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.
Highlights
The family Closteroviridae includes almost 60 plant virus species grouped in the genera Ampelovirus, Closterovirus, Crinivirus, and Velarivirus, plus a number of unassigned species (Fuchs et al, 2020)
The objectives of this work were (i) to generate infectious cDNA clones for Tomato chlorosis virus (ToCV) RNA1 and RNA2 containing a ribozyme at the 3 end, (ii) to identify the 3 ends of the virus genomes generated after agroinoculation with clones with or without the ribozyme, (iii) to quantify the effect of the ribozyme on the infection rate of N. benthamiana plants, and (iv) to determine whether a possible increase in infectivity due to the presence of the ribozyme would allow direct agroinfection of tomato plants
With the objective of improving the infectivity of available ToCV agroinfectious clones (Orílio et al, 2014), an hepatitis delta virus (HDV) ribozyme sequence was incorporated at the end of both RNA1 and RNA2 by cloning in the binary vector pJL89 (Lindbo, 2007)
Summary
The family Closteroviridae includes almost 60 plant virus species grouped in the genera Ampelovirus, Closterovirus, Crinivirus, and Velarivirus, plus a number of unassigned species (Fuchs et al, 2020). Closterovirids have several distinguishing characteristics: (i) semi-persistent transmission by insects, (ii) mostly phloem-limited, (iii) elongated particles built of several proteins, (iv) large single-stranded positive-sense RNA (ssRNA) genomes of 15–20 kb, (v) presence of a gene homologous to the HSP70 family of cell chaperones, and (vi) a number of duplicated and diverged genes (Agranovsky, 2016). Some closterovirids cause serious diseases in important crops including citrus, cucurbits, grapevine, sugar beet, and tomato. Criniviruses have segmented genomes encapsidated in separate long flexuous virions that are restricted to the phloem and companion cells (Kiss et al, 2013). Criniviruses are transmitted in nature by whiteflies (Hemiptera: Aleyrodidae) in a semipersistent manner (Tzanetakis et al, 2013)
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