Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience of Dr. Rosa's efforts are graduate and undergraduate students in the courses she teaches at Penn State (introductory plant pathology and plant virology)where she uses her research results to provide students with real examples of modern day plant virology. In addition, plant pathologists were presented research results from Dr. Rosa's lab in the Department of Plant Pathology at the University of Kentucky. The work from the Roossinck lab targets the virology research community, in particular those working on virus evolution, and the evolution of virulence in plant viruses. Changes/Problems:One of Rosa's graduate students, Natali Ozber, graduated and Rosa could not find funding to continue the work on virus movement protein function in plants. What opportunities for training and professional development has the project provided?Rosa Lab:Kaixi Zhao graduated with a Ph.D. in Plant Pathology in November 2019. Asifa Hameed (Ph.D. student, Entomology Department) passed her Ph.D. defense in September 2020. Other students who were trained in the Rosa lab during this period of performance were: Chauncy Hinshaw (Ph.D. student, Plant Pathology and Environmental Microbiology Department), Tareq Tariqul (Ph.D. student, Plant Pathology and Environmental Microbiology Department), Alex Varaksa (M.S. student, Plant Pathology and Environmental Microbiology Department), Powen Pai (undergraduate, Plant Science),Crosley Kudla-Williams (undergraduate, Plant Science), and Laine Huckeberger (undergraduate, Plant Science). Rimnoma Serge Ouedraogo completed the required work for his Ph.D. thesis, and defended his thesis in Burkina Faso in December of 2019. He received his Ph.D. with honors. How have the results been disseminated to communities of interest?Dr. Rosa: one article was published in 2020. Dr. Roossinck presented the following invited talks: "Virus Ecology: Lessons from 40 years of Research,"Oregon State University, October 2019 "Viruses on the Mutualism-Antagonism Lifestyle Continuum"Jacque Monod Virus Evolution workshop, Roscoff, France, October 2019 Dr. Ouedraogo presented his results in a seminar. What do you plan to do during the next reporting period to accomplish the goals?The Rosa lab will: Continue to identity the molecular mechanisms and pathways involved in plant defenses against biotic and abiotic stresses, and use plant mutants to narrow-down specific genes involved in these processes. Continue to collect and analyze data on the effect of weather variables such as temperature, light intensity, and rainfall on the effect of leafroll disease on grapevines in different climatic regions of Pennsylvania. We will investigate the interactions of plant pathogens and invasive sucking insects such as the spotted lanternfly on plant defenses in grapevines. Provide Forest Service with RPA technology to test elm associated phytoplasmas directly in the field, as proof that the technology we established for tospoviruses can be extended to other unculturable plant pathogens. Dr. Roossinck:Complete writing and publication of a paper on the structure of cucumber mosaic virus.

Impacts
What was accomplished under these goals? 1. Dr. Rosa. Nothing new to report for this objective. 2. Dr. Rosa. Organic compounds based on chlorophyll and commercialized by SunCor Canada were used to identify plant pathways that regulate responses to biotic and abiotic stresses via RNASeq. The regulation of a subset of the genes identified by RNASeq was confirmed by real time Polymerase chain reaction (PCR). A study on the effect of local weather conditions on grapevine productivity when grapes are infected with viruses have been established. 3. Dr. Rosa. We established that while plants serve as permissive hosts during mixed infections of orthotospoviruses, vectors serve as a bottleneck and favors the transmission of one virus over the second, while maintaining both viruses in the pathosystem. 4. Dr. Rosa. We have started the adaptation of the Recombinase Polymerase Amplification (RPA)assay we developed for tomato spotted wilt virus as technology to detect, aside from viruses, other unculturable plant pathogens (phytoplasmas) in forest settings. 5. Nothing to report from Dr. Roossinck's lab.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhao, K., C. Rosa. 2020. Thrips as the Transmission Bottleneck for Mixed Infection of Two Orthotospoviruses. Plants 2020, 9, 509. doi:10.3390/plants9040509
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Thapa,V., Tuner, G. and Roossinck, M.J. Phylogeographic analysis of Psuedogymnoascus destructans partitivirus-pa explains the spread dynamics of white-nose syndrome in North America. PLOS Pathogens

