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

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.


Introduction
Plants are threatened by a variety of abiotic and biotic stresses. Among biotic stresses, viruses are the most serious pathogens. Plants have evolved sophisticated mechanisms to defend a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 against viral infection by limiting virus replication and movement [1]. These mechanisms include resistance that is either induced or mediated by salicylic acid (SA), jasmonic acid (JA)-, and RNA interference (RNAi). All of these mechanisms play important roles in plant antiviral defense systems [2][3][4][5][6][7][8].
If they survive an initial pathogen attack, plants can exhibit enhanced resistance to subsequent infection by a broad range of pathogens. This induced resistance, which requires the endogenous plant hormone SA, is known as systemic acquired resistance (SAR) [9,10]. Exogenous application of certain natural or synthetic compounds [e.g., SA and Me (methyl) JA] can also induce resistance [11,12]. In plants, induced resistance is often associated with "cell priming" [13,14]. Priming enables cells to respond to less stimulus in a rapid and robust manner [15][16][17]. Priming is thought to be the basis of induced resistance to all plant pathogens [16,18]. Induced resistance can cause faster and stronger activation of defense responses when plants experience either biotic or abiotic stress [15]. A previous report indicated that MAPK3 and MAPK6 are critical for full priming of stress responses in Arabidopsis [19]. Previous studies have shown that MAPKs have an important role in defense against pathogens [20][21][22].
Transgenic methods offer great potential for enhancing the internal defense mechanisms of plants against viruses. However, consumers have not widely accepted genetically modified organisms (GMOs) because of concern about the effect of GMOs on human health. Recently, SA and JA were used in a nontransgenic approach to inhibit RNA viruses in tomato (Lycopersicon esculentum), hot pepper (Capsicum frutescens), and tobacco (Nicotiana benthamiana) [39]. It was reported that SA and JA inhibited not only virus replication but also cell-to-cell and long-distance movement of the virus [39,40]. One hypothesis is that SA and JA enhance plant resistance by triggering either induced resistance or SAR [18]. This kind of resistance needs the participation of MAPK [41][42][43].
Mitogen-activated protein kinase (MAPK) cascades are three-tiered signaling kinase modules [44,45]. Their main function is to transmit extracellular stimuli into intracellular responses [44]. MAPK  There is still uncertainty about the relationship between plant MAPKs and the SA-and JA-defense signal pathways in regard to antiviral activity. The objectives of this study were (i) to analyze the function of MAPK3 in the antiviral defense response of tomato to TYLCV and (ii) to learn more about the relationship between MAPK3 and the SA-and JA-defense signal pathways.

Plant materials and growth conditions
Tomato line 'Y19' (with Ty-1 and Ty-3 markers, S1 Fig), three transgenic lines with overexpression of SlMAPK3 (OE4, OE6, and OE7, Accession No. AY261514), and their wild-type (WT, 'M82') were used in this study. The three OE lines were from our lab and described previously [54]. The seeds were germinated on wet filter paper in Petri dishes in the dark at 28˚C for 3 d.

TYLCV inoculation
The TYLCV infectious clone was provided by Professor Zhou Xueping of Zhejiang University [55]. The clone was introduced into Agrobacterium GV3101 and then injected into the phloem of 6-week-old plants as described previously [56]. The injections were done with a 1.0 mL syringe at three points (10 cm apart) on the stem. The first injection point was 10 cm above the soil surface. Plants infected with empty vectors were used as controls. Virus infection was determined visually and confirmed through PCR (S2 Fig) [57]. Each treatment had 15-20 plants. Three biological replicates were performed for these experiments.

VIGS experiment
The pTRV1 and pTRV2 VIGS vectors were obtained from Dr. Dinesh-Kumar of Yale University [58]. Fragments of SlMPK1, SlMPK2, and SlMPK3 were amplified using specific primers containing XhoI (5' end) and SacI (3' end) sites and inserted into the pTRV2 vector. The TRV: PDS (phytoene desaturase) construct, which is used as a marker of VIGS silencing in plants, was made as described previously [59]. The constructs were introduced into Agrobacterium GV3101 by electroporation and injected into fully-expanded leaves of 3-week-old tomato plants according to Li et al. [59]. The primers used for construction of the vectors are listed in S1 Table. Silencing frequency (%) and silencing efficiency were calculated as described previously [59].

