Abstract
WRKY transcription factors are involved in plant defense responses against pathogens. However, no WRKYs have been reported yet in resistance of Nicotiana species to Alternaria alternata, a necrotrophic fungal pathogen causing brown spot disease. Here, we found that silencing NaWRKY3 lead to wild tobacco Nicotiana attenuata highly susceptible to A. alternata. Combination of transcriptome, electrophoretic mobility shift, ChIP-qPCR and dual-LUC analyses, we uncovered that NaWRKY3 bound to many defense genes’ promoter and activated their expression. Target genes included: 1) lipoxygenases 3, ACC synthase 1 and ACC oxidase 1, three key enzyme genes for JA and ethylene biosynthesis which were critical for A. alternata resistance; 2) feruloyl-CoA 6’-hydroxylase 1 (NaF6’H1), the key enzyme gene for phytoalexins against A. alternata, scopoletin and scopolin; and 3) three A. alternata resistance genes, long non-coding RNA (LncRNA L2), NADPH oxidase (NaRboh D) and berberine bridge-like (NaBBL28). Silencing LncRNA L2 reduced A. alternata-induced levels of JA and NaF6’H1 expression. NaRboh D-silenced plants were strongly impaired in ROS production and stomata closure responses. NaBBL28 was the first A. alternata resistance BBLs identified and was involved in HGL-DTGs hydroxylation. Finally, NaWRKY3 could bind to its own promoter but acted as a transcriptional repressor. Thus we demonstrated that NaWRKY3 is a fine-tuned master regulator of defense network against A. alternata in N. attenuata by regulating different signaling pathways and defense metabolites. For the first time, such an important WRKY was identified in Nicotiana species, providing new insight into defense mechanism of Nicotiana plants to A. alternata.
Introduction
To cope with pathogens, different layers of plant defense response are activated, including pathogen recognition, activation of phytohormone and reactive oxygen species (ROS) signaling pathways, production of antimicrobial metabolites and proteins, stomata closure, and physical reinforcement of cell walls through production of callose and lignin (Camejo et al., 2016; Zhang et al., 2018).
Phytohormones and Rboh-based ROS are important signaling molecules, playing pivotal roles in regulating plant defense response. The jasmonate (JA) and ethylene signaling pathways are usually associated with plant defense against necrotrophic pathogens (Glazebrook, 2005). Alternaria alternata (tobacco pathotype) is a necrotrophic fungal pathogen causing brown spot diseases in Nicotiana species (LaMondia, 2001). In wild tobacco Nicotiana attenuata, lipoxygenases 3 (NaLOX3), allene oxide synthase (NaAOS) and allene oxide cyclase (NaAOC) are three key enzyme genes of JA biosynthesis (Halitschke and Baldwin, 2003; Kallenbach et al., 2012; Sun et al., 2014). N. attenuata plants increased their JA levels in response to A. alternata infection at 1 dpi, and silencing AOC or receptor gene COI1 lead to plants highly susceptible to the fungus (Sun et al., 2014). Ethylene is produced from S-adenosyl-L-methionine (SAM) with the help of ACC synthase (ACS) and ACC oxidase (ACO). Both NaACS and NaACO are encoded by multi-gene families in N. attenuata (von Dahl et al., 2007). NaACSs and NaACOs are strongly up-regulated after A. alternata infection, and bigger lesions are developed in ACOs-silenced plants, suggesting that ethylene signaling pathway plays an essential role in the resistance of N. attenuata to A. alternata (Sun et al., 2017). Reactive oxygen species (ROS) act as important signaling molecules during pathogen attack (Camejo et al., 2016). Rboh, an integral membrane protein, is the homologue of NADPH oxidase generating O− during pathogen attack. NbRboh A and NbRboh B are essential for ROS production against oomycete pathogen Phytophthora infestans (Adachi et al., 2015). GbRboh5/18 enhances cottons’ resistance against Verticillium dahliae by elevating the levels of ROS (Chang et al., 2020). In N. attenuata, NaRboh D is the major ROS source after herbivore attack, and confers plant resistance to Spodoptora littoralis (Wu et al., 2013). However, whether this NaRboh D is involved in A. alternata resistance is unknown.