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Rosa's efforts targeted audience included: plant pathologists and plant virologists, graduate students in plant biology, plant pathology and plant science, by giving seminars, lectures, presenting at conferences and publishing the results of her lab work. She also presented her research goals to APHIS PGQP scientists. The work from the Roossinck lab targets the virology research community, in particular those working on virus evolution, and the evolution of virulence in plant viruses. Changes/Problems:The Roossinck lab will be phasing out this research as Dr. Roossinck will begin a phased retirement (35%) in 2020, and take full retirement at the end of 2020. Rosa did not encounter any problem during this reporting period. What opportunities for training and professional development has the project provided?Natali Ozber graduated with a Ph.D. in Plant Biology in Spring 2019. Juan Francisco Iturralde Martinez defended his M.S. thesis in Plant Pathology and submitted his thesis dissertation to his committee in Fall 2019. Other students who were trained in the Rosa lab during this period of performance were: Kaixi Zhao (Ph.D. student, Plant Pathology and Environmental Microbiology Department), Chauncy Hinshaw (Ph.D. student, Plant Pathology and Environmental Microbiology Department), Tareq Tariqul (Ph.D. student, Plant Pathology and Environmental Microbiology Department), Asifa Hameed (Ph.D. student, Entomology Department), Alex Varaksa (M.S. student, Plant Pathology and Environmental Microbiology Department), Simon Smith (undergraduate at Juniata College), Powen Pai (undergraduate, Plant Science) and Crosley Kudla-Williams (undergraduate, Plant Science). Rimnoma Serge Ouedraogo completed the required work for his PhD thesis and this has been submitted to his committee. He anticipates defending his thesis in Burkina Faso in November of 2019. How have the results been disseminated to communities of interest?Four articles were published in 2019. The results of this research were presented in poster format to the 10th Workshop in Virus Evolution, 2019, State College, Pennsylvania; and to the Plant Health 2019, American Phytopathological Society, Cleveland, OH. They were also published as conference abstract in Phytopathology. What do you plan to do during the next reporting period to accomplish the goals?Initiate the investigation to test if some of the hosts factors identified as candidate interactors with Ourmia melon virus movement protein are real interactors (Rosa). Identify molecular mechanisms and pathways involved in plant defenses against biotic and abiotic stresses, and test if bio-stimulants can boost plant defenses and how (Rosa). Finish analyzing data provided by the Rosa lab experiments using dual virus infection and publish them in peer reviewed journals (Rosa). Establish collaborations with APHIS PGQP to provide nanotechnology to entrap viruses of interest to PGQP (Rosa). The major goal for the Roossinck lab for the next reporting period is to complete the publication of all results.

Impacts
What was accomplished under these goals? 1. As proven in the Rosa lab, Ourmia melon virus movement protein has an unusual intracellular distribution, including ER, TGN, plasma membrane and plasmodesmata, and possibly interacts with a myriad of plant host factors. In this reporting period, we performed pull-down assays coupled with GC/MS/MS to identify candidate host factors in Arabidopsis thaliana that interact with the Ourmia melon virus movement protein. Some of these factors are involved in vesicular trafficking (ARA 6), transport (V-type proton ATPase subunit B), chaperones (BiP1/2) and proteolysis (SASP). 2. Tomato spotted wilt virus (TSWV) infection rate and severity increases under draught conditions, and the administration of Jasmonic Acid inhibits viral infection under normal and draught conditions. Small RNA profile is altered in plants infected by TSWV under induced RNAi protection, compared with TSWV infected control plants. 3. Plant responses including the production of virus specific small RNAs and the regulation of plant miRNAs vary in plants infected by Tomato spotted wilt virus or Impatiens necrotic spot virus, or by both viruses, revealing that plants respond differentially when infected by one virus or two. These responses are specific for each virus species, and are intermediate in plants infected with the two viruses. 4. An assay for in-situ detection of Tomato spotted wilt virus from crude plant extracts using Reverse Transcriptase- Recombinase Polymerase Amplification (RT-RPA) in endpoint and real-time was developed by the Rosa lab. This assay can be used by growers directly in the field, since it does not require the use of equipment and it works at room temperature. The developed assay resulted in superior sensitivity and can work on different strains of TSWV and different plant species. 5. We solved the structure of the Cucumber mosaic virus Fny-strain virus particle to 3.5 angstroms by cryo-EM. The RNA was clearly visualized in the structure. Interestingly, the residues that are changed by selection after some silent mutations are not on the internal surface, but rather on the exterior of the capsid.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ouedraogo, R.S., J.S. Pita, I.P. Somda, O. Traore, and M.J. Roossinck. 2019. Impact of cultivated hosts on the recombination of Cucumber mosaic virus. Journal of Virology, 93: e01770-18
