Identification of the tomato pathogen-induced ortholog of Arabidopsis thaliana S5H
To identify the enzyme responsible for the conversion of SA into GA in tomato, a Blastp analysis was performed in Sol Genomics databases (https://solgenomics.net/) by using the AtS5H/DMR6 sequence (At5g24530). A phylogenetic tree was built including the closest tomato sequences (Supplemental Fig. S1). The Solyc03g080190 was selected as a candidate for SH5 role in tomato, since it resulted to be one of the closest in the phylogenetic tree, and it presented the highest identity percentage (67.35%), in contrast to the 62.43% of identity displayed by the Solyc06g073080 sequence, also near in the phylogenetic tree. The Blastp analysis of the Solyc03g080190 in The Arabidopsis Information Resource (www.arabidopsis.org) confirmed the selected candidate, being AtS5H/DMR6 the closest gene to Solyc03g080190 (SlS5H) in Arabidopsis thaliana. Finally, this sequence coincided with At5g24530 tomato ortholog proposed by EnsemblePlants (http://plants.ensembl.org/index.html) and with SlDMR6-1, which has been recently proposed as the DMR6 ortholog in tomato and which displayed salicylic acid 5-hydroxylase activity [24].
According to available transcriptome data, SlDMR6-1 expression has been described to be induced in response to several pathogens [24]. To confirm the pathogen triggered SlS5H induction, and to extend the study of expression to other pathogens which provoke the accumulation of SA and GA, tomato plants were subjected to infection either with Citrus Exocortis Viroid (CEVd), Tomato Spotted Wilt Virus (TSWV), Tomato Mosaic Virus (ToMV) or a virulent and an avirulent strain of Pseudomonas syringae pv. tomato DC3000 (Pst) (see Materials and Methods). Leaf samples were collected at the indicated time points and SlS5H expression levels were analysed by qRT-PCR in those tomato-pathogen interactions (Fig. 1 A to D). The induction of SlS5H was observed upon all the pathogen infections in the analysed samples, reaching levels up to 5 times higher in CEVd-infected or Pst-infected plants than those observed in the non-infected plants, and around 2 times higher in the case of TSWV or ToMV infections. It is worthy to note that these induction patterns correlated with symptomatology, producing infection with CEVd the most severe disease symptoms and being ToMV infection practically symptomless [39]. Regarding the bacterial infection, SlS5H expression levels were higher in the tomato plants inoculated with the virulent bacteria (Pst DC3000 ΔAvrPto) as compared to the avirulent infection at 48 h post inoculation (hpi), therefore confirming this tendency.
To study the induction of SlS5H by its own substrate, SA treatments were performed, and samples were collected at different time points (see Materials and Methods). As Fig. S2A shows, a statistically significant induction of SlS5H was detected by qRT-PCR at 6 h post treatment (hpt) when compared with non-treated plants, presenting the maximal induction at 1 hpt. PR1 activation was used as a positive control for the treatments, presenting a significant induction at 24 hpt (Fig. S2B).
All these data confirm that SlS5H is involved in the plant response to pathogens, extending its putative role to different tomato-pathogen interactions.
Overexpression of Sl5H in Nicotiana benthamiana decreases SA levels in vivo
To confirm the S5H biochemical activity in vivo, Nicotiana benthamiana plants were agroinoculated with a construction carrying the SlS5H cDNA containing a His tag under the 35S CaMV promoter. These plants (pGWB8-SlS5H) and the corresponding control plants inoculated with the empty plasmid (pGWB8) were then embedded with SA (see Materials and Methods). The accumulation of the recombinant protein was confirmed by western blot analysis in pGWB8-SlS5H plants, and levels of free and total SA were measured (Fig. S3). As expected, levels of free and total SA were almost 3 times lower in pGWB8-SlS5H plants, being these differences statistically significant, and thus confirming that SA is a substrate for SlS5H in vivo. However, no differences in neither the GA nor in 2,3-DHBA accumulation were detected between pGWB8 and pGWB8-SlS5H Nicotiana benthamiana plants (Fig. S3D).
Silencing SlS5H increases resistance to CEVd in tomato
To gain further insights into the in vivo role of SlS5H, silenced transgenic Moneymaker tomato plants were generated by following an RNAi strategy (see Materials and Methods). The generated tomato lines RNAi_SlS5H were characterized, and several independent transgenic lines were confirmed. Homozygous lines RNAi_SlS5H 14 and RNAi_SlS5H 16 both carrying one copy of the transgene, were selected for further studies.
