Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species

Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Zn-finger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.

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Identification of Key Residues Required for RNA Silencing Suppressor Activity of p23 Protein from a Mild Strain of Citrus Tristeza Virus

Viruses ◽

10.3390/v11090782 ◽

2019 ◽

Vol 11 (9) ◽

pp. 782 ◽

Cited By ~ 1

Author(s):

Zhuoran Li ◽

Yizhong He ◽

Tao Luo ◽

Xi Zhang ◽

Haoliang Wan ◽

...

Keyword(s):

Rna Silencing ◽

Citrus Tristeza Virus ◽

Silencing Suppressor ◽

Mild Strain ◽

High Sequence Identity ◽

Suppressor Activity ◽

Protein Levels ◽

Tristeza Virus ◽

Citrus Tristeza ◽

Citrus Trees

The severe strain of citrus tristeza virus (CTV) causes quick decline of citrus trees. However, the CTV mild strain causes no symptoms and commonly presents in citrus trees. Viral suppressor of RNA silencing (VSR) plays an important role in the successful invasion of viruses into plants. For CTV, VSR has mostly been studied in severe strains. In this study, the N4 mild strain in China was sequenced and found to have high sequence identity with the T30 strain. Furthermore, we verified the functions of three VSRs in the N4 strain, and p23 was found to be the most effective in terms of local silencing suppressor activity among the three CTV VSRs and localized to both nucleus and plasmodesmata, which is similar to CTV T36 strain. Several conserved amino acids were identified in p23. Mutation of E95A/V96A and M99A/L100AA impaired p23 protein stability. Consequently, these two mutants lost most of its suppressor activity and their protein levels could not be rescued by co-expressing p19. Q93A and R143A/E144A abolished p23 suppressor activity only and their protein levels increased to wild type level when co-expressed with p19. This work may facilitate a better understanding of the pathogenic mechanism of CTV mild strains.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-11-0110 ◽

2011 ◽

Vol 24 (10) ◽

pp. 1119-1131 ◽

Cited By ~ 36

Author(s):

Silvia Ambrós ◽

Choaa El-Mohtar ◽

Susana Ruiz-Ruiz ◽

Leandro Peña ◽

José Guerri ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Time Course ◽

Citrus Tristeza Virus ◽

Systemic Infection ◽

Genetic System ◽

Herbaceous Host ◽

System A ◽

Tristeza Virus ◽

Efficient Transmission ◽

Citrus Tristeza

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

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Latency of Systemic Infection in YoungField-Grown Sweet Orange Trees Following Graft-Inoculation with Citrus tristeza virus

International Organization of Citrus Virologists Conference Proceedings (1957-2010) ◽

10.5070/c59rb677sf ◽

2002 ◽

Vol 15 (15) ◽

Author(s):

T. R. Gottwald ◽

S. M. Garnsey ◽

T. D. Riley

Keyword(s):

Citrus Tristeza Virus ◽

Sweet Orange ◽

Systemic Infection ◽

Orange Trees ◽

Tristeza Virus ◽

Citrus Tristeza

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Citrus tristeza virus: Host RNA Silencing and Virus Counteraction

Methods in Molecular Biology - Citrus Tristeza Virus ◽

10.1007/978-1-4939-9558-5_14 ◽

2019 ◽

pp. 195-207 ◽

Cited By ~ 2

Author(s):

Susana Ruiz-Ruiz ◽

Beatriz Navarro ◽

Leandro Peña ◽

Luis Navarro ◽

Pedro Moreno ◽

...

Keyword(s):

Rna Silencing ◽

Citrus Tristeza Virus ◽

Tristeza Virus ◽

Citrus Tristeza

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Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins

Virology ◽

10.1016/j.virol.2017.11.014 ◽

2018 ◽

Vol 514 ◽

pp. 192-202 ◽

Cited By ~ 6

Author(s):

Sung-Hwan Kang ◽

Osama O. Atallah ◽

Yong-Duo Sun ◽

Svetlana Y. Folimonova

Keyword(s):

Citrus Tristeza Virus ◽

Virus Genome ◽

Protease Domain ◽

Functional Diversification ◽

Rna Sequences ◽

Domain Duplication ◽

Encoded Proteins ◽

Tristeza Virus ◽

Citrus Tristeza

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Citrus Tristeza Virus: Survival at the Edge of the Movement Continuum

Journal of Virology ◽

10.1128/jvi.00515-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6546-6556 ◽

Cited By ~ 42

Author(s):

Svetlana Y. Folimonova ◽

Alexey S. Folimonov ◽

Satyanarayana Tatineni ◽

William O. Dawson

Keyword(s):

Citrus Tristeza Virus ◽

Cell Movement ◽

Systemic Infection ◽

Sour Orange ◽

Citrus Species ◽

Long Distance ◽

Susceptible Host ◽

Long Distance Movement ◽

Tristeza Virus ◽

Citrus Tristeza

ABSTRACT Systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. There is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. We examined the systemic distribution of Citrus tristeza virus (CTV) in citrus species with a range of susceptibilities. By using a “pure” culture of CTV from a cDNA clone and green fluorescent protein-labeled virus we show that both cell-to-cell and long-distance movement are unusually limited, and the degree of limitation varies depending on the citrus host. In the more-susceptible hosts CTV infected only a small portion of phloem-associated cells, and moreover, the number of infection sites in less-susceptible citrus species was substantially decreased further, indicating that long-distance movement was reduced in those hosts. Analysis of infection foci in the two most differential citrus species, Citrus macrophylla and sour orange, revealed that in the more-susceptible host the infection foci were composed of a cluster of multiple cells, while in the less-susceptible host infection foci were usually single cells, suggesting that essentially no cell-to-cell movement occurred in the latter host. Thus, CTV in sour orange represents a pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature.

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Chimeric cDNA Sequences from Citrus tristeza virus Confer RNA Silencing-Mediated Resistance in Transgenic Nicotiana benthamiana Plants

Phytopathology ◽

10.1094/phyto-96-0819 ◽

2006 ◽

Vol 96 (8) ◽

pp. 819-827 ◽

Cited By ~ 11

Author(s):

Gourgopal Roy ◽

Mysore R. Sudarshana ◽

Diane E. Ullman ◽

Shou-Wei Ding ◽

Abhaya M. Dandekar ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Rna Silencing ◽

Citrus Tristeza Virus ◽

Potato Virus X ◽

Northern Hybridization ◽

Transcriptional Gene Silencing ◽

Small Interfering Rnas ◽

Cdna Sequences ◽

Tristeza Virus ◽

Citrus Tristeza

RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3′ untranslated region (UTR); and pCTV2 contained CP, p23, and 3′ UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.

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