Phytoalexins, as direct chemical weapons, are produced by plants de novo in response to pathogen attack. Camalexin is the most prominent phytoalexin in Brassicaceae (He et al., 2019; Wu, 2020; Zhou et al., 2022). Capsidiol is an important phytoalexin against A. alternata in N. attenuata (Song et al., 2019). Scopoletin, known as anti-carcinogenic and anti-viral natural coumarin, is demonstrated as a phytoalexin against A. alternata regulated by JA and ethylene signaling pathways (Sun et al., 2014; Sun et al., 2017).
WRKYs are the biggest and most important transcription factor family in plants involved in plant immunity. They are characterized by the conserved WRKYGQK and zinc finger motifs, recognizing cis-elements W-boxes in the promoter of target genes and activating or inhibiting their expression (Wani et al., 2021). AtWRKY33 is required for resistance to necrotrophic fungal pathogen Botrytis cinerea via regulating phytohormones and camalexin (Birkenbihl et al., 2012). AtWRKY57 is a repressor of B. cinerea resistance due to activating JA signaling pathway repressors JAZ1 and JAZ5 (Jiang and Yu, 2016). AtWRKY46, AtWRKY70, and AtWRKY53 positively regulate basal resistance to Pseudomonas syringae, while AtWRKY8 represses resistance to this pathogen (Hu et al., 2012). However, to date no WRKYs have been reported to play a role in the defense response of Nicotiana species to A. alternata.
Currently, it is still unclear how phytohormone and ROS signaling pathways, and phytoalexins are coordinately regulated in Nicotiana species during A. alternata attack. In this study, we identified the transcription factor NaWRKY3 as a master transcriptional regulator of defense network against A. alternata in N. attenuata. NaWRKY3 enhanced the expression of target genes involved in different layers of plant defense, including those for directly defense response like phytoalexins, and immune signals like phytohormones and ROS from respiratory burst oxidase, A. alternata resistance LncRNA L2 and NaBBL28, while repressed its own transcription. For the first time, we identified such an important WRKY involved in so many defense responses in N. attenuata, thus providing new insight into defense mechanism of Nicotiana species to A. alternata.
Results
NaWRKY3 is required for A. alternata resistance in N. attenuata
Previously, NaWRKY3 was reported to be involved in herbivore resistance in N. attenuata (Skibbe et al., 2008). We also found that NaWRKY3 transcripts were significantly up-regulated in N. attenuata after A. alternata inoculation during transcriptome analysis (Song et al., 2019). This result was also confirmed by real time PCR that it was expressed at low levels in healthy, uninfected source-sink transition leaves (0 leaves) of wild-type (WT) plants; but increased significantly at 1 day post inoculation (dpi) and reached around 10-fold at 3 dpi (Fig. 1a).
(a) Mean (±SE) relative A. alternata-induced NaWRKY3 transcripts as measured by qPCR in five replicates of source-sink transition leaves (0 leaves) at 1 and 3 dpi. Asterisks indicated levels of significant difference between WT and irWRKY3 plants with the same treatments (Student’s t-test: ***P<0.005)
(b) Photographs of infected WT and irWRKY3 leaves were taken at 4 dpi (upper panel); Mean (±SE) diameter of necrotic lesions of 0 leaves of WT and irWRKY3 plants at 4 dpi (n = 20; lower panel). Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: ***, p < 0.005).
(c) Heatmap of DEGs in three replicates of 0 leaves of WT and irWRKY3 plants after A. alternata infected at 1 dpi.
To determine the function of NaWRKY3 in A. alternata resistance, stable transgenic plants silenced with NaWRKY3 (irWRKY3), which were generated previously by Skibbe et al. (2008), were used for A. alternata inoculation. In these plants, NaWRKY3 transcripts were decreased by around 87% in mock control, and 96% at 3 dpi (Fig. 1a). As expected, the 0 leaves of irWRKY3 plants showed bigger lesions than those of WT at 4 dpi (Fig. 1b), which marked increased susceptibility to A. alternata. Thus, our results show that NaWRKY3 plays an important role in defense against A. alternata.
Transcriptome analysis of irWRKY3 plants after A. alternata inoculation
We performed a transcriptome analysis through RNA sequencing to obtain a deep view of the transcriptional reprogramming mediated by NaWRKY3 upon A. alternata challenge. A total of 12 samples were collected from 0 leaves of WT and irWRKY3 plants with or without A. alternata at 1 dpi, respectively.