To extend the role of SlS5H in plant defence, the tomato-CEVd interaction was selected, since GA -the proposed product of S5H activity- had been described to accumulate at very high levels in CEVd-infected tomato plants [33, 36,37,38]. Therefore, wild type (WT) and RNAi_SlS5H transgenic plants were inoculated with CEVd and checked for the development of symptoms. The characteristic symptomatology of CEVd-infected tomato plants consists of epinasty, stunting, leaf rugosity, midvein necrosis and chlorosis [40]. As Fig. 2A shows, differences in the percentage of plants displaying symptoms were observed in both RNAi_SlS5H transgenic lines with respect to the parental tomato plants. Particularly, transgenic line RNAi_SlS5H 16 displayed only 35% of plants showing symptoms at 1.9 weeks post inoculation (wpi), while almost 75% of non-transgenic plants exhibited them. Moreover, at 2.3 wpi all the WT plants displayed symptoms while around 20% of the RNAi_SlS5H remained symptomless. These results indicate a delay in symptom appearance in CEVd-infected RNAi_SlS5H tomato plants.
To confirm the differences in the disease development observed, a scale of the disease severity was established, scoring symptoms from mild (mild epinasty) to very severe (midvein necrosis and chlorosis), at different time points (see Materials and Methods). As Fig. 2B shows, differences were observed between WT and RNAi_SlS5H transgenic tomato plants from 2.3 to 3.6 wpi. Moreover, RNAi_SlS5H 16 transgenic line did not display very severe symptoms at 3 wpi, whilst 30% parental plants exhibited severe symptoms at the same time point. Therefore, the observed differences in symptom severity confirmed the partial reduction in the susceptibility of RNAi_SlS5H tomato plants to CEVd infection. Interestingly, transgenic plants appeared to display a higher internode length when compared to the control plants. This phenotype was quantified as significant in further studies (Fig. S6B).
Finally, to confirm the enhanced resistance, the presence of pathogen was measured at 3 weeks post-inoculation (wpi), detecting a statistically significant decrease in the CEVd accumulation in both RNAi_SlS5H transgenic lines (Fig. 3A).
Our results appear to indicate S5H silencing reduces tomato susceptibility to CEVd, confirming the role of SlS5H in the plant defence response.
Silencing SlS5H causes an activation of plant defence upon CEVd infection
To analyse SlS5H expression levels in the RNAi transgenic plants infected with CEVd, qRT-PCR from WT, RNAi_SlS5H 14 and RNAi_SlS5H 16 plants, were performed at 2 weeks after the inoculation with CEVd (Fig. 3B). Levels of SlS5H expression were significantly lower in both mock and viroid infected transgenic lines, than the corresponding WT tomato plants. To find out if RNAi_SlS5H transgenic lines exhibited an activation of the defensive response against CEVd, the expression of the pathogenesis related protein 1 (PR1; accession X71592), which has been described as a classical marker of plant defence rapidly induced in CEVd-infected tomato plants [41, 42], was also studied by qRT-PCR at 2 wpi. As expected, PR1 was induced by CEVd in WT plants, but also in both RNAi_SlS5H transgenic lines (Fig. 3C). Interestingly, levels of expression of PR1 were already higher in mock-infected SlS5H-silenced tomato plants. These results appear to indicate that SlS5H silencing provokes the activation of the SA-mediated plant defence.
To better characterise the plant response of the RNAi_SlS5H transgenic lines upon CEVd infection, the expression of several defence genes was also studied at 3 wpi both in mock and infected plants. Regarding the gene silencing mechanisms that are activated by CEVd in WT plants, a significant reduction in DCL1 and DCL2 induction was observed in CEVd-infected RNAi_SlS5H lines when compared to the corresponding WT plants, thus indicating a correlation between the amount of CEVd and the induction of these two dicers (Fig. S4A and B). However, no significant differences were observed in the RDR1 induction pattern in the transgenic plants, which displayed a significant induction of this gene upon viroid infection (Fig. S4C). As far as jasmonic acid (JA) response is concerned, a statistically significant reduction of the JA-induced proteinase inhibitor TCI21 [43], was observed in both mock-inoculated RNAi_SlS5H lines when compared with WT (Fig. S4D), indicating that the final JA-mediated response is repressed in these tomato transgenic plants.
Therefore, we have observed that RNAi_SlS5H transgenic lines display constitutive TCI21 repression as well as PR1 overexpression, thus suggesting an increase of SA levels in these transgenic lines.
Levels of SA and GA are altered in RNAi_SlS5H transgenic tomato plants upon CEVd infection
Free and total levels of SA and its hydroxylated product GA were quantified in the wild-type and RNAi_SlS5H transgenic lines upon viroid infection at 3 wpi (Fig. 3D and E). As expected, free and total SA and GA levels were higher in all the CEVd-infected plants.
