Glutathione Can Compensate for Salicylic Acid Deficiency in Tobacco to Maintain Resistance to Tobacco Mosaic Virus

Abstract

Earlier studies showed that the artificial elevation of endogenous glutathione (GSH) contents can markedly increase the resistance of plants against different viruses. On the other hand, salicylic acid (SA)-deficient NahG plants display enhanced susceptibility to viral infections. In the present study, the biochemical mechanisms underlying GSH-induced resistance were investigated in various tobacco biotypes displaying markedly different GSH and SA levels. The endogenous GSH levels of Nicotiana tabacum cv. Xanthi NN and N. tabacum cv. Xanthi NN NahG tobacco leaves were increased by infiltration of exogenous GSH or its synthetic precursor R-2-oxo-4-thiazolidine-carboxylic acid (OTC). Alternatively, we also used tobacco lines containing high GSH levels due to transgenes encoding critical enzymes for cysteine and GSH biosynthesis. We crossed Xanthi NN and NahG tobaccos with the GSH overproducer transgenic tobacco lines in order to obtain F₁ progenies with increased levels of GSH and decreased levels of SA. We demonstrated that in SA-deficient NahG tobacco the elevation of in planta GSH and GSSG levels either by exogenous GSH or by crossing with glutathione overproducing plants confers enhanced resistance to Tobacco mosaic virus (TMV) manifested as both reduced symptoms (i.e. suppression of hypersensitive-type localized necrosis) and lower virus titers. The beneficial effects of elevated GSH on TMV resistance was markedly stronger in NahG than in Xanthi NN leaves. Infiltration of exogenous GSH and OTC or crossing with GSH overproducer tobacco lines resulted in a substantial rise of bound SA and to a lesser extent of free SA levels in tobacco, especially following TMV infection. Significant increases in expression of pathogenesis related (NtPR-1a, and NtPRB-1b), and glutathione S-transferase (NtGSTtau, and NtGSTphi) genes were evident in TMV-inoculated leaves in later stages of pathogenesis. However, the highest levels of defense gene expression were associated with SA-deficiency, rather than enhanced TMV resistance. In summary, elevated levels of glutathione in TMV-infected tobacco can compensate for SA deficiency to maintain virus resistance. Our results suggest that glutathione-induced redox changes are important components of antiviral signaling in tobacco.

Keywords: NahG; Tobacco mosaic virus; glutathione; salicylic acid; tobacco; virus resistance.

Figure 1

(A) Reduced (GSH) and oxidized (GSSG) glutathione levels and (B) free and bound salicylic acid (SA) levels in Nicotiana tabacum cv. Xanthi NN and its SA-deficient NahG line 1 and 2 days after infiltration (DAI) with 2 mM GSH or 2 mM R-2-oxothiazolidine-4-carboxylic acid (OTC). As a control, we infiltrated NahG and Xanthi plants with tap water. Ø = untreated tobacco. Asterisks (*, **) indicate statistically significant differences between GSH, GSSG, free and bound SA levels of control (water injected) and GSH or OTC treated leaf halves of the respective genotype at p ≤ 0.05 and p ≤ 0.01 (Student’s t-test). Columns represent means ± SD from three independent biological experiments. Asterisk (*) in case of untreated plants (Ø) indicates statistically significant differences (Student’s t-test, p = 0.05) between the NahG and Xanthi tobacco genotypes.

Figure 2

(A) Symptoms of Tobacco mosaic virus (TMV) inoculation, (B) relative expression of TMV coat protein gene (TMV-CP) and (C) levels of reduced (GSH) and oxidized (GSSG) glutathione in leaves of Nicotiana tabacum cv. Xanthi NN and its SA deficient NahG line 5 days after TMV inoculation. Right leaf halves infiltrated with 2 mM GSH (A, upper photos) or 2 mM R-2-oxothiazolidine-4-carboxylic acid (OTC) (A, lower photos). Left leaf halves infiltrated with pH 6.8 tap water as a control. GSH or OTC infiltrations were performed 2 days before TMV inoculations. Columns represent means ± SD from three independent biological experiments. Asterisks (*, **) indicate statistically significant differences in TMV-CP expression and GSH or GSSG levels of control (water injected) and GSH or OTC-treated leaf halves at p ≤ 0.05 and p ≤ 0.01 (Student’s t-test).

