Progress 01/15/22 to 01/14/23
Outputs
Target Audience:Our results obtained in our project is of interest to stakeholders, industry and scientists. We expect to develop an ELISA kit for CLRDV and that companies specialized in the commercialization of ELISA kits such as Agdia and Nano Diagnostics get an interest to commercialize it so that plant diagnostic clinics in the southern United States and other countries benefit fromthis product. We have active and constant communication with Cotton Inc and the National Cotton Council and our results are being opportunely delivered to them. Changes/Problems:It has been challenging for our team to recruita technician on the project. After two rounds of advertising for the position, no suitable candidate was found. However, with a postdoctoral associate, a graduate student, and two junior trainees, the research has advanced significantly.I now plan to hire a scholar to assist with the work via experiential learningthrough the ORISE program, which is available to USDA ARS scientists. What opportunities for training and professional development has the project provided?Currently, this project is being actively managed by the PI, Michelle Heck, and a post-doctoral research scholar that was hired through Cornell University, Dr. Alejandro Olmedo-Velarde. Alejandro has been leading the development of the different research lines for this project and has been mentoring Michael West-Ortiz, a recent PhD student in Michelle Heck laboratory, who has a project related to the characterization of virus populations in CLRDV-infected plants as part of his PhD. Indeed, Michael has obtained first-hand experience characterizing plant viruses both at the biological and molecular level. Additionally, Alejandro has mentored a Cornell undergrad, Hayk Shakhzadyan, and a TST-BOCES New Visions Life Sciences student from Ithaca High School, Emma Pollock. Both Hayk and Emma were trained by Alejandro in biological assays working with CLRDV and the cotton aphid vector as well as in the necessary serological and molecular tools to study this complex pathosystem such as ELISA and RT-PCR. Alejandro had the opportunity to attend to the Cotton Pathology Tour that took place in Memphis, TN in September 2022 where he not only learnedmore about cotton pathology and CLRDV by observing diseased cotton field, and collecting samples from CLRDV-symptomatic plants from fields in TN, AR and MS; but also he had the opportunity to network with other plant virologists working with CLRDV as well as cotton pathologists and breeders. The graduate studentMichael recently had the opportunity to attend to the WERA20 meeting in Beltsville, MD, organized by USDA APHIS, where he presented his results in his project regarding the characterization of virus populations from CLRDV-infected plants from different states. Since several plant virologists working with different fruit trees and ornamentals attend to the yearly meeting of WERA20, Michael had a unique opportunity to showcase his CLRDV-related work and also network with plant virologists working in the academia and government. How have the results been disseminated to communities of interest? Alejandro and Michelle actively participate in a 'CLRDV monthly discussion group' that takes place through zoom and is led by the National Program Leader Tim Widmer. This monthly discussion involves researchers from academia and government as part of the 'CLRDV task force'. Alejandro and Michelle have opportunely shared their results with the group which were related to the current discussion topic. Also, Alejandro presented to the group the aims and results obtained in the project as of February 2023. Michelle has given several different talks, including at the USDA ARS All Hands meeting at the Cotton Beltwide Conference, in New Orleans, LA in Jan. 2023. Alejandro also gave several presentations regarding his CLRDV-related research in different seminars: USDA-All Center Meeting for the Robert Holley Center in December 2022 Research seminar to the Boyce Thompson Institute in April 2023. The finding of the new DNA virus in cotton was communicated to Drs. Tim Widmer and Roy Scott (National Program Leaders) and Don Parker, Vice President of the Technical Services of the National Cotton Council. Michael presented a short talk regarding his research on the characterization of virus populations in CLRDV-infected plants to the scientific community attending the WERA20 meeting in Beltsville, MD. The scientific community was mainly comprised by virologists working in fruit crops and ornamentals in other states in both government and academia. What do you plan to do during the next reporting period to accomplish the goals? Using the nanobodies against CLRDV which bind to the coat protein and N-RTD and the CLRDV infectious clones developed, we expect to be able to enrich for virus fractions containing both plant and aphid proteins interacting with CLRDV. Using mass spectrometry techniques on these fractions will allow us to identify cotton and cotton aphid proteins that bind to the virus. Using the constructs to express both nanobodies and CLRDV N-RTD (wild-type and mutants), transgenic lines of cotton will be obtained by outsourcing its production in the Plant Transformation Lab at NC State University.
Impacts
What was accomplished under these goals?