In CEVd-inoculated plants, levels of total SA in both RNAi_SlS5H transgenic infected lines resulted to be significantly higher when compared with those observed in control infected plants, reaching 1000 nmol/g fresh weight whilst levels in control infected plants barely reached 400 nmol/g fresh weight (Fig. 3D). SA levels in non-pathogenic (mock) conditions were slightly higher in both transgenic lines, being statistically not significant when compared with wild-type plants.
Once detected the over-accumulation of SA, we studied the levels of GA, which is the product of the S5H activity. As Fig. 3E shows, a drastic reduction of total GA levels was observed upon viroid-infected in both RNAi_SlS5H transgenic lines as compared to the levels detected in the infected WT tomato plants (10-fold). Interestingly, this significant reduction was also observed in free GA corresponding to mock conditions. The presence of 2,3-DHBA was measured in all samples, and the levels were negligible. The higher levels of SA and the lower accumulation of GA found in the CEVd-infected RNAi_SlS5H transgenic lines confirm the decrease of the salicylate 5-hydroxylase activity in vivo and explain the observed enhanced resistance and activation of the SA-mediated plant defence in these transgenic plants.
Infection with Pseudomonas syringae pv. tomato DC3000 produces specific phenolic accumulation pattern in RNAi_SlS5H transgenic tomato plants
Bacterial infection of tomato plants produces lower accumulation of SA when compared with levels produced by CEVd infection [37]. To study the role of SlS5H in this tomato-pathogen interaction, WT and RNAi_SlS5H transgenic tomato plants were infected with the virulent strain of Pst, and bacterial counting from infected leaves was carried out at 24 h after infection (hpi). As shown in Fig. 4 A, a significant 10-fold decrease in the number of colony forming units (CFUs) was observed in infected tissues of the different transgenic lines with respect to their genetic background, confirming that transgenic plants RNAi_SlS5H also exhibited resistance against Pst.
SlS5H silencing in tomato plants upon bacterial infection was also confirmed by qRT-PCR, being differences in SlS5H expression in bacterial infected RNAi_SlS5H transgenic plants statistically significant compared to the expression levels observed in the infected WT (Fig. 4B). Similar to what was observed upon viroid infection; PR1 expression was already higher in mock-inoculated SlS5H-silenced tomato plants and was significantly induced by bacteria in both WT and RNAi_SlS5H transgenic lines (Fig. 4C).
In a similar manner to that performed with CEVd-infected plants, SA and GA levels were measured in both WT and RNAi_SlS5H transgenic plants upon bacterial infection (Fig. 4D and E). In Pst-tomato interaction, the levels of free and total SA were induced by the hemibiotrophic pathogen in all the analysed plants. As previously observed in mock-inoculated plants for CEVd infection, no statistically significant differences were observed in mock-inoculated transgenic plants, when compared with WT. Nevertheless, the slight overaccumulation of free SA in both mock-inoculated transgenic plants could explain the PR1 induction observed in these plants (Fig. 4C). Strikingly unlike what was observed upon CEVd infection, the bacteria produced a significant reduction of free and total SA levels in both RNAi_SlS5H transgenic lines when compared with levels observed in Pst-infected WT plants (Fig. 4D). Regarding GA, only WT tomato plants showed a statistical accumulation of total GA levels after bacterial infection, thus indicating that the product of SlS5H is also reduced in RNAi_SlS5H transgenic plants upon bacterial infection (Fig. 4E).
The reduction of SA levels observed in RNAi_SlS5H transgenic lines upon bacterial infection indicates that SA undergoes a specific catabolic process upon pathogen infection in these transgenic plants.
A metabolomic analysis of the RNAi_SlS5H transgenic tomato plants upon viroid and bacterial infection reveals differences in SA metabolism upon pathogen attack
To better understand the SA metabolism in RNAi_SlS5H transgenic lines upon each infection, a metabolomic study based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was performed. For viroid infection, 8-day-old tomato plants were used, and samples were collected 3 weeks after CEVd inoculation (wpi), while bacterial infection was carried out on 5-week-old tomato plants and the harvesting time was 24 h after Pst infiltration (hpi). Then, hydroalcoholic extracts from control and infected RNAi_SlS5H tomato leaves were analysed by UPLC-MS, and multivariate data analysis was employed to deal with the large number of mass data. Specifically, a principal component analysis (PCA) was first applied to identify metabolic changes after viroid and bacterial infection of tomato plants (Fig. 5A). An extensive separation between both tomato interactions was observed by PC1 due to the different experimental conditions: temperature and plant age. Reaching the PC3, the metabolic changes in tomato leaves produced by both pathogens were explained, being greater those caused by viroid inoculation. To exclude the differences due to this biological variability, two different PCA were therefore applied separately on each tomato-interaction.