Silencing NaWRKY3 resulted a massive transcriptional reprogramming. A. alternata infected 0 leaves of irWRKY3 plants up-regulated 3669 genes and repressed 4921 genes at 1 dpi when compared to WT plants with the same treatments (Fig. 1c). We especially interested in genes related to defense that were down-regulated in irWRKY3 plants, including genes involved in JA, ethylene, ROS, long non-coding RNA, phytoalexins and berberine bridge-like. We thus selected them for further analysis as below.
NaWRKY3 is required for JA accumulation and the expression of JA synthesis gene NaLOX3 after A. alternata inoculation
The JA levels were increased form 0.55 ng/g to 70 ng/g fresh weight in 0 leaves of WT plants after 1 dpi, but A. alternata-induced JA levels in irWRKY3 plants were only 58% of those of WT plants with the same treatments (Fig. 2a). Similarly, A. alternata-induced JA-Ile was also significantly attenuated in irWRKY3 plants (Fig. 2a).
(a) Mean (±SE) relative A. alternata-induced JA/JA-Ile levels and NaLOX3 transcripts in five replicates of 0 leaves of WT and irWRKY3 plants at 1 dpi. JA and JA-Ile levels were determined by LC-MS/MS, NaLOX3 transcripts were measured by qPCR. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: ***P<0.005).
(b) Schematic diagram of the promoter of NaLOX3. Black triangles indicated the W-box motifs. Short orange lines indicated DNA probes used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(c) EMSA showed that His-NaWRKY3 bind to the promoter of NaLOX3 directly. Labeled probes incubated without His-NaWRKY3 served as a negative control. 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(d) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by qPCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control, and asterisks indicated levels of significant difference between negative controls and experimental group (Student’s t-test: **P<0.01).
(e) Schematic diagram of constructs for effectors and reporters (left panel), and mean (±SE) relative NaLOX3pro::LUC transient transcription activity activated by NaWRKY3 in N. benthamiana plants (right panel). The Agrobacterium with NaLOX3pro::LUC reporter was co-injected with those with indicated effector into leaves of N. benthamiana. After 36 h, values represented the NaLOX3pro::LUC activity relative to the internal control (35S::REN activity) were obtained. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; ***P<0.005).
Lipoxygenase 3 (NaLOX3) was an important enzyme in JA biosynthesis (Halitschke and Baldwin, 2003). Transcriptome data indicated NaLOX3 was decreased in irWRKY3 plants at 1 dpi. Indeed, real-time PCR revealed that levels of A. alternata-induced NaLOX3 in 0 leaves of irWRKY3 plants were only 38% of that of WT plants (Fig. 2a), indicating that NaWRKY3 was required for the fungus-induced expression of NaLOX3. Meanwhile, another enzyme gene NaAOS for JA biosynthesis did not alter its expression in irWRKY3 plants (Supplemental Fig. 1a).
Biotin-labeled probes were synthesized according to W-boxes in the promoter of NaLOX3 (Fig. 2b). Recombinant NaWRKY3 fused with 6x His was purified for electrophoretic mobility shift assay (EMSA). Our results indicated that NaWRKY3 could specifically bind to two selected probes (LOX3P1 and LOX3P2), but not to their mutated ones (Biotin-Probe-m). However, these bindings were greatly impaired when 200 times of unlabeled cold probes were applied (Fig. 2c). These results indicated that NaWRKY3 could specifically bind to NaLOX3 promoter directly in vitro.
To further confirm the binding of NaWRKY3 and NaLOX3 promoter in vivo, NaWRKY3-eGFP over-expressed transgenic plants were used for ChIP-qPCR. Primers were designed according to the binding sites of NaWRKY3 obtained from EMSA results (Fig. 2b and 2c). Our results showed that DNA regions which were immuno-precipitated by anti-GFP antibody, were more enriched around the W-boxes of NaLOX3 promoter than those of Actin II (Fig. 2d), suggesting that NaWRKY3 could directly bind to the promoter of NaLOX3 in vivo.
