plant Pathology Society (in Maceio Brazil), and at the National Agricultural University in Fuzhou, China. Goodin presented seminars in the Plant Pathology Departments at the Universities of Kentucky and Florida, Auburn University, and South Dakota State University over the course of the reporting period. Results from these projects were discussed in two freshman classes at the University of Kentucky in order to stimulate interest in STEM-related careers and to increase science literacy among undergraduates. Changes/Problems:Chenopodium quinoa is not a particulalry tractable system for molecular genetics studies of virus-plant interactions (Objective 1). Therefore, we are adpating this virus for work in Nicotiana benthamiana system, which is much easier to mainupulate genetically and biochemically. To initiate this transition we are construcing a recombinant genetics system that will allow replication of CoRSV in N. benthamiana. What opportunities for training and professional development has the project provided?The molecular characterization of the second strain of Potato yellow dwarf virus, hereafter referred to as CYDV, was conducted by graduate student Mr. Chanyong Jang. An undergraduate research assistant, Ms. Kelly Sovacool, under supervision of Dr. Jaromczyk generated the reference sequence database for C. quinoa. Both students were able to present their research findings at professional meetings. Taken together, this aims of this project has resulted in training at both the graduate and undergraduate levels. Three undergraduates enrolled in the Agricultural & Medical Biotechnology program at the University of Kentucky we instrumental in the screening and identification of novel nuclear proteins, and thereby enhancing their undergraduate research experience. How have the results been disseminated to communities of interest?Mr. Chanyong Jang, graduate student in the Goodin lab gave an oral presentation at the 2016 South Eastern Regional Virology Conference. Goodin gave invited presentations at the 2016 Annual meeting of the American Phytopathological Society, the Brazilian Plant Pathology Society (in Maceio Brazil), and at the National Agricultural University in Fuzhou, China. Goodin presented seminars in the Plant Pathology Departments at the Universities of Kentucky and Florida, Auburn University, and South Dakota State University over the course of the reporting period. Results from these projects were discussed in two freshman classes at the University of Kentucky in order to stimulate interest in STEM-related careers. What do you plan to do during the next reporting period to accomplish the goals?We are constructing developing a reverse-genetics system for PYDV and CoRSV, which will allow biologically relevant domains in these viruses to be mapped. We are continuing to characterize novel nuclear proteins in the context of virus infections in order to discect molecualar basis of virus-plant interactions.

Impacts
What was accomplished under these goals? The genome of a second strain of Potato yellow dwarf virus was determined using next-generation sequencing, and published (See Jang et al., 2017). We have also constructed and screened an N. benthamiana cDNA library to identify plant proteins that associate with the nucleus. While this strategy is a deviation from our stated two objectives, it has provided >180 novel proteins that are associated with the plant nucleus, where CoRSV and PYDV replicate. The localization patterns of many of these proteins changes in response to virus infection. Therefore, in addition to changes in gene expression (Objective 2), we can extend these studies to changes in localization.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Jang C, Wang R, Wells J, Leon F, Farman M, Hammond J, Goodin MM. Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus. J Gen Virol. 2017 Jun;98(6):1526-1536. doi: 10.1099/jgv.0.000771. Epub 2017 Jun 21. PubMed PMID:28635588; PubMed Central PMCID: PMC5656794.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Mapping of functional domains in the matrix protein of Potato yellow dwarf virus (2017) G. Anderson, R. Wang, C. Jang, M.Goodin. Journal of General Virology
• Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Protein Localization and Interaction Studies in Plants: Toward Defining Complete Proteomes by Visualization (2017) M. Goodin. Advances in Virus Research
• Type: Book Chapters Status: Submitted Year Published: 2018 Citation: The rise and rise of Nicotiana benthamiana: a plant for all reasons. (2017) J. Bally, H. Jung, C. Mortimer, F. Naim, J. Philips, R. Hellens, M. Goodin, & P. M. Waterhouse. 2018. Annual Review of Phytopathology