In particular, the first two components of the PCA score plot of viroid-tomato interaction (Fig. S5A) divided the observations by the infection (mock vs. infected plants; PC1) and genotype (WT vs. transgenic plants; PC2). In order to elucidate the SA metabolism in tomato plants against CEVd, a PCA of both infected WT and transgenic RNAi_SlS5H plants was performed (Fig. 5B). For the identification of the metabolites accumulated in the infected SlS5H silenced lines (14 and 16), the positive PC1 loading plot was analysed. Interestingly, the glycosylated form of SA (SAG) was the most accumulated compound in the transgenic plants (fold change transgenic lines vs. WT: 3.0; p-value 0,009). These results are in accordance with the total SA accumulation measured by HPLC-fluorescence in CEVd infected RNAi_SlS5H plants (Fig. 3D).
In the case of bacteria-tomato interaction, the PC3 of PCA (Fig. S5B) explained the different metabolic content of transgenic tomato plants from WT, while PC1 clearly discriminated the metabolome of the infected RNAi_SlS5H line 16. Similarly to CEVd interaction, the PCA of infected tomato plants was required to investigate the role of SA in the bacterial infected tomato plants (Fig. 5C). By analysing the negative PC1 and PC2 loading plot, the metabolites accumulated in both transgenic lines were identified. In contrast to CEVd infection, SAG accumulation was not induced by Pst in both transgenic lines according to the results obtained using fluorescence-based detection (Fig. 4D). In tomato-bacteria interaction, some of the compounds over-accumulated in the transgenic infected leaves were identified as feruloyldopamine (fold change between transgenic lines and WT: 5.3; p-value 0.003), feruloylquinic acid (fold change: 5.1; p-value 0.006), feruloylgalactarate (fold change: 3.4; p-value 0.01) and 2-hydroxyglutarate (fold change: 1.4; p-value 0.04).
SlS5H silencing reveals differences in SA biosynthesis gene expression upon pathogen attack
To study differential expression of genes participating in SA biosynthesis and how they are affected by silencing SlS5H, qRT-PCR were performed for ICS (Solyc06g071030 or XM_019214145), PAL (Solyc09g007890 or NM_001320040), EPS1 (Solyc08g005890 or XP_004244447), SAMT [44], and the glycosyltransferases of phenolic compounds GAGT [31] and Twi1 [45]in samples corresponding to CEVd or Pst infections for both WT and RNAi_SlS5H transgenic plants (Fig. 6).
As Fig. 6 A shows, a significant induction in ICS was observed in WT plants upon CEVd infection, being that induction impaired in the transgenic lines, thus suggesting that the SA biosynthesis is down-regulated when the SA hydroxylation is prevented, which may lead to SA over-accumulation. Contrasting with CEVd infection, a clear reduction of ICS expression was detected upon bacterial infection, in both WT and transgenic lines. However, this decrease of expression was not statistically significant in the transgenic plants (Fig. 6B).
As far as PAL pattern of expression is concerned, whilst CEVd infection caused a significant reduction in both transgenic lines compared to infected WT, no significant differences caused by Pst infection were observed (Fig. 6 C and D).
The last step in the SA biosynthesis through the isochorismate pathway involves the conversion of isochorismate-9-glutamate into SA, being performed by EPS1. Whilst WT plants appear not to display any significant induction of EPS1 by CEVd, bacterial infection clearly provoked the induction of this gene. This pattern was completely opposite in both RNAi_SlS5H transgenic lines, since they showed a slight induction of EPS1 upon CEVd infection, being impaired in the expression of EPS1 upon bacterial infection (Fig. 6E, F).
Moreover, a reduction of SAMT expression upon CEVd infection was measured in all the genotypes, with no significant differences observed between WT and transgenic plants. In contrast, SAMT induction caused by bacterial infection was lower in transgenic plants, showing statistically significant differences between the induction observed in infected WT and RNAi_SlS5H 16 transgenic plants (Fig. 6H).
Finally, a far as the glycosyltransferases of phenolic compounds GAGT and Twi1 are concerned, whilst no statistical differences were observed in the transgenic plants infected with Pst, a slight increase in the induction of both glycosyltransferases was observed upon viroid infection (Fig. 6I-L).
Although the differences observed in both viroid and bacterial infections were not statistically significant for all the analysed genes, a noticeable variation in the pattern of expression of several genes participating in SA metabolism was detected upon infection by these two pathogens, thus suggesting that SA homeostasis has specific differences for each tomato-pathogen interaction.
