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

Silvia Ambrós,Susana Ruiz-Ruiz,Pedro Moreno,Leandro Peña

doi:10.3389/fmicb.2013.00165

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.

Highlights

Citrus tristeza virus (CTV), a member of genus Closterovirus, is one of the more economically important plant viruses
AGROINOCULATION OF CTV WITH ONCOGENIC A. tumefaciens STRAINS PRODUCES VIRUS-INFECTED TUMOURS AND SYSTEMIC INFECTION IN Nicotiana benthamiana (NB) BUT NOT IN MEXICAN LIME In an attempt to simplify the newly developed genetic system for CTV (Ambrós et al, 2011) we tried direct agroinoculation of citrus with C58 and A281, two oncogenic strains of A. tumefaciens that produce good tumours in citrus plants, transfected with a suitable binary vector carrying the cDNA of CTV isolate T36 (CTV-T36) isolate (Satyanarayana et al, 2001; Ambrós et al, 2011) and an appropriate plant selectable marker to monitor cell transformation
For this purpose we developed the plasmid pCH20-GUSi, with a gus-intron marker gene (Figures 1A,B) that ensures that positive GUS expression was derived from transformed plant cells and not from residual bacteria, and the vector pCH20-GUSiCTV containing the expression cassette of CTV-T36 (Figure 1C)

Summary

Introduction

Citrus tristeza virus (CTV), a member of genus Closterovirus, is one of the more economically important plant viruses. CTV virions (2000 × 10–12 nm) are composed of a singlestranded, positive-sense genomic RNA (gRNA) of about 20 kb and two coat proteins of 25 (CP) and 27 (CPm) kDa that encapsidate about 97 and 3% of the gRNA, respectively (Bar-Joseph and Lee, 1989; Satyanarayana et al, 2004; Gowda et al, 2009). P33 is required in CTV-infected plants to exclude superinfection by isolates of the same strain (Folimonova et al, 2010; Folimonova, 2012), and the expression ratio between p33 and p13 or p18 seems to determine the stem pitting symptom (Tatineni and Dawson, 2012). Proteins p25, p20, and p23 have been shown to act as silencing suppressors in Nicotiana benthamiana (NB) and N. tabaccum plants (Lu et al, 2004), with p23 being a pathogenicity determinant www.frontiersin.org

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