Signaling molecules and hormonal treatment
Tomato plants at the 5-leaf stage were treated by foliar spraying with either 10 mM H 2 O 2 , 100 μM MeJA, 100 μM SA, 100 μM ABA, or water (i.e, mock spray) [60]. The top leaves of the plants were collected at 0, 3, 6, 12, 24, 48, 72, and 96 h. The leaves were immediately frozen in liquid N and then stored at -80˚C for further analysis.

DNA / RNA isolation and quantitative PCR (qPCR)
Systemic leaves were collected from three TYLCV-inoculated plants and three uninoculated ones. The leaves were immediately frozen in liquid N and kept at -80˚C. Total DNA was extracted from the leaves using the cetyltrimethyl-ammonium bromide (CTAB) method [61]. Quantitative PCR (qPCR) was used to detect TYLCV in the total DNA samples as described previously [62]. The β-actin gene was used as a control for qPCR detection of TYLCV [62,63]. Total RNA was isolated using an RNA extraction kit (Invitrogen, USA). The cDNA was synthesized using MultiScribe reverse transcriptase (Takara, China). Quantitative real-time RT-PCR (qRT-PCR) was performed using SYBR Premix Ex Taq II (Takara, China) on an iQ5 Real-Time PCR Detection System (BIO-RAD, USA). The expression of the SlMAPK genes and the defense-related genes was determined using qRT-PCR. The elongation factor 1-α (SlEF1α) gene was used as an internal reference [59,64]. Three biological replicates were performed for these experiments. The gene specific primers for qRT-PCR are listed in S1 Table. Disease evaluation in transgenic plants Transgenic plants overexpressing SlMAPK3 (OE4, OE6 and OE7) were inoculated with TYLCV as described above. The TYLCV contents were detected by qPCR [62]. Three biological replicates were performed for these experiments. The number of flowers was counted at 45 dpi. Disease severity was evaluated using a rating scale of 0 to 4, in which, 0 = no disease symptoms, 1 = slight symptoms visible only on close inspection, 2 = symptoms apparent at a distance of two-thirds of a meter from the plant, 3 = severe symptoms over the entire plant, and 4 = severe symptoms and stunting [65]. Intermediate scores (e.g., 0.5, 1.5, and 2.5) were used to allow more precise rating [30].

Physiological parameters and enzyme activity
The activities of SlMAPK1, SlMAPK2 and SlMAPK3 were determined using an ELISA kit (Shanghai Biological Technology Co., Ltd.). Leaf H 2 O 2 was measured by the method of Jiang and Zhang (2001) [66]. Leaf chlorophyll and superoxide O 2 were measured according to the methods of Porra et al. (1989) [67] and Wang and Luo (1990) [68], respectively. The activities of the antioxidant enzymes catalase (CAT), peroxidase (POD), acerbate peroxidase (APX), and superoxide dismutase (SOD) were assayed from leaves as described previously [69].

Statistical analysis
Analysis of variance (ANOVA) was conducted using SPSS version 12.0 software. The significance of differences between means was determined by Tukey's test. Data are presented as means ± standard error (SE). Double ( ÃÃ ) and single ( Ã ) asterisks indicate significant differences relative to controls at P <0.01 and P <0.05, respectively. Different letters indicate significant differences compared to control at P <0.05.

