Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus

Abstract

Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants.

Fig 1. Accumulation of phenolic compounds in Money Maker plants infected with citrus exocortis viroid (CEVd) or tomato spotted wilt virus (TSWV).

Levels of free A) and total B) gentisic acid (GA) and salicylic acid (SA) in mock-inoculated and infected Money Maker tomato leaves. Plants were inoculated with either CEVd or TSWV and samples were collected at the indicated times (weeks post-inoculation, wpi) for both infected (I) and mock-inoculated (M) leaves, and then analyzed by HPLC-fluorescence detection. Total SA is the sum of free SA and SA glucoside and total GA is the sum of free GA and GA xyloside. Data correspond to the mean ± SD of three biological replicates of one representative experiment. The experiments were repeated at least 3 times.

Fig 2. Accumulation of phenolic compounds in NahG plants infected with citrus exocortis viroid (CEVd) or tomato spotted wilt virus (TSWV).

Levels of free and total gentisic acid (GA), salicylic acid (SA) and catechol (CA) in infected NahG tomato leaves. Plants were inoculated with either CEVd or TSWV and samples were collected at the indicated times (weeks post-inoculation, wpi), and then analyzed by HPLC-fluorescence detection. Total phenolics are the sum of free and glycosylated forms. Data correspond to the mean ± SD of three biological replicates of one representative experiment. The experiments were repeated at least 3 times.

Fig 3. Disease severity of infected Money Maker and NahG tomato plants.

Symptoms scale provoked by A) Citrus Exocortis Viroid (CEVd) and B) Tomato Spotted Wilt Virus (TSWV). Symptomatology was scored at 2, 2.5, 3, and 4 weeks post-inoculation (wpi) with CEVd and at 6, 8, 10, 13, and 15 days post-inoculation (dpi) with TSWV using the following scale: symptomless (white), moderate (grey), severe (dark gray), and very severe (black). Data of a representative experiment are shown. A Kruskal-Wallis analysis was performed and different letters indicate significant differences (p < 0.05) between the Money Maker and NahG plants. The experiments were repeated at least 3 times.

Fig 4. Growth of representative Money Maker and NahG tomato plants following inoculation with citrus exocortis viroid (CEVd) and tomato spotted wilt virus (TSWV).

A) Mock-inoculated Money Maker plant (Control), Money Maker plant 4 weeks after CEVd-inoculation (CEVd), and Money Maker plant 15 days after TSWV-inoculation (TSWV). B) Mock-inoculated NahG plant (Control), NahG plant 4 weeks after CEVd-inoculation (CEVd), and NahG plant 15 day after TSWV-inoculation (TSWV).

Fig 5. Disease development in Money Maker and NahG plants infected with A) CEVd or B) TSWV.

Plants were treated either with water (continuous line) or BTH (discontinuous line). Evolution of the number of tomato plants showing symptoms at the indicated days post-inoculation (dpi). Data correspond to one representative experiment.

Fig 6. Growth of NahG plants following treatment with BTH and CEVd or TSWV inoculation.

A) Representative phenotype observed in infected plants 4 weeks after CEVd inoculation (CEVd), and in equivalent plants that had been pre-treated with 1 mM of BTH (CEVd+BTH). B) Representative phenotype observed in TSWV-infected plants 15 days after inoculation (TSWV), and in equivalent plants that had been pre-treated with 1 mM of BTH (TSWV+BTH).

Fig 7. Detection of CEVd levels in infected Money Maker, NahG and BTH-treated NahG plants.

Panels A and B show the Northern blot analyses to detect Citrus Exocortis Viroid (CEVd) and CEVd-specific small RNAs (VdsRNAs), respectively, at 2, 2.5 and 4 weeks after inoculation (wpi). RNAs were separated in a 5% or 17% denaturing polyacrylamide gel, then transferred and hybridized with radioactive negative strand CEVd riboprobes. CEVd and its corresponding small RNAs are denoted with an arrow.

Fig 8. Detection of TSWV levels in infected Money Maker, NahG and BTH-treated NahG plants.

Quantitative ELISA to detect TSWV at 0.5, 1 and 2 wpi. A polyclonal antibody against TSWV (BR-01, Loewe) and an alkaline phosphatase conjugated antibody were used as primary and secondary antibodies, respectively. Immunodetection was carried out by using p-nitrophenyl phosphate as a substrate and the absorbance was measured at 405 nm. Data correspond to the mean ± SD of three biological replicates of one representative experiment.

Fig 9. Effect of Citrus Exocortis Viroid (CEVd) and Tomato Spotted Wilt Virus (TSWV) infections on levels of p-coumaroyltyramine (CT), feruloyltyramine (FT), caffeoylputrescine (CaP), and feruloylputrescine (FP) in Money Maker and NahG tomato leaves.

Plants were inoculated with either CEVd or TSWV and samples were collected at the indicated times (weeks post-inoculation, wpi) for both infected and mock-inoculated leaves, and then analyzed by HPLC/ESI-MS. Data correspond to the mean ± SD of three biological replicates of one representative experiment. The experiments were repeated at least 3 times.

Fig 10. Accumulation of Pathogenesis-Related (PR) proteins in Money Maker and NahG tomato plants upon viroid and virus infection.

Plants were inoculated with either Citrus Exocortis Viroid (CEVd, A) or Tomato Spotted Wilt Virus (TSWV, B), and samples were collected at the indicated times (weeks post-inoculation, wpi) for both infected and mock-inoculated plants. Crude extracts were separated by SDS-PAGE and the presence of PR1 and P23 was detected by immunoblot. Upper panels correspond to Coomassie Blue stained SDS-PAGE gels (14% acrylamide). Lower panels show the immunostaining of P23 (23 kDa) and PR1 (14 kDa).

Fig 11. Time course of ethylene production in NahG tomato leaves infected with Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV) and their corresponding controls.

Samples were collected at the indicated times (weeks post-inoculation, wpi) for both mock-inoculated (Mock) and infected (Inf) plants, and ethylene was measured with a gas chromatograph fitted with a flame ionization detector (FID). Data correspond to the mean ± SD of three biological replicates of one representative experiment. The experiments were repeated at least 3 times.