SlMAPK3 enhances tolerance to tomato yellow leaf curl virus (TYLCV) by regulating salicylic acid and jasmonic acid signaling in tomato (Solanum lycopersicum)

Abstract

Several recent studies have reported on the role of mitogen-activated protein kinase (MAPK3) in plant immune responses. However, little is known about how MAPK3 functions in tomato (Solanum lycopersicum L.) infected with tomato yellow leaf curl virus (TYLCV). There is also uncertainty about the connection between plant MAPK3 and the salicylic acid (SA) and jasmonic acid (JA) defense-signaling pathways. The results of this study indicated that SlMAPK3 participates in the antiviral response against TYLCV. Tomato seedlings were inoculated with TYLCV to investigate the possible roles of SlMAPK1, SlMAPK2, and SlMAPK3 against this virus. Inoculation with TYLCV strongly induced the expression and the activity of all three genes. Silencing of SlMAPK1, SlMAPK2, and SlMAPK3 reduced tolerance to TYLCV, increased leaf H2O2 concentrations, and attenuated expression of defense-related genes after TYLCV infection, especially in SlMAPK3-silenced plants. Exogenous SA and methyl jasmonic acid (MeJA) both significantly induced SlMAPK3 expression in tomato leaves. Over-expression of SlMAPK3 increased the transcript levels of SA/JA-mediated defense-related genes (PR1, PR1b/SlLapA, SlPI-I, and SlPI-II) and enhanced tolerance to TYLCV. After TYLCV inoculation, the leaves of SlMAPK3 over-expressed plants compared with wild type plants showed less H2O2 accumulation and greater superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activity. Overall, the results suggested that SlMAPK3 participates in the antiviral response of tomato to TYLCV, and that this process may be through either the SA or JA defense-signaling pathways.

Fig 1. The RNA expression and protein activity of SlMAPK1, SlMAPK2 and SlMAPK3 were analyzed in ‘Y19’ tomato leaves following TYLCV infection.

Samples were collected from the uppermost leaves at different times after TYLCV inoculation. The samples were used to extract RNA, which was reverse transcripted into cDNA to detect the relative expression of SlMAPK1 (A), SlMAPK2 (B) and SlMAPK3 (C) by qRT-PCR. The tomato SlEF1α gene was used as an internal control [59, 64]. The expression levels are relative to 0 h post infection. The activities of SlMAPK1 (D), SlMAPK2 (E) and SlMAPK3 (F) were determined with an ELISA kit (Shanghai Biological Technology Co., Ltd.). Values are means ± standard error (SE), replicated thrice. The treatments were compared with the control using Tukey’s test. * Significant at P<0.05, ** Significant at P<0.01.

Fig 2. SlMAPK3-silencing reduced the tolerance of ‘Y19’ tomato to TYLCV.

(A) The percentage of SlMAPK-silenced (TRV: SlMAPK1-, TRV: SlMAPK2-, and TRV: SlMAPK3) and non-silenced (TRV:00-, control) plants exhibiting TYLCV symptoms at 0, 14, and 35 dpi. Each treatment had 15–20 ‘Y19’ plants, replicated thrice. (B) Disease index of TYLCV in SlMAPK-silenced (TRV: SlMAPK1-, TRV: SlMAPK2-, and TRV: SlMAPK3) and non-silenced (TRV: 00-, control) plants at 0, 14, and 35 dpi. Each treatment had 15–20 ‘Y19’ plants, replicated thrice. (C) Relative TYLCV content in SlMAPK-silenced (TRV: SlMAPK1-, TRV: SlMAPK2-, and TRV: SlMAPK3) and non-silenced (TRV: 00-, control) plants at 3, 7, 14, 21, and 28 dpi. Leaf samples were collected from all plants, whether or not they displayed symptoms. The leaf samples were mixed within a treatment and then analyzed to determine total DNA. Three biological replicates were performed. The relative TYLCV content in the samples was determined using qPCR. The results are means ± standard error (SE), replicated thrice. The treatments were compared with the control using Tukey’s test. Different letters indicate significant differences at P<0.05.

Fig 3. Defense-related gene expression decreased when SlMAPK1, SlMAPK2, SlMAPK3-silenced ‘Y19’ tomato plants were infected with TYLCV.

(A-E) The relative expression of the SA-mediated defense marker genes SlPR1 (A), SlPR1b (B) and the JA-mediated defense marker genes SlLapA (C), SlPI-I (D) and SlPI-II (E) at 14 dpi with TYLCV. Values are means ± standard error (SE), replicated thrice. The treatments were compared with the control using Tukey’s test. Different letters indicate significant differences at P<0.05.

Fig 4. Exogenous application of signaling molecules increased SlMAPK3 expression in ‘Y19’ tomato leaves.

Tomato seedlings were treated with either 100 μM SA, 100 mM MeJA, 10 mM H₂O₂, 100 μM ABA, or water (i.e. mock treatment). The tomato SlEF1α gene was used as an internal control [59, 64]. Values are means ± standard error (SE), replicated thrice. The expression levels are relative to 0 h. The treatments were compared with the control using Tukey’s test. * Significant at P<0.05.

Fig 5. Overexpression of SlMAPK3 in ‘M82’ tomato enhanced tolerance to TYLCV.

(A) Phenotype analysis of wild type (WT, ‘M82’) and SlMAPK3-overexpressed (OE-4, OE-6 and OE-7) plants at 10, 30, and 45 dpi with TYLCV. (B) The relative TYLCV content in WT and SlMAPK3-overexpressed lines at 1, 10, 30, and 45 dpi. Values are means ± standard error (SE), replicated thrice. Tomato β-actin was used as an internal control for qRT-PCR [62, 63]. The expression levels are relative to 1 dpi. (C) Leaf chlorophyll contents in WT and SlMAPK3-overexpressed lines. Values in B-D are means ± SE of at least three replicates. (D) The average number of flowers on WT and SlMAPK3-overexpressed plants at 45 dpi. Values are means ± SE of at least three replicates. Significant differences between the OE lines and the WT lines were compared using Tukey’s test. * Significant at P<0.05, ** Significant at P<0.01.

Fig 6. H₂O₂ accumulation, antioxidant enzyme activity, and defense-related gene expression in wild type (WT) and SlMAPK3-overexpressed (OE4, OE6, OE 7) lines after infection with TYLCV.

(A-F) H₂O₂ accumulation and antioxidant enzyme activity at 0, 10, 30, and 45 dpi. The second and third uppermost leaves were collected from each plant and analyzed for H₂O₂ content (A), SOD activity (B), POD activity (C), CAT activity (D), APX activity (E), and O₂^- content (F). Each bar represents the mean of three replicates ± standard error (SE). (G-K) Expression analysis of defense-related genes, SlPR1 (G), SlPR1b (H), SlLapA (I), SlPI-I (J) and SlPI-II (K) at 14 dpi with TYLCV. The SlEF1α gene was used as an internal control [59, 64]. Data are means ± SE (N = 3 to 9) of three independent experiments. Double asterisk (**) and single asterisk (*) indicate significant differences relative to controls at P< 0.01 (Tukey’s test) and P<0.05 (Tukey’s test), respectively.