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

Outputs
Target Audience:A wide range of audiences were reached during this reporting period. Our published survey of the distribution and population structure of Coffee ringspot virus was the first to illustrate to the scientific community that this virus infects plants in one hundred percent of the area surveyed, which compromises 75% of the total coffee growing region in Brazil where 40% of the world supply of coffee is produced. These findings were elaborated at a workshop on viral evolution held at The Pennsylvania State University, where world experts in virus population genetics were in attendance. The survey itself was conducted with assistance from two students enrolled in the Agricultural Biotechnology Program at the University of Kentucky, and a graduate student from the Department of Plant Pathology at the Universidade Federal de Lavras. This provided opportunities to engage communities of students in Brazil and the United States. Invited seminars related to this work were given at both the 2015 and 2016 Annual Meetings of the American Phytopathological Society, which were attended by the full spectrum of academic and industry professionals. Changes/Problems:A downturn in the Brazilian economy resulted in an inability to renew funding that supported collaborations with research partners in Brazil. This will severely impact our work on CoRSV. What opportunities for training and professional development has the project provided?The molecular characterization of the second strain of Potato yellow dwarf virus, hereafter referred to as CYDV, was conducted by graduate student Mr. Chanyong Jang. An undergraduate research assistant, Ms. Kelly Sovacool, under supervision of Dr. Jaromczyk generated the reference sequence database for C. quinoa. Both students were able to present their research findings at professional meetings. Taken together, this aims of this project has resulted in training at both the graduate and undergraduate levels. How have the results been disseminated to communities of interest?Ms. Kelly Sovacool presented a poster of this work at the 2016 National Council on Undergraduate Research. Mr. Chanyong Jang gave an oral presentation at the 2016 South Eastern Regional Virology Conference. Goodin gave invited presentations at the 2016 Annual meeting of the American Phytopathological Society, the Brazilian Plant Pathology Society (in Maceio Brazil), and at the National Agricultural University in Fuzhou, China. What do you plan to do during the next reporting period to accomplish the goals?The immediate plan to complete the characterization of CYDV and publish a manucript related to this work in the Journal of General Virology. Both of the project aims would benefit from the availability of infectious clones of both CoRSV and CYDV. Therefore, this work is in progress to generate recombinant genetic systems for these two viruses.

Impacts
What was accomplished under these goals? At the time this project was initiated, a reference genome for the construction of a Chenopodium quinoa sequence database was not available. Together with Dr. Jerzy Jaromczyk in the University of Kentucky's Computer Science Department, we have established a reference database generated from next-generation sequence data of CoRSV-infected C. quinoa. Relevant to Objective 2, the sequence of a second strain of Potato yellow dwarf virus was determined using NGS technology. A manuscript describing this work will be published next year.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Ramalho TO, Figueira AR, Wang R, Jones O, Harris LE, Goodin MM. Detection and survey of coffee ringspot virus in Brazil. Arch Virol. 2016 Feb;161(2):335-43.doi: 10.1007/s00705-015-2663-0.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Tripathi D, Raikhy G, Goodin MM, Dietzgen RG, Pappu HR. In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins. PLoS One. 2015 Mar 17;10(3):e0118973. doi: 10.1371/journal.pone.0118973.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Gong X, Hurtado O, Wang B, Wu C, Yi M, Giraldo M, Valent B, Goodin M, Farman M. pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins. Mol Plant Microbe Interact. 2015 Feb;28(2):107-21.

Progress 01/01/15 to 09/30/15

Outputs
Target Audience:Presentations to the following target audiences were made during the present reporting period. March, 2015: Eighth Workshop in Virus Evolution, The Pennsylvania State University, State College, PA April 2015: Departmental Seminar: Universidade Federal de Lavras (Brazil). Title "Characterization of coffee ringspot virus in Brazil" October 2015: 2nd international SFB796 meeting on "Mechanisms of microbial host cell manipulation: from plants to humans", Erlangen, Germany, Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training in next-generation sequencing data analysis has been provided to one graduate student in the Plant Pathology Department (Mr. Chanyong Jang) How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will evaluate changes in plant gene expresion levels in response to virus infection. Further, we will determine the amount of sequence space occupied by virus-derived mRNA transcripts within infected cells.

Impacts
What was accomplished under these goals? We have generated three Illumina RNA-Seq databases to determine the changes in gene expression in response to infection of plants by coffee ringspot virus (relative to Objective 1). These data are currently being processed by collaborators in the Computer Science Department (University of Kentucky).

Publications