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Walls, J., Rajotte, E. G., & Rosa, C. (2019). The past, present, and future of Barley yellow dwarf management. Agriculture, 9(1), 23.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ozber, N., Margaria, P., Turina, M., Anderson, C. T., & Rosa, C. (2019). Dissecting the intercellular trafficking of the movement protein of Ourmia melon virus. bioRxiv. DOI: 10.1101/724716.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Iturralde Martinez, J. F., & Rosa, C. (Supervised Student Author) (2019). In-situ detection of Tomato spotted wilt orthotospovirus from crude plant extracts using Reverse Transcriptase- Recombinase Polymerase Amplification (RT-RPA) in endpoint and real-time. bioRxiv. DOI: 10.1101/720623.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ouedraogo, R.S., S. Hafenstein, and M.J. Roossinck Importance of CMV genome RNA structure and CP stability revealed by cryo-TEM. 10th Workshop in Virus Evolution, 2019, State College, Pennsylvania
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhao, K., & Rosa, C. (2019). "Characterization of the small RNA profiles of Impatiens necrotic spot orthotospovirus from infected Nicotiana benthamiana," Phytopathology. (109, 27-28).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhao, K., & Rosa, C. (2019). "Characterization of the small RNA profiles of Impatiens necrotic spot orthotospovirus from infected Nicotiana benthamiana," Northeastern APS, American Phytopathological Society, State College, PA, Accepted. Regional.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Zhao, K., & Rosa, C. (2019). "Characterization of the small RNA profiles of Impatiens necrotic spot orthotospovirus from infected Nicotiana benthamiana," Plant Health 2019, American Phytopathological Society, Cleveland, OH, Accepted. National.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Hinshaw, C. R., Lopez-Uribe, M. M., & Rosa, C. "Identification of host factors interacting with the movement protein of Ourmia melon virus in Arabidopsis," Plant Health 2019, American Phytopathological Society, Cleveland, OH, Accepted. National.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Ozber, N., Margaria, P., Anderson, C. T., Turina, M., & Rosa, C. (2019). "Identification of host factors interacting with the movement protein of Ourmia melon virus in Arabidopsis," Plant Health 2019, American Phytopathological Society, Cleveland, OH, Accepted. National.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Iturralde Martinez, J. F., & Rosa, C. (2019). "In-situ detection of Tomato spotted wilt orthotospovirus from crude plant extracts using Reverse Transcriptase-Recombinase Polymerase Amplification (RT-RPA) in endpoint and real-time," Plant Health 2019, American Phytopathological Society, Cleveland, OH, Accepted. National.

Progress 07/01/18 to 09/30/18

Outputs
Target Audience:During this current reporting period, the target audience reached included plant virologists and viral evolutionists, in relation to our work on Cucumber mosaic virus, virologists, graduate students and postdocs working at Penn State, and participants of the International Congress of Plant Pathology held in Boston in 2018. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?R. Serge Ouedraogo has continued his training towards a PhD degree in Plant Virology. He plans to return to West Africa to make a greater contribution to International Agriculture when he completes his degree (to be awarded by the University of Burkino Faso). N. Ozber, K. Zhao, F. Iturralde, J. Walls have progressed their training toward their graduate degrees, and showcased posters on their research at the International Congress of Plant Pathology, where they received valuable feedback and gained invaluable experience. N. Ozber also gave a research talk at the Virology@PSU group. How have the results been disseminated to communities of interest?Dissemination includes a research talk presented at the American Society for Virology annual meeting in July 2018, by Mr. Ouedraogo, one publication in Journal of Virology, 5 posters presentation at the International Congress of Plant Pathology in Boston, one research talk at the Virology@PSU group. What do you plan to do during the next reporting period to accomplish the goals?We will continue to elucidate the plant factors necessary for short distance movement of viruses in plants, focusing on plant structural proteins and secretory pathways. We will also investigate if movement proteins cause changes in plant plasmodesmata. We will assess if tomato mutants in genes involved in the plant jasmonic pathway are more or less resistant to viral infection, when subjected to drought stress. We will measure thrips ability for transmission of co-infecting viruses. We are planning to couple the use of nanotechnology for virus enrichment with field diagnosis tools and with next generation sequencing. We have initiated cryo-EM work to examine the changes in viral structure when both silent mutations are made in the RNA.