A dual-LUC reporter assay was employed to investigate whether NaWRKY3 activated the expression of NaLOX3 promoter. The expression of LUC (reporter) was driven by the promoter of NaLOX3 (NaLOX3pro), and REN driven by 35S promoter acted as an internal control (Fig. 2e). When NaWRKY3 was over-expressed under the control of 35S promoter (effector; 35S::NaWRKY3), a significant increasing in LUC/REN ratio was observed at 36 h, indicating that NaWRKY3 could activate NaLOX3 promoter (Fig. 2e).
Taken together, our results indicate that NaWRKY3 is a transcriptional activator of JA synthetic gene NaLOX3 and regulates the expression of NaLOX3 by directly binding to W-boxes within its promoter.
NaWRKY3 regulates an A. alternata resistance lncRNA L2
Several long non-coding RNAs (lncRNAs) were found to be dramatically up-regulated in WT plants at 1 dpi, but were not in irWRKY3 plants during transcriptome analysis. qPCR results confirmed that XR_002068323.1 (L2), XR_002067418.1 (L6) and XR_002066427.1 (L4) were strongly induced in 0 leaves of WT plants after A. alternata inoculation at 1 and 3 dpi, but these inductions were greatly reduced in irWRKY3 plants at both time points (Supplemental Fig. 2 and Fig. 3c). We thus silenced these three lncRNAs via VIGS separately (Supplemental Fig. 3a). Our results revealed that L2 was required for N. attenuata resistance to A. alternata, as bigger lesions were developed in L2-silenced plants (Supplemental Fig. 3b). Thus, L2 was chosen for further analysis.
To further explore the role of L2, stable transformed RNAi (RNA interference) plants were generated. We obtained two T2 transgenic L2 RNAi lines, irL2-1 and irL2-3. qPCR showed L2 expression were significantly decreased in both RNAi lines at 1 dpi (Fig. 3a). As expected, the enhanced susceptibility to A. alternata was observed in irL2-1 and irL2-3 plants (Fig. 3b). Interestingly, A. alternata-induced NaF6’H1 expression, JA and JA-Ile levels were decreased in irL2-1 and irL2-3 plants (Fig. 3a). These results indicated that silencing L2 impaired A. alternata-induced JA and NaF6’H1 expression.
(a) Mean (±SE) relative A. alternata-induced transcripts of lncRNA L2 and NaF6’H1, and JA and JA-Ile levels as measured in five replicate 0 leaves of WT and two lncRNA L2 RNAi lines at 1 dpi. Asterisks indicated levels of significant difference between WT and lncRNA L2 RNAi lines (Student’s t-test: *P<0.05, **P<0.01, ***P<0.005).
(b) Mean (±SE) diameter of necrotic lesions of 20 biological replicate 0 leaves of WT and irL2 (irL2-1 and irL2-3) plants at 4 dpi (n = 20). Asterisks indicated the levels of significant differences between WT and lncRNA L2 RNAi plants with the same treatments (Student’s t-test: *P<0.05, **P<0.01).
(c) Mean (±SE) relative A. alternata-induced lncRNA L2 transcripts as measured by qPCR in five replicate leaves of WT and irWRKY3 plants at 1 and 3 dpi. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: *P<0.05).
(d) Schematic diagram of the promoter of lncRNA L2. Black triangle indicated the W-box motif. Short orange line indicated DNA probe used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(e) EMSA showed that His-NaWRKY3 could bind to the promoter of lncRNA L2 directly. 50- and 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(f) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by real time PCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control, and asterisks indicated levels of significant difference between negative controls and experimental group (Student’s t-test: **P<0.01).
(g) Schematic diagram of constructs for effectors and reporters (left panel), and mean (±SE) relative L2pro::LUC transient transcription activity activated by NaWRKY3 in N. benthamiana plants (right panel). The Agrobacterium with L2pro::LUC reporter was co-injected with those with indicated effector into leaves of N. benthamiana. After 36 h, values represented the L2pro::LUC activity relative to the internal control (35S::REN activity) were obtained. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; *P<0.05).
Next, we performed EMSA and ChIP-qPCR to investigate the regulation of L2 by NaWRKY3. EMSA results indicated that NaWRKY3 could specifically bind to the probe L2P9 which was designed from L2 promoter containing a W-box, as it could not bind to the mutated L2P9 (Biotin-Probe-m; Fig. 3d and 3e), and the binding of NaWRKY3 to L2P9 was attenuated when 50 times of cold unlabeled probes were additionally added and abolished when 200 times of cold probes were applied (Fig. 3e). Further ChIP-qPCR assay using anti-GFP antibody and NaWRKY3-eGFP over-expressed transgenic plants indicated that NaWRKY3 could bind to the promoter of L2 in vivo (Fig. 3f).