This year focused heavily on team building and establishing the research tools required for the project.A postdoctoral scientist, Dr. Alejandro Olmedo-Velarde, was hired to work on the project.A Cornell graduate student, Michael West-Ortiz, was recruited to work on the project. Additionally, a Cornell Universityundergraduate student Hayk Shakhzadyan,and an Ithaca High School student in the TST BOCES New Visions Program were recruited to work on the project. A reliable diagnostic RT-PCR assay for CLRDV was developed. Using full and almost-full genome sequences of CLRDV available in GenBank as of September 2022, a multiple nucleotide sequence alignment was performed which allowed us to identify conserved regions along the RdRp and CP genes. By using the freely-available web-based primer design software, Primer3 (https://primer3.ut.ee), several primer sets targeting the RdRp and CP genes were designed. After optimization, single-tube nested RT-PCR assays were implemented to be specific for CLRDV RdRp and CP genes. These diagnostic RT-PCR assays have been used for reliable detection of the virus in our experiments. Camelid antibodies (nanobodies) against the CP and N-RTD of the virus were produced in collaboration with the Center for Molecular Medicine atthe University of Kentucky. In collaboration with Joshua Chappie lab (Cornell University), recombinant virus proteins (CP and N-RTD) were purified from batches ofEscherichia colihighly expressing them. The pure proteins were sent to the Center for Molecular Medicine (University of Kentucky) for nanobody production. A total of five nanobodies were received: two against CLRDV CP and three against CLRDV N-RTD. Their binding activity against the virus proteins was validated using indirect ELISA as well as DAS-ELISA (see below). Two different ELISA protocols were developed and are in validation for diagnosis of CLRDV. Using the five nanobodies detailed above, and anti-camelid conjugate HRP, an indirect ELISA assay was developed to be specific for CLRDV proteins: CP and N-RTD. Using the five nanobodies detailed above, and anti-PLRV conjugate AP, which was found to cross-react with CLRDV, a DAS-ELISA assay was developed to be specific for CLRDV proteins: CP and N-RTD. Optimization of both ELISA formats (indirect and DAS) is being performed so that an ELISA kit can be potentially commercialized and benefit plant diagnostic clinics in Southern states where cotton is grown. An invention disclosure was reviewed by the USDA ARS Life Sciences Patenting Committee and recommended for a provisional patent submission, which is currently in preparation. An infectious clone for CLRDV (Argentina isolate) was developed. Using the genome of an Argentinian isolate of the virus (GenBank GU167940.1), which was synthesized by CodexDNA, an infectious clone was developed by inserting this genome sequence into the binary vector pJL89. The binary vector pJL89 has been widely used for the development of virus infectious clones. Through agrobacterium-mediated delivery usingAgrobacterium tumefaciensLBA4404, it has been demonstrated this infectious clone can infectNicotiana benthamianaand a variety of cultivars ofGossypium hirsutum. Infectivity of this infectious clone will be further evaluated in three other commercial cotton species:G. barbadense,G. arboretumandG. herbaceum. Also, an infectious clone of an American isolate of CLRDV is being developed using the same approach detailed above. Heck Lab APHIS PPQ permits have been modified to reflect a de-facto introduction of the Argentinian CLRDV isolate. The infectious clone and nanobody tools will be used to identify aphid and plant proteins involved in CLRDV infection and transmission. It has been determined that different colonies of the cotton aphid,Aphis gossypii, originary from different locations possess different vector competence for CLRDV. Using leaf disc assays, it was determined that colonies originary from New York, and Mississippi transmitted a CLRDV isolate from Mississippi with an efficiency of 25%; whereas colonies that originated from Alabama and Tennessee, presented a transmission efficiency of 12.5%. These results are being validated by repeated experimentations. Molecular constructs are being developed to produce transgenic cotton expressing the wild-type NRTD from CLRDV (TX isolate), and mutants, as well as two out of the five nanobodies detailed above. Once the constructs are completed, they will be sent to the Plant Transformation Lab at North Carolina State University to produce transgenic cotton (G. hirsutum) lines.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Heck, M. Cotton leafroll dwarf virus research - establishing an emerging pathosystem research program. USDA ARS All Hands Meeting, Cotton Beltwide Conference, New Orleans, LA. 10 Jan 2023.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Olmedo-Velarde, A. Approaches to study aphid-transmitted viruses in cotton. Boyce Thompson Institute Monday Morning Seminar Series. Ithaca, NY, 10 April 2023.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Pollock, E. Aphid-virus interactions in cotton. New Visions Life Sciences Symposium. Cornell University, Ithaca, NY 15 May 2023.
