A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update

Abstract

In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.

Closteroviridae: the beginning

“…In retrospect, allowing me to continue the project at that stage was both remarkably generous and far-sighted. Improvements in TLP purification, including (i) the finding that young bark of only certain citrus species is the best source of TLP, (ii) the use of a careful extraction procedure and precipitation of TLP using polyethylene glycol, and (iii) the use of different combinations of buffers for extraction and resuspension allowed us to obtain sufficiently purified TLP particles to establish their viral-like composition and biophysical nature (Bar-Joseph et al, 1972 ), the infectivity of which was demonstrated by Garnsey et al ( 1977 ).…”

Plant Virus–Derived Vectors: Applications in Agricultural and Medical Biotechnology

“…Agroinoculation of citrus trees to obtain infection with different CTV expression vectors was found to be difficult. Thus, to avoid this difficulty, inoculation of citrus plants was carried out by stem-slashing or bark flap inoculation into 1-1.5-year-old Citrus macrophylla seedlings (68, 84) using virions purified from infected N. benthamiana plants (85,86). Direct agroinoculation of young leaves with citrus leaf blotch virus full-length cDNA clones was successful but only on Etrog citron (87); therefore, the virus species, virus construct, Agrobacterium strain, and host genotype all contribute to the success rate of agroinoculation.…”

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

“…Currently, agroinfectious clones are available for eight closterovirids, the closteroviruses beet yellows virus (BYV) ( Prokhnevsky et al, 2002 ), citrus tristeza virus (CTV) ( Gowda et al, 2005 ; Ambrós et al, 2011 , 2013 ), and grapevine leafroll-associated virus 2 (GLRaV-2) ( Liu et al, 2009 ; Kurth et al, 2012 ), the criniviruses lettuce infectious yellows virus (LIYV) ( Wang et al, 2009 ), lettuce chlorosis virus (LCV) ( Chen et al, 2012 ), ToCV ( Orílio et al, 2014 ; Zhao et al, 2016 ), and cucurbit chlorotic yellows virus (CCYV) ( Shi et al, 2016 ), and the ampelovirus grapevine leafroll-associated virus 3 (GLRaV-3) ( Jarugula et al, 2018 ).…”