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Infectious Clones of Tomato Chlorosis Virus: Toward Increasing Efficiency by Introducing the Hepatitis Delta Virus Ribozyme.

ABSTRACT: Tomato chlorosis virus (ToCV) is an emergent plant pathogen that causes a yellow leaf disorder in tomato and other solanaceous crops. ToCV is a positive-sense, single stranded (ss)RNA bipartite virus with long and flexuous virions belonging to the genus Crininivirus (family Closteroviridae). ToCV is phloem-limited, transmissible by whiteflies, and causes symptoms of interveinal chlorosis, bronzing, and necrosis in the lower leaves of tomato accompanied by a decline in vigor and reduction in fruit yield. The availability of infectious virus clones is a valuable tool for reverse genetic studies that has been long been hampered in the case of closterovirids due to their genome size and complexity. Here, attempts were made to improve the infectivity of the available agroinfectious cDNA ToCV clones (isolate AT80/99-IC from Spain) by adding the hepatitis delta virus (HDV) ribozyme fused to the 3' end of both genome components, RNA1 and RNA2. The inclusion of the ribozyme generated a viral progeny with RNA1 3' ends more similar to that present in the clone used for agroinoculation. Nevertheless, the obtained clones were not able to infect tomato plants by direct agroinoculation, like the original clones. However, the infectivity of the clones carrying the HDV ribozyme in Nicotiana benthamiana plants increased, on average, by two-fold compared with the previously available clones.

SUBMITTER: Navas-Hermosilla E

PROVIDER: S-EPMC8351799 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTomato leaf curl Palampur virus (ToLCPMV) is a bipartite begomovirus which has been reported from India and Iran but infectious clones have not been obtained. We have previously shown the association of Zucchini yellow mosaic virus (ZYMV), a potyvirus, with severe leaf curl disease of muskmelon in Pakistan. However, the severity of symptoms in the field and yield losses led us to believe that some other agent, such as a begomovirus, could be associated with the disease.ResultsA bipartite begomovirus associated with a severe yellow leaf curl disease on muskmelon in Pakistan has been characterized. Analysis of the complete nucleotide sequence of the DNA A and DNA B components of the begomovirus showed that it has the highest DNA sequence identity with ToLCPMV. However, the gene encoding the nuclear shuttle protein (NSP) was truncated in comparison to previously characterised isolates. Agrobacterium-mediated inoculation of Nicotiana benthamiana with the ToLCPMV clones obtained here did not result in symptoms. However, inoculation of plants with the DNA A component of ToLCPMV and the DNA B component of Tomato leaf curl New Delhi virus (ToLCNDV) lead to systemic infection with leaf curl symptoms. This suggested that the lack of infectivity of the ToLCPMV clones was due to the defect in DNA B. The DNA B of ToLCPMV was able to move systemically when inoculated with DNA A of the either virus. Agro-infiltration of muskmelon with the DNA A and DNA B components of ToLCPMV did not lead to symptomatic infection whereas inoculation with the DNA A with the DNA B of ToLCNDV resulted in a hypersensitive response (HR) along the veins. Additionally, agro-infiltration of muskmelon with a construct for the expression of the NSP gene of ToLCNDV under the control of the cauliflower mosaic virus 35S promoter induced a HR, suggesting that this is the gene causing the HR.ConclusionsBoth ToLCPMV and ZYMV are associated with muskmelon leaf curl disease in Pakistan. However, the ToLCPMV variant identified in association with ZYMV has a defective NSP. The results suggest that a variant with a defective NSP may have been selected for in muskmelon, as this protein is an avirulence determinant in this species, and possibly that infection requires the synergistic interaction with ZYMV.

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Infectious Clones of Tomato Chlorosis Virus: Toward Increasing Efficiency by Introducing the Hepatitis Delta Virus Ribozyme.

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