Figure 3

Disease symptoms of Tobacco mosaic virus (TMV) inoculation (A) and relative expression levels of TMV coat protein gene (TMV-CP) (B) in different tobacco lines 5 days after inoculation. Upper part (A) from left to right: Nicotiana tabacum cv. Burley NN TRI-2 (glutathione overproducer), N. tabacum cv. Burley NN TRI-2 X cv. Xanthi NN NahG F₁ hybrid (glutathione overproducer X salicylic acid deficient), N. tabacum cv. Xanthi NN NahG (salicylic acid deficient). Lower part (A) from left to right: N. tabacum cv. Burley NN CEMK-9 (glutathione overproducer), N. tabacum cv. Burley NN CEMK-9 X cv. Xanthi NN NahG F₁ hybrid (glutathione overproducer X salicylic acid deficient), N. tabacum cv. Xanthi NN NahG (salicylic acid deficient). Representative results of three independent biological experiments are shown. Relative expression levels of TMV-CP (B) as detected by real time RT-qPCR. The following tobacco lines were used: Burley NN and Xanthi NN (wild type plants); Burley NN CEMK-9 and TRI-2 (GSH overproducer lines); Xanthi NN NahG (SA-deficient line); CEMK-9 X NahG F₁, TRI-2 X NahG F₁, CEMK-9 X Xanthi F₁ and TRI-2 X Xanthi F₁ (F₁ generation plants). Columns represent means ± SD from three independent biological experiments. Asterisks (*, p ≤ 0.05 and **, p ≤ 0.01) indicate statistically significant differences in TMV-CP expression by Student’s t-test between Burley – CEMK-9, Burley – TRI-2, as well as NahG – CEMK-9 X NahG F₁, NahG – TRI-2 X NahG F₁ and Xanthi – CEMK-9 X Xanthi F₁, Xanthi – TRI-2 X Xanthi F₁ in a pairwise manner.

Figure 4

Reduced (GSH) and oxidized (GSSG) glutathione levels in uninoculated, mock-inoculated and Tobacco mosaic virus (TMV)-inoculated Nicotiana tabacum cultivars/lines. Detection of glutathione was performed 5 days after inoculation. For the description of tobacco lines see the legend of Figure 3 . Columns represent means ± SD from three independent biological experiments. Asterisks (*, p ≤ 0.05 and **, p ≤ 0.01) indicate statistically significant differences by Student’s t-test in GSH or GSSG levels between Burley and its corresponding transgenic lines (CEMK-9 and TRI-2) as well as Xanthi and NahG versus their corresponding F₁ hybrids in case of uninoculated, mock and TMV inoculated plants, respectively.

Figure 5

Free and bound salicylic acid (SA) levels in uninoculated, mock-inoculated and Tobacco mosaic virus (TMV)-inoculated Nicotiana tabacum cultivars/lines. Detection of SA was performed 5 days after inoculation. For the description of tobacco lines see the legend of Figure 3 . Columns represent means ± SD from three independent biological experiments. Asterisks (*, p ≤ 0.05 and **, p ≤ 0.01) indicate statistically significant differences by Student’s t-test in free and bound SA levels between cv. Burley and corresponding transgenic lines (CEMK-9 and TRI-2) as well as Xanthi and NahG versus their corresponding F₁ hybrids in case of uninoculated, mock and TMV inoculated plants, respectively.

Figure 6

Expression levels of two pathogenesis related genes (NtPR-1a and NtPRB-1b) in uninoculated, mock-inoculated and Tobacco mosaic virus (TMV)-inoculated Nicotiana tabacum cultivars/lines. Analyses of gene expression were performed 5 days after TMV-inoculation by real time RT-qPCR. For the description of tobacco lines see the legend of Figure 3 . Columns represent means ± SD from three independent biological experiments. Asterisks (*, p ≤ 0.05 and **, p ≤ 0.01 ) indicate statistically significant differences (Student’s t-test) in gene expression between cv. Burley and corresponding transgenic lines (CEMK-9 and TRI-2) and cv. Xanthi NahG and cv. Xanthi versus their corresponding F1 hybrids in case of uninoculated, mock and TMV inoculated plants, respectively.

Figure 7

Expression levels of two glutathione S-transferase (GST) genes (NtGSTtau and NtGSTphi) in uninoculated, mock-inoculated, and Tobacco mosaic virus (TMV)-inoculated Nicotiana tabacum cultivars/lines. Detection of GST gene expression was performed 5 days after TMV-inoculation by real time RT-qPCR. For the description of tobacco lines see the legend of Figure 3 . Columns represent means ± SD from three independent biological experiments. Asterisks (*, p ≤ 0.05 ) indicate statistically significant differences (Student’s t-test) in gene expression between cv. Burley and corresponding transgenic lines (CEMK-9 and TRI-2) and cv. Xanthi NahG and cv. Xanthi versus their corresponding F₁ hybrids in case of uninoculated, mock and TMV inoculated plants, respectively.