Impacts
What was accomplished under these goals? In this timeframe, we were able to make good progress on all the Objectives we had set in our original project proposal, and to present our results either in published papers or at conferences (see 'products' section). Specifically, and summarizing our accomplishments based on the order of the original proposed objectives, we made significant advances in the elucidation of plant virus movement for Ourmia melon virus (OuMV), a virus with a mixed 'tubule guided,' and 'non-tubule guided' cell to cell movement. In fact, our experiments confirmed by confocal microscopy and by the use of chemical inhibitors that the OuMV movement protein interacts with the plant endoplasmic reticulum, as well as with the plant secretory pathway. Looking at plant responses during the very early stages of virus infection (4 hours post inoculation), we found that tomatoes respond to inoculation of Tomato spotted wilt virus (TSWV) by downregulating genes in the jasmonic acid pathway, as expected, but we surprisingly found that this hormonal pathway is also interconnected to the abscisic acid pathway by the expression of a ABA gene that has not been found to be involved in this cross-talk before. Since this cross-talk was particularly evident in virus resistant plants, we need further investigation of this phenomenon to confirm our preliminary results. We also looked at differences in behavior of vectors exposed to plants infected by single or a mixture of tospoviruses and found that the vectors can distinguish between the two types of infection and will choose to oviposit more in plants infected with two viruses, compared to plants infected with one virus, or healthy plants. This result could have implication on virus acquisition by first instars and on transmission of mixed infection. Our work on the use of nanotechnology in the form of carbon-filter-devices for capture of viral particles is also proceeding well, and we were able to use the nanodevices to capture particles of tospoviruses from infected tomatoes and to use them for real time amplification. We optimized aspects of the nanotube assembly and gained more standardized and predictable devices that we successfully used in TSWV infected tomatoes fields in California. Thus, we can now produce devices that work also under field-conditions. We finally investigated the role of large-scale synonymous substitutions present in the RNA 3 of Cucumber mosaic virus (CMV) and showed how they facilitate amino acid mutations in the virus coat protein. The capsid protein of CMV enable virus transmission via aphids. We also found that plant hosts influence CMV recombination.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mochizuki, R. Ohara, M.J. Roossinck. 2018. Large-scale synonymous substitutions in cucumber mosaic virus RNA 3 facilitate amino acid mutations in the coat protein. J. Virol. 92:e01007-18
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: R.S. Ouedraogo, J.S. Pita, I.P Somda, O. Traore, M.J. Roossinck. 2018. Impact of cultivated hosts on the recombination of Cucumber mosaic virus. J. Virol.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Natali Ozber, Charles Anderson, Massimo Turina and Cristina Rosa. Dissecting the intercellular trafficking of the movement protein of Ourmia melon virus. Poster 25-P. ICPP, Boston July 30, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Juan Francisco Iturralde Martinez, Cristina Rosa, Christopher Rotella, Mauricio Terrones and Edwin Rajotte. Application of a carbon-nanotube filter that traps and concentrates virions to improve the limits of detection of Tomato spotted wilt virus in tomato. Poster 327-P. ICPP, Boston July 30, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kaixi Zhao and Cristina Rosa. Are vectors the bottleneck for Orthotospoviruses' fitness during mixed-infection? Poster 391-P. ICPP, Boston July 30, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Joseph Walls III and Cristina Rosa. Involvement of hormone pathways in early onset of TSWV resistance. Poster 1199-P. ICPP, Boston August 1, 2018.