SlS5H silencing causes repression of the JA defence response
Once confirmed that SlS5H silencing provokes an activation of the SA-mediated defence, we studied the possible cross-talk with the JA-mediated response. To that purpose, control and RNAi_SlS5H transgenic lines were wounded, and the induction of TCI21 was studied at 24 h after wounding (Fig. 7A). As expected, TCI21 was highly induced by wounding in WT plants, whilst RNAi_SlS5H wounded plants displayed only a slight induction of TCI21, which was comparable to non-wounded plants, thus indicating that JA response is repressed in these transgenic plants. Interestingly, a higher induction of PR1 was detected in the SlS5H-silenced plants both in non-wounded and upon wounding when compared with the WT plants (Fig. 7B), reinforcing the observed activation of SA-mediated defence in the RNAi_SlS5H transgenic plants.
Since JA response appeared to be repressed in RNAi_SlS5H transgenic lines, the phenotype against the necrotrophic fungal pathogen Botrytis cinerea was explored (Fig. 8A). Although no statistically significant differences were found in the size of necrotic lesions between the transgenic lines and the corresponding parental plants (Fig. 8B), both RNAi_SlS5H transgenic lines displayed an increase in the susceptibility against Botrytis cinerea, as suggested by the increase in the yellowish area shown by the transgenic plants. To better quantify this effect, chlorophyll content was measured in control and transgenic plants infected with the fungus. As Fig. 8C shows, RNAi_SlS5H transgenic lines accumulated significant lower levels of chlorophyll B and total chlorophylls, therefore confirming the observed increased susceptibility of RNAi_SlS5H transgenic lines against Botrytis cinerea.
Silencing of SlS5H results in early senescence
Previous research on salicylate hydroxylase activity in Arabidopsis reported that the deficiency in this enzyme provokes an advanced senescent response [21, 23]. However, no noticeable phenotypic differences have been reported in SlDMR6-1 tomato mutants [24].
To study the developmental phenotype of RNAi_SlS5H transgenic tomato plants, 5 individuals for each genotype were grown for 10 weeks, and the percentages of leaflets displaying different senescence stages were recorded. As Fig. 9 A shows, RNAi_SlS5H transgenic plants displayed and earlier chlorosis, even leading to the leaf collapse. Particularly, in 10-week-old plants we observed that control plants still possess approximately 50% of the green leaves, while hardly any leaf of the transgenic lines remained green, turning the entire observed leaves to different intensities of yellow and brown, eventually leading to leaf fall (Fig. 9B).
To better characterize the phenotype of the RNAi_SlS5H transgenic tomato plants, we measured weight and conductivity at 10 weeks after germination, observing a significant weight reduction in the transgenic plants (Fig. S6A). Moreover, electrolyte leakage, which is a hallmark of cell death, was also significantly increased in both transgenic lines silencing SlS5H (Fig. S6C). Finally, the chlorophyll content was also measured, observing significant lower levels of chlorophyll B and total chlorophylls in RNAi_SlS5H transgenic tomato leaves (Fig. S6D).
To reinforce the data obtained by phenotypic visualization, a gene expression analysis for PR1 as well as the senescence markers SAG12 (AT5G45890 tomato ortholog; Solyc02g076910) and NOR [46] was carried out by qRT-PCR at 4 weeks after germination. As shown in Fig. 9C, a significant increase of PR1 expression was observed in RNAi_SlS5H transgenic lines with respect to control plants, which correlates with the higher SA levels previously observed in mock-inoculated plants (Fig. 4D). In parallel with PR1 expression levels, both senescence markers SAG12 (Fig. 9D) and NOR (Fig. 9E) were differentially upregulated in the RNAi_SlS5H transgenic leaves, confirming the developmental phenotype of early senescence observed in these plants.
Levels of free and total SA and GA were measured at different stages of development (Fig. 10). To that purpose, samples were collected at the indicated time points and free and total levels of both phenolics were measured by HPLC in both control and RNAi_SlS5H transgenic tomato plants. In general, levels of SA were higher in the transgenic plants impaired in 5-hydroxylation, being differences in total SA with control plants statistically significant at 10 weeks after germination. However, levels of GA were found to be lower at every time points, being total GA levels statistically significant from 6 weeks after germination, which confirms that the reduction of GA in these transgenic plants is consistent and reproducible in all the analysed samples.
Therefore, our results appear to indicate that over-accumulation of SA in the RNAi_SlS5H transgenic tomato plants provokes toxicity, which may explain their lower growth rates and early senescence.