Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus
Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family <i>Closteroviridae</i>,...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-11-01
|
| Series: | Pathogens |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-0817/12/11/1314 |
| _version_ | 1797458105611583488 |
|---|---|
| author | Mehdi Shabanian Caihong Li Ali Ebadi Valerian Dolja Baozhong Meng |
| author_facet | Mehdi Shabanian Caihong Li Ali Ebadi Valerian Dolja Baozhong Meng |
| author_sort | Mehdi Shabanian |
| collection | DOAJ |
| description | Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family <i>Closteroviridae</i>, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch’s postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses. |
| first_indexed | 2024-03-09T16:33:20Z |
| format | Article |
| id | doaj.art-6f49e3aa68a643079b62c046e0b84fda |
| institution | Directory Open Access Journal |
| issn | 2076-0817 |
| language | English |
| last_indexed | 2024-03-09T16:33:20Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pathogens |
| spelling | doaj.art-6f49e3aa68a643079b62c046e0b84fda2023-11-24T14:59:42ZengMDPI AGPathogens2076-08172023-11-011211131410.3390/pathogens12111314Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a VirusMehdi Shabanian0Caihong Li1Ali Ebadi2Valerian Dolja3Baozhong Meng4Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, CanadaDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, CanadaDepartment of Horticulture, College of Agriculture and Natural Resources, University of Tehran, Karaj 31587-11167, IranDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USADepartment of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, CanadaGrapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family <i>Closteroviridae</i>, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch’s postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses.https://www.mdpi.com/2076-0817/12/11/1314grapevine leafroll disease<i>Closteroviridae</i>GLRaV-3 cDNA clonesRT-qPCRWestern blotvacuum agro-infiltration |
| spellingShingle | Mehdi Shabanian Caihong Li Ali Ebadi Valerian Dolja Baozhong Meng Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus Pathogens grapevine leafroll disease <i>Closteroviridae</i> GLRaV-3 cDNA clones RT-qPCR Western blot vacuum agro-infiltration |
| title | Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus |
| title_full | Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus |
| title_fullStr | Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus |
| title_full_unstemmed | Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus |
| title_short | Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus |
| title_sort | optimization of a protocol for launching grapevine infection with the biologically active cdna clones of a virus |
| topic | grapevine leafroll disease <i>Closteroviridae</i> GLRaV-3 cDNA clones RT-qPCR Western blot vacuum agro-infiltration |
| url | https://www.mdpi.com/2076-0817/12/11/1314 |
| work_keys_str_mv | AT mehdishabanian optimizationofaprotocolforlaunchinggrapevineinfectionwiththebiologicallyactivecdnaclonesofavirus AT caihongli optimizationofaprotocolforlaunchinggrapevineinfectionwiththebiologicallyactivecdnaclonesofavirus AT aliebadi optimizationofaprotocolforlaunchinggrapevineinfectionwiththebiologicallyactivecdnaclonesofavirus AT valeriandolja optimizationofaprotocolforlaunchinggrapevineinfectionwiththebiologicallyactivecdnaclonesofavirus AT baozhongmeng optimizationofaprotocolforlaunchinggrapevineinfectionwiththebiologicallyactivecdnaclonesofavirus |