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. 2010 Jan;11(1):55-67.
doi: 10.1111/j.1364-3703.2009.00572.x.

The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3'-terminal region of the viral genome

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The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3'-terminal region of the viral genome

Maria R Albiach-Marti et al. Mol Plant Pathol. 2010 Jan.

Abstract

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) causes some of the more important viral diseases of citrus worldwide. The ability to map disease-inducing determinants of CTV is needed to develop better diagnostic and disease control procedures. A distinctive phenotype of some isolates of CTV is the ability to induce seedling yellows (SY) in sour orange, lemon and grapefruit seedlings. In Florida, the decline isolate of CTV, T36, induces SY, whereas a widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3' half of the genome of an infectious cDNA of T36. Eleven T36/T30 hybrids replicated in Nicotiana benthamiana protoplasts. Five of these hybrids formed viable virions that were mechanically transmitted to Citrus macrophylla, a permissive host for CTV. All induced systemic infections, similar to that of the parental T36 clone. Tissues from these C. macrophylla source plants were then used to graft inoculate sour orange and grapefruit seedlings. Inoculation with three of the T30/T36 hybrid constructs induced SY symptoms identical to those of T36; however, two hybrids with T30 substitutions in the p23-3' nontranslated region (NTR) (nucleotides 18 394-19 296) failed to induce SY. Sour orange seedlings infected with a recombinant non-SY p23-3' NTR hybrid also remained symptomless when challenged with the parental virus (T36), demonstrating the potential feasibility of using engineered constructs of CTV to mitigate disease.

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Figures

Figure 1
Figure 1
(a) Graphic of the nucleotide identity between the T36 and T30 genomes and schematic representation of the genome organization of wild‐type Citrus tristeza virus (CTV) (Karasev et al., 1995). PRO, MT, HEL and RdRp indicate protein domains of papain‐like protease, methyltransferase, helicase and RNA‐dependent RNA polymerase, respectively. HSP70h, CPm and CP indicate open reading frames (ORFs) encoding a homologue of heat shock protein 70 and the minor and major coat proteins, respectively. (b) Schematic representation of the T36/T30 hybrid constructs. Black boxes indicate the T36 sequence and grey boxes indicate the T30 sequences substituted within the T36 genome.
Figure 2
Figure 2
Deletion mutants of Citrus tristeza virus (CTV) infectious clone pCTV9 used for the construction of the T36/T30 hybrids. (a) Schematic representation of the pCTV9 infectious clone and the deletion mutants. Arrows indicate the position of the unique restriction endonuclease sites used for cloning purposes. Nucleotide deletions in the pCTV9 sequence are indicated by dotted lines with the location of the deletions indicated in square brackets. (b) RNA accumulation in Nicotiana benthamiana protoplasts infected with in vitro RNA transcripts from pCTV9 and the self‐replicating mutant pΔ6‐20. Northern blots were performed using positive‐stranded, RNA‐specific riboprobes to the 3′ end of the T36 genome. gRNA and sgRNAs indicate genomic RNA and 3′‐coterminal subgenomic RNAs, respectively.
Figure 3
Figure 3
Accumulation of viral RNA in Nicotiana benthamiana protoplasts inoculated with in vitro transcripts of Citrus tristeza virus (CTV) T36 infectious clone CTV9 and the 11 T36/T30 hybrid constructs (line T), and accumulation of viral RNA in a second set of protoplasts inoculated with the virion extracts from transcript‐infected protoplasts of line T (line P). Northern blots were performed using positive‐stranded, RNA‐specific riboprobes to the 3′ end of the T36 genome. gRNA and sgRNAs indicate genomic RNA and 3′‐coterminal subgenomic RNAs, respectively.
Figure 4
Figure 4
Accumulation of Citrus tristeza virus (CTV) RNA in bark extracts from Citrus macrophylla plants infected with isolate T30, the T36 infectious clone CTV9 and five T36/T30 hybrid constructs. Northern blot hybridization was performed using positive‐stranded, RNA‐specific riboprobes to the 3′ end of the T36 genome. gRNA, sgRNAs and D‐RNAs (arrows) indicate genomic RNA, 3′‐coterminal subgenomic RNAs and defective RNAs, respectively.
Figure 5
Figure 5
Development of seedling yellows (SY) syndrome in Citrus tristeza virus (CTV)‐infected plants. (a) SY symptoms in sour orange (1) and Duncan grapefruit (2) seedlings compared with a healthy sour orange plant (3). (b) SY symptoms in T36/T30 hybrid [P23‐3′NTR] (1), isolate T30 (2), healthy (3) and T36 infectious clone CTV9 (4) and T36/T30 hybrid [HSP70h‐P61] (5) sour orange seedlings. (c) Average intensity of SY symptoms (plus standard error of the mean) rated on a scale of 0–4 in sour orange seedlings infected with T36/T30 hybrids [P13] (1), [P18‐3′NTR] (2), [P23‐3′NTR] (3), [P61] (4) and [HSP70h‐P61] (5), and controls healthy (6), T30 isolate (7) and T36 CTV9 (8). Bars accompanied by the same letter were not statistically significantly different (Mann–Whitney U‐tests and sequential Bonferroni correction).
Figure 6
Figure 6
Viral RNA and protein accumulation in bark extracts from sour orange plants infected with Citrus tristeza virus (CTV) isolates T30, T36 CTV9 and five T36/T30 hybrid constructs. (a) Analysis of CTV RNA accumulation using Northern blot hybridization with a positive‐stranded, RNA‐specific riboprobe of the 3′ end of the T36 genome. CTV gRNA and sgRNAs indicate genomic and 3′‐coterminal subgenomic RNAs, respectively. (b) Immunoblot analysis of CTV coat protein (CP) (1) and p23 protein (2) obtained from each of the T36/T30 hybrids and controls. The protein band under CP corresponds to a truncated CP, which was generated during the protein extraction process (Albiach‐Martíet al., 2000a).
Figure 7
Figure 7
Ability of the T36/T30 hybrid [P23‐3′NTR] to protect against the development of seedling yellows (SY) symptoms in sour orange seedlings when challenged with the SY‐inducing isolate T36 CTV9. (a) Symptoms in plants inoculated with T36/T30 hybrid [P23‐3′UTR] (1), hybrid [P23‐3′UTR] and then challenged with T36 CTV9 (2) and T36 CTV9 (3). (b) Average SY symptom intensity (plus standard error of the mean) rated on a scale of 0–4 in sour orange plants, healthy (1) or inoculated with T36 CTV9 (2), isolate T30 (3), hybrid [P23‐3′NTR] (4) and hybrid [P23‐3′UTR] and then challenged with T36 CTV9 (5). Bars accompanied by the same letter were not statistically significantly different (one‐way anova and Bonferroni test).

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References

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