Cloning and phylogenetic analysis of SlERF01
The full-length CDS of SlERF01 was cloned by PCR using cDNA derived from tomato (Table S1 for PCR primers). The CDS of SlERF01 encodes a 240 amino acid protein that has one AP2/ERF domain and belongs to the ERF TF B-3 family (Fig. 1a). Database analysis of conserved protein sequences showed that SlERF01 contains an ERF domain, a putative nuclear localization signal (NLS) and a putative activation domain (AD), among which only the ERF domain is conserved between SlERF01 and other ERF proteins (Fig. 1b). Further analysis showed that SlERF01 shares low similarity with other ERF proteins in terms of their whole putative protein sequences; however, sequence alignment showed a high degree of homology in the ERF domain regions. Thus, the phylogenetic analysis results showed that SlERF01 may encode a novel ERF protein that participates in the disease resistance response.
Subcellular localization of SlERF01
A SlERF01-GFP fusion was constructed. The SlERF01::GFP fusion gene was transformed into the A. tumefaciens GV3101 strain with the empty GFP vector as a negative control, and N. benthamiana leaves were then infected. The results showed that SlERF01 was localized in the nucleus (Fig. 2).
SlERF01 improves disease resistance against S. lycopersici in tomato
To identify the function of SlERF01 in S. lycopersici resistance in tomato, overexpression (OE) and TRV-mediated VIGS vectors were constructed for further analysis. Finally, 3 SlERF01-overexpressing tomato lines with the highest expression (lines 5, 11 and 15) and 3 TRV lines with the lowest expression (lines 3, 7 and 8) were generated for further analysis (Fig. 3). Overexpression of SlERF01 resulted in a typical HR phenotype at 3 dpi with S. lycopersici, and the susceptibility symptoms of SIERF01-overexpressing transgenic plants (OE) were significantly less severe than those of susceptible plants. The transgenic line exhibited enhanced resistance to S. lycopersici infection compared with that of the empty vector control (35s::00).
Furthermore, the HR was found to be weaker and slower in SlERF01-silenced plants (TRV) than in empty vector plants (TRV::00). Typical disease lesions were observed on SlERF01-silenced plants at 3 dpi, and no obvious susceptible symptoms were observed on the leaves from the TRV::00 plant (Fig. 3a). Furthermore, necrotic lesions and perforated center symptoms were evident on the leaves of susceptible plants. These results indicated that SlERF01 promoted S. lycopersici resistance in tomato.
The effects of disease resistance in tomato were also evaluated by examining HR-related cell death and accumulation of H2O2, lignin, and callose by staining with trypan blue, DAB, TB and AB, respectively. For trypan blue staining, a strong HR at 3 dpi with S. lycopersici was observed in SlERF01-overexpressing (35s::SlERF01) plants. In contrast, no visible HR was observed in the empty vector (35s::00) plants at 3 dpi; the hyphae gradually grew, and the lesions were aggravated and transparent. In contrast to the overexpression plants, the leaves from the SlERF01-silenced plants were sensitive to S. lycopersici infection. HR was impaired in TRV::SlERF01 plants compared to TRV::00 plants at 3 dpi with S. lycopersici; hyphal spreading was observed, and the lesions were aggravated and perforated. However, a strong HR was observed on the leaves from TRV::00 plants (Fig. 4). These results showed that SlERF01 can trigger HR in tomato leaves
In addition, H2O2 production was observed in tomato leaves from 35s::SlERF01 overexpression plants by DAB staining (Fig. 4). At 3 dpi, H2O2 accumulation was not detectable in TRV::SlERF01 plants compared to the TRV::00 empty vector plants. This result indicated that SlERF01 can induce H2O2 generation as a defensive response to S. lycopersici infection.
To further explore the potential mechanism, lignin and callose production was analyzed in the 35s::SlERF01 overexpression plants, TRV::SlERF01 plants and empty vector (35s::00 and TRV::00) plants at 3 dpi. The accumulation of lignin and callose in the leaves of 35s::SlERF01 overexpression plants was higher than that in the leaves of 35s::00 empty vector plants at 3 dpi (Fig. 4). However, the intensities and areas of fluorescence in the leaves of TRV:: SlERF01-silenced plants were weaker than those in the leaves of TRV::00 plants. Based on all of the above results, we conclude that SlERF01 overexpression enhances the resistance of tomato to S. lycopersici compared to that of control plants.
Silencing of SlERF01 decreased the expression levels of the defense-related gene PR1 after infection with S. lycopersici
In previous transcriptome sequencing experiments, we found that the differentially expressed genes SlERF01 and PR1 were significantly upregulated in the pathway “Plant hormone signal transduction”. Here, qRT-PCR was used to identify the regulatory relationship among SlERF01 and PR in the “Plant hormone signal transduction” pathway. As shown in Fig. 7, once SlERF01 was silenced, the expression level of PR1 was significantly suppressed compared to that of TRV::00. Therefore, we proposed SlERF01 enhance disease resistance to S. lycopersici by regulating the expression of the PR1 gene in tomato.
SlERF01 may require the SA and JA signaling pathways to enhance disease resistance in tomato
The above results show that overexpression of SlERF01 can improve disease resistance against S. lycopersici in tomato. In addition, our previous study showed that SlERF01 is involved in the significantly enriched KEGG pathway “Plant hormone signal transduction”. qRT-PCR was used to determine whether the transcript levels of SlERF01 were associated with SA- and JA-induced resistance in resistant plants during SlERF01 infection. Compared with the control (water-sprayed plants), plants treated with 0.2 mM exogenous SA exhibited an approximately 34-fold increase in SlERF01 transcript levels at 24 h (Fig. 5). After SA treatment, SlERF01 was significantly up-regulated and reached its peak expression at 24h, showed the gene expression pattern in response to SA induction in resistant plants MO. In the control material MT, SlERF01 was up-regulated in 12h and 48h after treatment with SA, the rapid decline in 24h, showing the irregular change. So in control material MT, SlERF01 was up-regulated at the different time points but did not show the gene expression pattern in response to SA induction.
Treatment with JA also significantly enhanced the expression of SlERF01, with the peak expression level being 28-fold higher than that of the control. These results showed that SlERF01 could be significantly upregulated by SA and JA treatment. In resistant material MO, SlERF01 showed the gene expression pattern in response to JA induction. However, SlERF01 was not significantly up-regulated at different time points in MT and did not response to JA induction.
It is well established that SA and JA play important roles in the plant defense response to pathogens. To analyze the hormonal response to S. lycopersici infection, LC-MS was performed to measure the JA and SA contents in T1-generation SlERF01-overexpressing plants. The SA and JA levels of T1-generation SlERF01-overexpressing tomato plants were significantly higher than those of the control plants after inoculation with S. lycopersici (Fig. 6). After inoculation with S. lycopersici, the SA levels in the SlERF01-overexpressing plants were 5-fold higher than those in the empty vector plant, and the JA levels were approximately 3-fold higher than those in the empty vector plant (Fig. 6). Thus, overexpression of SlERF01 could significantly enhance the production of SA and JA, once again indicating that SlERF01 probably participates in both the SA and JA signaling pathways to improve the disease resistance to S. lycopersici in tomato.