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:Cotton leafroll dwarf virus is a polerovirus infecting US grown cotton. There is no cure for viral infection in plants and the economic impacts in cotton are not known. Research in this grant have focused on briding an organismal and molecular understanding of virus-host-vector interactions.There have been several target audiences for our work during this reporting period. Dr. Heck has been participating in a national CRLDV working/coordinationgroup reporting research results from this project. Included are scientists and administrators from USDA ARS Office of National Programs, Auburn University, Mississippi State University and the University of Georgia. Dr. Heck gave a seminar on her research in the Department of Plant and Environmental Sciences and at the University of California atRiverside. Importantly, industry stakeholders have been included in project interactions, including Cotton, Inc. and an industry partner interested in commercializing a diagnostic antibody test kit for detection of Cotton leafrolldwarf virus. Additionally, graduate and undergraduate students at Cornell University have been a major audience, working on developing research for this pathosystem. Changes/Problems:There is no major change to the approach, but I did experience a difficulty in hiring personnel on this project due to internal challenges within USDA ARS regarding hiring and the covid-19 pandemic. Fortunately, I recently found an outstanding postdoctoral candidate to hire on this proejct.I will reach out to my program officer to discuss plans for hiring the postdoc. What opportunities for training and professional development has the project provided?To date, one postdoctoral associate, two graduate students and an undergraduate student have been trained on this project. The PI, Dr. Heck, attended a two week long leadership training offered by American University. While grant funds were not used to support that training, the training has already benefitted Dr. Heck's ability to lead the research on this project. How have the results been disseminated to communities of interest?Dr. Heck has given a number of different seminars to Universities and updates to the Office of National Programs. She is an active participant in the monthly CLRDV working group meetings organized by the USDA ARS Office of National Programs (which also includes University cooperators). What do you plan to do during the next reporting period to accomplish the goals?Hiring personnel on this project has been slow due to the Covid-19 pandemic. Excitingly, anewpostdoctoral associate has been recruited to work on the project and will be starting work on the project in August 2022.
Impacts
What was accomplished under these goals?
Research has focusedon the architecture, uptake, and insect-mediated transmission of Cotton leafroll dwarf virus (CLRDV), a polerovirus that is related to the aphid transmittedluteovirusesand enamoviruses (collectively referred to asP/E/L viruses).P/E/L virusesinfect and decimate economically important staple food crops. These pathogens replicate exclusively in the vasculature of plant hosts and are transmitted by aphids during feeding. Truncation and mutagenesis experiments suggest that coat protein (CP) and the N-terminal portion of an extended readthrough domain on the viral capsid surface (N-RTD) is required for this process. To identify the molecular determinants of luteovirid transmission by aphids, wegenerated soluble N-RTD and CP constructs from cotton leafroll dwarf virus(CLRDV) that can be purified on the milligram scale for structural studies. Wehave obtained preliminary X-ray diffraction data for the N-RTD of CLRDV, and are currently optimizing crystalization conditionsfor structural determination in collaboration with Professor Joshua Chappie at Cornell University. Concurrent with structural studies, we single domain antibodies (from alpacas)against our recombinantpotato leafroll virus(PLRV) N-RTD and CLRDV CP as a strategy to neutralize viral transmission.Using MicroScale Thermophoresis (MST) and Analytical Size Exclusion Chromatography, wecharacterized the interactions and tested the affinity of these nanobodies for the N-RTDs and CPs of PLRV and CLRDV. Research is ongoing and these findings will yield novel insights into the structural organization of P/E/L viruses and provide new tools to probe the mechanisms of vector-mediated viral transmission. Work is ongoing in collaboration with CodexDNA, a synthetic biology company, to generate an infectious clone for CLRDV. Laboratory studies with CLRDV have demonstrated that the virus is slow to move systemically in greenhouse growncotton and aphid transmission is not efficient.An infectious clone will greatly accelerate molecular research into CLRDV interactions with its cotton host and aphid vector. Research on aphid transmission parameters is also ongoing. Two assays are currently being developed: a leaf disk assay and a whole plant assay. The leaf disk assay is high throughput and will allow a rapid assessment of CLRDV transmission by aphids. The whole plant assay is more laborious but will allow a deeper understanding of aphid-plant interactions and CLRDV induced pathology in cotton.
Publications
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