Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts

During infection, Citrus tristeza virus (CTV) produces a non-coding subgenomic RNA referred to as low-molecular-weight tristeza 1 (LMT1), which for a long time has been considered as a by-product of the complex CTV replication machinery. In this study, we investigated the role of LMT1 in the virus infection cycle using a CTV variant that does not produce LMT1 (CTV-LMT1d). We showed that lack of LMT1 did not halt virus ability to replicate or form proper virions. However, the mutant virus demonstrated significantly reduced invasiveness and systemic spread in Nicotiana benthamiana as well as an inability to establish infection in citrus. Introduction of CTV-LMT1d into the herbaceous host resulted in elevation of the levels of salicylic acid (SA) and SA-responsive pathogenesis-related genes beyond those upon inoculation with wild-type (WT) virus (CTV-WT). Further analysis showed that the LMT1 RNA produced by CTV-WT or via ectopic expression in the N. benthamiana leaves suppressed SA accumulation and up-regulated an alternative oxidase gene, which appeared to mitigate the accumulation of reactive oxygen species. To the best of our knowledge, this is the first report of a plant viral long non-coding RNA being involved in counter-acting host response by subverting the SA-mediated plant defense.

Download Full-text

Viral-Like Symptoms Induced by the Ectopic Expression of the p23 Gene of Citrus tristeza virus Are Citrus Specific and Do Not Correlate with the Pathogenicity of the Virus Strain

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0435 ◽

2005 ◽

Vol 18 (5) ◽

pp. 435-445 ◽

Cited By ~ 50

Author(s):

Carmen Fagoaga ◽

Carmelo López ◽

Pedro Moreno ◽

Luis Navarro ◽

Ricardo Flores ◽

...

Keyword(s):

Citrus Tristeza Virus ◽

Ectopic Expression ◽

Sour Orange ◽

Citrus Species ◽

Mexican Lime ◽

Symptom Intensity ◽

P23 Gene ◽

Tristeza Virus ◽

Leaf Symptoms ◽

Citrus Tristeza

Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that correlated with accumulation of p23 protein irrespective of the source strain. CTV inoculation of transgenic limes showing CTV-like leaf symptoms and high p23 accumulation did not modify symptoms initially, with the virus titer being as in inoculated nontransgenic controls; however, at later stages, symptoms became attenuated. Transformation with p23-T36 of CTV-susceptible sweet and sour orange and CTV-resistant trifoliate orange also led to CTV-like leaf symptoms that did not develop when plants were transformed with a truncated p23 version. In transgenic citrus species and relatives other than Mexican lime, p23 was barely detectable, although symptom intensity correlated with levels of p23 transcripts. The lower accumulation of p23 in sweet and sour orange compared with Mexican lime also was observed in nontransgenic plants inoculated with CTV, suggesting that minimal p23 levels cause deleterious effects in the first two species. Conversely, transgenic expression of p23 in CTV nonhost Nicotiana spp. led to accumulation of p23 without phenotypic aberrations, indicating that p23 interferes with plant development only in citrus species and relatives.

Download Full-text

Post-Transcriptional Gene Silencing of the p23 Silencing Suppressor of Citrus tristeza Virus Confers Resistance to the Virus in Transgenic Mexican Lime

Biotechnology and Sustainable Agriculture 2006 and Beyond ◽

10.1007/978-1-4020-6635-1_33 ◽

2007 ◽

pp. 211-213

Author(s):

Carmen Fagoaga ◽

Carmelo López ◽

Alfonso Hermoso de Mendoza ◽

Pedro Moreno ◽

Luis Navarro ◽

...

Keyword(s):

Gene Silencing ◽

Citrus Tristeza Virus ◽

Transcriptional Gene Silencing ◽

Silencing Suppressor ◽

Mexican Lime ◽

Post Transcriptional Gene Silencing ◽

Tristeza Virus ◽

Citrus Tristeza

Download Full-text

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.

Download Full-text

Citrus tristeza virus p23: Determinants for Nucleolar Localization and Their Influence on Suppression of RNA Silencing and Pathogenesis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-12-0201-r ◽

2013 ◽

Vol 26 (3) ◽

pp. 306-318 ◽

Cited By ~ 32

Author(s):

Susana Ruiz-Ruiz ◽

Nuria Soler ◽

Jesús Sánchez-Navarro ◽

Carmen Fagoaga ◽

Carmelo López ◽

...

Keyword(s):

Amino Acids ◽

Rna Silencing ◽

Citrus Tristeza Virus ◽

Ectopic Expression ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Nucleolar Localization ◽

Deletion Mutants ◽

Tristeza Virus ◽

Citrus Tristeza

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.

Download Full-text