Agroinoculation of <i>Citrus tristeza virus</i> Causes Systemic Infection and Symptoms in the Presumed Nonhost <i>Nicotiana benthamiana</i>

Ambrós, Silvia; El-Mohtar, Choaa; Ruiz-Ruiz, Susana; Peña, Leandro; Guerri, José; Dawson, William O.; Moreno, Pedro

doi:10.1094/mpmi-05-11-0110

Cited by 53 publications

(69 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The bacterial cultures carrying pCTV9⌬p33-RFP binary plasmids were used for infiltration of Nicotiana benthamiana plants as described earlier (46,49). One week after infiltration, CTV9⌬p33-RFP virions were extracted from the infiltrated leaves, concentrated by centrifugation on a sucrose cushion, and used for inoculation of the bark flap of Citrus macrophylla seedlings according to the procedure described earlier (50).…”

Section: Methodsmentioning

confidence: 99%

A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

Bergua

Zwart

El-Mohtar

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels-the cellular and the whole-organism levels-by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondaryinfection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.

show abstract

Section: Methodsmentioning

confidence: 99%

A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

Bergua

Zwart

El-Mohtar

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The catalase intron of the GUS marker gene blocks its expression in transformed A. tumefaciens ensuring that glucuronidase activity is possible only in eukaryotic cells. The CTV9R expression cassettes (with or without an intron in the ORF 1a) were Not I excised from the original BAC vectors (Ambrós et al, 2011) and ligated vía Not I to the pCH20-GUSi plasmid to yield the pCH20-GUSi-CTV vector. E. coli DH10B transformants of these plasmids were selected on LB plates with kanamycin (50 mg/l) and sucrose (5%), and purified plasmid used to transform A. tumefaciens strains by electroporation.…”

Section: Methodsmentioning

confidence: 99%

“…Other details as in (A) . (C) Schematic representation and relative orientation of the CTV-T36 expression cassette containing the full-genome cDNA clone CTV9R from the agroinfectious BAC CTV-vector (Ambrós et al, 2011). Shadowed boxes represent the double enhanced 35S promoter (35S × 2), the Hepatitis delta virus ribozyme (Rbz) and the NOS- t present in the cassette.…”

Section: Methodsmentioning

confidence: 99%

“…To overcome these problems we developed an improved genetic system based on agroinfiltration of NB leaves with binary plasmids carrying a cDNA of the CTV-T36 genome and different silencing suppressors and performed a time-course analysis of CTV accumulation in those leaves (Ambrós et al, 2011). Unexpectedly, we found that agroinoculated plants of this species, presumed to be “non-host,” were systemically invaded by CTV-T36 with high viral titers, particularly in the upper leaves in which the virus eventually invaded some non-phloem tissues and incited typical disease symptoms.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Ambrós¹,

Ruiz-Ruiz²,

Peña³

et al. 2013

Front. Microbiol.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…In natural infections it is confined to the phloem of most species of the genera Citrus and Fortunella within the subfamily Aurantioideae (Bar-Joseph et al, 1989; Moreno et al, 2008), although when inoculated experimentally via Agrobacterium tumefaciens it may incite systemic infection and symptoms into the presumed non-host Nicotiana benthamiana (Ambrós et al, 2011). CTV may cause three different syndromes depending on the virus isolate, the citrus genotype, and the scion/rootstock combination.…”

Section: Introductionmentioning

confidence: 99%

Citrus tristeza virus p23: a unique protein mediating key virus–host interactions

Flores¹,

Ruiz-Ruiz²,

Soler³

et al. 2013

Front. Microbiol.

Self Cite

View full text Add to dashboard Cite

The large RNA genome of Citrus tristeza virus (CTV; ca. 20 kb) contains 12 open reading frames, with the 3 -terminal one corresponding to a protein of 209 amino acids (p23) that is expressed from an abundant subgenomic RNA. p23, an RNA-binding protein with a putative zinc-finger domain and some basic motifs, is unique to CTV because no homologs have been found in other closteroviruses, including the type species of the genus Beet yellows virus (despite both viruses having many homologous genes). Consequently, p23 might have evolved for the specific interaction of CTV with its citrus hosts. From a functional perspective p23 has been involved in many roles: (i) regulation of the asymmetrical accumulation of CTV RNA strands, (ii) induction of the seedling yellows syndrome in sour orange and grapefruit, (iii) intracellular suppression of RNA silencing, (iv) elicitation of CTV-like symptoms when expressed ectopically as a transgene in several Citrus spp., and (v) enhancement of systemic infection (and virus accumulation) in sour orange and CTV release from the phloem in p23-expressing transgenic sweet and sour orange. Moreover, transformation of Mexican lime with intron-hairpin constructs designed for the co-inactivation of p23 and the two other CTV silencing suppressors results in complete resistance against the homologous virus. From a cellular point of view, recent data indicate that p23 accumulates preferentially in the nucleolus, being the first closterovirus protein with such a subcellular localization, as well as in plasmodesmata. These major accumulation sites most likely determine some of the functional roles of p23.

show abstract

Agroinoculation of Citrus tristeza virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana

Cited by 53 publications

References 60 publications

A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

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

Citrus tristeza virus p23: a unique protein mediating key virus–host interactions

Contact Info

Product

Resources

About