Results
The RNA expression and protein activity of SlMAPK1, SlMAPK2 and SlMAPK3 was induced after infection with TYLCV The first step in this experiment was to analyze changes in SlMAPK expression after TYLCV infection. A preliminary study indicated using PCR showed 100% inoculation success using this method (data not shown). SlMAPK1, SlMAPK2, and SlMAPK3 expression was induced by TYLCV infection; however their expression levels were different (Fig 1A-1C). SlMAPK1, SlMAPK2, and SlMAPK3 expression reached peaks between 12 and 24 h post infection and then declined. The expression of SlMAPK1 and SlMAPK2 in TYLCV infected plants was 7.4 and 5.9 fold greater at 12 h than at 0 h post inoculation. SlMAPK3 expression was 56.5 fold greater at 24 h than at 0 h post inoculation. (Fig 1C).
The activities of SlMAPK1, SlMAPK2, and SlMAPK3 were analyzed by ELISA at different times after TYLCV inoculation. SlMAPK1, SlMAPK2, and SlMAPK3 were activated by TYLCV inoculation at 12 h after TYLCV inoculation (Fig 1). The activities of SlMAPK1 and SlMAPK3 were significantly greater than that of the control between 12 and 72 h after TYLCV inoculation. In comparison, SlMAPK2 activity was greater than that of the control between 12 and 48 h after TYLCV inoculation. These results indicated that SlMAPK1, SlMAPK2 and SlMAPK3 all responded to TYLCV infection at both the RNA and protein levels; however, the expression and activity levels were different. Among the three, SlMAPK3 had the strongest response to TYLCV infection.

Silencing of SlMAPK3 reduced tolerance to TYLCV
The second step of the experiment was to silence the SlMAPK genes by inoculating seedlings at The percentage of TRV: SlMAPK3-infiltrated plants exhibiting TYLCV symptoms increased to 56% at 14 dpi and 68% at 35 dpi (Fig 2A). These values were significantly greater than those of the control (TRV: 00). The percentage of TRV: SlMAPK1 and TRV: SlMAPK2-infiltrated plants exhibiting symptoms was not significantly different from the control. The disease index of TRV: SlMAPK3-infiltrated plants was 38.5 at 14 dpi and 42.0 at 35 dpi ( Fig 2B). These values were significantly greater than those of the control (2.2 at 14 dpi and 8.8 at 35 dpi).
The relative TYLCV content, which was determined by qPCR, increased with the development of disease ( Fig 2C). The relative TYLCV contents of SlMAPK2-and SlMAPK3-silenced plants were significantly greater than those of the control at 14, 21, and 28 dpi. SlMAPK3silenced plants had the highest relative TYLCV content in this study. It is interesting to note that the relative TYLCV content was significantly greater in SlMAPK1-silenced plants than in the control at 14 dpi. These data showed that SlMAPK silencing, particularly SlMAPK3 silencing, reduced resistance and increased susceptibility to TYLCV. Thus, SlMAPK3 might have an important role in regulating resistance against TYLCV in tomato.

Silencing of SlMAPKs reduced defense-related gene expression
The third step in the experiment was to compare defense-related gene expression in SlMAPKsilenced and non-silenced plants. The SA-and JA-mediated signaling pathways regulate the SlMAPK3-mediated response/tolerance to virus expression of certain defense marker genes. Specifically, the SA-mediated pathway regulates SlPR1 and SlPR1b. The JA-mediated signaling pathway regulates SlLapA, SlPII, and SlPIII. Therefore, the expression of these genes was analyzed to examine a possible molecular mechanism related to reduced TYLCV resistance in SlMAPK1-, SlMAPK2-and SlMAPK3-silenced plants. To do this, defense-related gene expression in TRV: SlMAPK1-, TRV: SlMAPK2-and TRV: SlMAPK3-silenced plants was compared with that in TRV-empty vector (TRV: 00)-infiltrated plants. SlMAPK-silencing had no effect on the activity of the SA-and JA-mediated defense genes (Fig 3). In contrast, SlMAPK-silenced plants with TYLCV inoculation exhibited significant differences in the expression of both SlPRP1 and SlPR1b at 14 dpi. The expressions of SlPRP1 and SlPR1b were greatest in TRV: 00 and least in TRV: SlMAPK3 (Fig 3B and 3C). Similar patterns were observed for SlLapA, SlPII, and SlPIII expression (Fig 3D and 3F). Overall, SlMAPK silencing, especially TRV:SlMAPK3 silencing, significantly reduced the expression  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.

Exogenous application of various signalling molecules induced SlMAPK3 expression
The fourth step in the experiment was to measure SlMAPK3 expression in tomato leaves after exogenous application of various signaling molecules. As shown in Fig 4, all four types of signaling molecules significantly increased SIMAPK3 expression at 12 h after application. Among the signaling molecules, exogenous MeJA had the most striking effect, increasing SIMAPK3 expression at each sampling time between 6 and 96 h after application. SlMAPK3 expression in the MeJA treatment reached a maximum 13.4 fold increase at 96 h. Exogenous SA significantly increased SlMAPK3 expression at 12, 24, 48, and 96 h. The maximum increase (5.2 fold) was observed at 12 h. Exogenous ABA and H 2 O 2 significantly increased SlMAPK3 expression at 12 h by 3.6 and 2.9 fold, respectively. The results indicated that SlMAPK3 responded significantly, but with different expression patterns, to exogenous MeJA and SA. This suggested that SlMAPK3 could be involved in stress-activated signaling pathways regulated by SA and MeJA.

SlMAPK3 overexpression enhanced tolerance to TYLCV
To further confirm the role of SlMAPK3 in antiviral defense, the TYLCV tolerance of three overexpression lines (i.e., OE4, OE6, and OE7) was compared with that of a WT line. No visible disease symptoms were observed on any of the plants at 10 dpi (Fig 5A). The WT line had typical TYLCV symptoms at 30 dpi, whereas the OE lines remained normal and developed flowers. At 45 dpi, the OE lines exhibited TYLCV symptoms but produced normal flowers. The WT line exhibited severe disease symptoms and was unable to produce normal flowers. Disease severity ratings reflected the patterns described above (Table 1). There was no difference in the disease ratings at 10 dpi. However, the ratings were significantly greater in the WT line than in the OE lines at 45 dpi.
The relative TYLCV contents were the same in all lines at 10 dpi ( Fig 5B). However, relative TYLCV contents were significantly less in the OE lines than in the WT line at 10, 30, and 45 dpi. At 45 dpi, relative TYLCV contents increased in the OE lines and produced visible symptoms (Fig 5B). One visible symptom of TYLCD is leaf yellowing due to a reduction in the number of chloroplasts per cell [70]. Leaf chlorophyll contents were significantly greater in the OE lines than in the WT line at 30 and 45 dpi (Fig 5C). The number of normal flowers was significantly greater in the OE lines than in the WT line at 45 dpi (Fig 5D). Overall, these results showed that the appearance of TYLCD symptoms was delayed in plants with SlMAPK3 overexpression.

SlMAPK3 over-expression enhanced antioxidant capacity and defenserelated gene expression
Previous studies have shown that biotic and abiotic stresses damage plants through accumulation of ROS during oxidative stress [71,72]. In this experiment, there were no clear differences SlMAPK3-mediated response/tolerance to virus   (Fig 6A and 6B). Plants have evolved complicated antioxidant defense systems to clear excess ROS and maintain cellular ROS homeostasis [71,72,73]. The enzymes CAT, SOD, APX, and POD are involved in the antioxidant defense system. There were no observable differences in enzyme activity between the WT and OE lines prior to TYLCV inoculation (Fig 6C-6F). However, the OE lines compared with the WT plants had significantly greater enzyme activity after TYLCV inoculation. These results suggested that SlMAPK3 overexpression inhibited ROS production and had major influence on antioxidant capacity in transgenic plants.
The expression of five genes related to plant defense was also determined to increase insight into the molecular mechanisms underlying enhanced tolerance to TYLCV in the OE lines after inoculation. Without TYLCV inoculation, the relative expression of all five defense genes were greater in the OE lines than in the WT line (Fig 6). Inoculation with TYLCV significantly upregulated the genes in all lines; the greatest increases were observed in the OE lines (Fig 6G-6K). This demonstrated that SlMAPK3 over-expression enhanced the transcript levels of SAand MeJA-mediated defense-related genes in both TYLCV inoculated and uninoculated plants.

Discussion
SlMAPK3 participated in an antiviral response to TYLCV and was induced by SA and JA hormones. VIGS-silencing of SlMAPK3 increased viral infection compared with non-silenced plants. SlMAPK3 silencing also reduced expression of defense-related genes in the SA-and JAmediated pathways. Compared with WT plants, over-expression of SlMAPK3 in transgenic plants enhanced the expression of defense-related genes in the SA-and JA-mediated pathways and increased tolerance to TYLCV. These results suggest that MAPK3 participates in the defense response to TYLCV. We propose that the antiviral role of MAPK3 could be attributed to induced resistance triggered by the SA and JA signal pathways.
In this study, the genes downstream of the MAPKs changed with the down-regulation of MAPK in silenced plants (Fig 3). This agrees with a previous report that PI-II and PI-I were down-regulated in SpMPK3-silenced plants [84]. SlMAPK1-, SlMAPK2-and SlMAPK3silenced plants all exhibited increased TYLCV content and reduced tolerance to TYLCV; however, SlMAPK3-silenced plants had the greatest disease incidence (Fig 2). These results were consistent with a previous study in which suppression of both MPK3 and MPK6 in transgenic Arabidopsis resulted in significant decreases in the induction of defense-related gene expression and pathogen resistance compared with wild-type plants [82]. Disease development and wilting symptoms of Ralstonia solanacearum also appeared more often in MPK3 silenced plants [81]. The VIGS-silencing experiment implied that the role of SlMAPK3 in the defense response was somewhat different from that of SlMAPK1 and SlMAPK2. This agrees with a previous report which showed that MPK3 functioned differently than MPK1 and MPK2 in the response of tomato to wounds caused by Manduca sexta (Lepidoptera) [83]. These results suggested that although they belong to the same gene family, the response of SlMAPK3 to TYLCV was different from that of SlMAPK1 and SlMAPK2.
Tomato is infected with TYLCV by viruliferous whiteflies. Many factors (e.g., whitefly gender and development stage) influence the efficacy and stability of the inoculation. For this reason, we used an artificial system of TYLCV infection by agroinfiltration in this study. It should be noted that TYLCV transmission by whiteflies is circular and persistent [85]. Prolonged infection may result in a different response compared with our study. Previous research indicated that the abundance of mammalian extracellular signal-regulated kinase (ERK)-like protein (representing MAPKs) remained high for 1 d and then decreased slowly for 40 d after whitefly infestation [86]. This suggested that (i) ERK-like proteins negatively regulate the defense response or (ii) TYLCV can suppress the activity of the ERK-like protein after 1 dpi. Additional research needs to be done to determine SlMAPKs expression patterns in tomato inoculated by whiteflies. If there is decrease in SlMAPKs levels during prolonged infection, it would suggest that the MAPK response to stress was complete within a short time after infection.
Upstream signaling components such as ROS, auxin, abscisic acid, and phosphatidic acid have been reported to be involved in MAPK activation [87]. In our results, the WT line compared with the OE lines accumulated more H 2 O 2 , leading to decreases in total leaf chlorophyll ( Fig 5C). The ROS can not only induce hypersensitive response (HR) due to damage after pathogen infection, but also inhibit the spread of cell death to neighboring cells by programmed cell death. Previous studies have reported that TYLCV infection does not induce HR in normal plants [88]. It is possible that the immune system becomes weaker in SlMAPK3silenced plants, leading to HR-like lesions. Similarly, Faoro and Gozzo reported in their review that compatible viruses can also cause systemic necrosis leading to plant death [40,89]. In addition, the activity of the antioxidant enzyme APX was enhanced after TYLCV inoculation in SlMAPK3-overexpressed plants compared with WT plants (Fig 6E). Recent studies reported that TYLCV down regulated APX1/2 transcription through mitigation of its regulator heat shock transcription factors HSFA2 under a combination of virus and heat stresses [90]. It is possible that the antiviral response of plants to a single stress is different than that to a combination stresses.
In plant immune systems, MPK3/MPK6 activation and rapid ROS burst are two independent, early signaling events [91]. Accumulation of H 2 O 2 could lead to SA synthesis [92]; however TYLCV also enhanced SA accumulation during the early stages of infection [62]. High levels of SA along with H 2 O 2 could activate local PR gene expression [93]. In effector-triggered immunity (ETI), the MPK3 and SA signaling pathways compensate each other regarding PR1 expression and pathogen inhibition [94]. Similarly, VIGS of MPK1, MPK2, and MPK3 in tomato resulted in reductions in JA-mediated defense gene expression and defense responses [84]. We observed that the expression of SA-and JA-mediated defense-related genes was reduced in silenced plants after TYLCV inoculation. The largest decreases were in SlMAPK3silenced plants (Fig 3). Exogenous SA and JA application induced SlMAPK3 expression. This suggested that MAPK3 was linked with both pathways; however, it may be that MAPK3 had a role in balancing or reducing antagonism between SA and JA signaling. Modulated interactions between SA and JA may contribute significantly to induced resistance [95]. Molecular characterization of MAPK3 in Nicotiana attenuate demonstrated that WIPK and SIPK were orthologs of AtMPK3 and AtMPK6, respectively, and were involved in JA and SA signaling pathways and biosynthesis [76]. However, OsMPK3 transcripts increased after treatment with JA but not SA [50].
In Arabidopsis, AtMPK3 and AtMPK6 positively regulated SA signaling [96]. After stress, phosphorylated active AtMPK3 and AtMPK6 were both correlated with enhanced expression of the PR-1 gene [19]. Over-expression of a tobacco MPK3 homolog (WlPK) resulted in enhanced JA levels and expression of the JA responsive gene P1-II [97]. Similarly, AhMPK3 over-expression increased PI-II expression, the amount of a basic pathogenesis-related protein, and plant resistance [98]. We observed that SlMAPK3 transgenic plants compared with WT plants had greater expression of SA-and JA-defense-related genes and less TYLCV content. The reduction of TYLCV in SlMAPK3 over-expressed plants may be due to increases in either the accumulation of SA and JA or the expression of defense-related genes in both pathways. Exogenous application of both SA and JA induced stronger resistance in tobacco against virus attack compared with application of SA or JA alone [39]. Previous reports indicate that SA not only has significant roles in the RNA silencing mechanism but also delays accumulation of RNA pathogens [99], perhaps due to pre-induction of RNA silencing-related genes by SA or GA [100]. The SA could act as an enhancer of RNA-silencing antiviral defense. An SA-mediated defense mechanism and an RNA-silencing mechanism acted together to reduce plum pox virus (PPV) infection in tobacco [101]. Based on these previous reports as well as our own findings, we propose that (i) MAPK3 could be an essential component in inducing "priming" of cells in virus-infected plants and (ii) MAPK3 plays an important role in the development of induced resistance against viruses by coordinating the expression of defense genes in SA and JA-mediated pathways. It should be noted that RNA silencing is thought to be an important antiviral defense mechanism. There is a possibility that SlMAPK3 also acts as an upstream signaling kinase, triggering the RNA silencing pathway against TYLCV. Further in-depth study is required to test this hypothesis.

Conclusions
SlMAPK (SlMAPK1, SlMAPK2 and SlMAPK3) transcription and activity in tomato leaves was strongly induced by TYLCV infection, with SlMAPK3 having the highest expression and activity among the three genes. Functional analyses by VIGS and overexpression showed that SlMAPK3 may participate in regulating the defense response against TYLCV by modulating SA and JA-mediated defense responses in tomato.