The activation of L2 promoter by NaWRKY3 was also tested by dual-LUC reporter assay. A significant increasing in LUC/REN ratio was observed when NaWRKY3 was over-expressed for 36 h (Fig. 3g), suggesting that NaWRKY3 could activate the promoter of L2.
Thus, we identify lncRNA L2, a direct target gene of NaWRKY3. We demonstrate that this lncRNA is required for A. alternata resistance likely by affecting JA and scopoletin responses.
Silencing NaWRKY3 impairs expressions of ethylene biosynthetic genes and ethylene emission during A. alternata inoculation
After A. alternata inoculation, 0 leaves of N. attenuata emitted around 12 nL/g FW ethylene in 24 h (Fig. 4a). However, A. alternata-elicited ethylene production was reduced by 70% in irWRKY3 plants (Fig. 4a), suggesting NaWRKY3 was involved in this fungus-induced ethylene signaling.
(a) Mean (±SE) relative A. alternata-induced ethylene production, and NaACS1/NaACO1 transcripts in five replicate 0 leaves of WT and irWRKY3 plants. Ethylene levels were determined by GC-MS/MS, NaACS1 and NaACO1 transcripts as measured by qPCR. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: ***P<0.005).
(b) Schematic diagram of the promoter of NaACS1 and NaACO1. Black triangle indicated the W-box motif. Short orange lines indicated DNA probes used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(c) and (d) EMSA showed that His-NaWRKY3 could bind to the promoter of NaACS1 and NaACO1 directly. 50- and 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(e) and (f) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by real time PCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control, and asterisks indicated levels of significant difference between negative controls and experimental group (Student’s t-test: *P<0.05).
(g) and (h) Schematic diagram of constructs for effectors and reporters (upper panel), and mean (±SE) relative NaACS1pro::LUC (g) and NaACO1pro::LUC (h) transient transcription activity activated by NaWRKY3 in N. benthamiana plants (lower panel). The Agrobacterium with NaACS1pro::LUC (g) and NaACO1pro::LUC (h) reporter was co-injected with those with indicated effector into leaves of N. benthamiana. After 36 h, values represented the NaACS1pro::LUC (g) and NaACO1pro::LUC (h) activity relative to the internal control (35S::REN activity) were obtained. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; *P<0.05, ***P<0.005).
Indeed, several ethylene synthetic enzyme genes were found to be decreased in infected irWRKY3 plants during transcriptome analysis. qPCR results showed A. alternata-induced NaACS1 and NaACO1 were strongly impaired in irWRKY3 plants (Fig. 4a). EMSA results indicated that NaWRKY3 protein could specifically bind to the probes designed from the W-boxes of NaACS1 and NaACO1 promoters (Fig. 4b-d). Further ChIP-qPCR assay showed that the DNA from NaACS1 and NaACO1 regions around the EMSA probes were more enriched by anti-GFP, indicating NaWRKY3 can bind directly to the promoters of NaACS1 and NaACO1 in vivo (Fig. 4e and 4f).
To determine whether NaWRKY3 was able to activate NaACS1 and NaACO1 promoter, a dual-LUC reporter assay was employed. As expected, the expression of the LUC reporter driven by NaACS1pro and NaACO1pro were activated by NaWRKY3 in leaves of N. benthamiana plants (Fig. 4g and 4h).
Thus, our data demonstrates that NaWRKY3 acts as a positive regulator of A. alternata-induced ethylene by directly binding to NaACS1 and NaACO1 promoter and activating their expression.
NaWRKY3 is required for A. alternata-induced phytoalexins
Scopoletin and scopolin are phytoalexins in N. attenuata regulated by JA and ethylene signaling pathways for A. alternata resistance (Sun et al., 2014; Sun et al., 2017). The levels of scopoletin and scopolin accumulated in irWRKY3 plants were greatly reduced at both 1 and 3 dpi (Fig. 5a). Similarly, A. alternata-induced expression of NaF6’H1, the key enzyme gene of the scopoletin biosynthesis, was also strongly impaired in the irWRKY3 plants (Fig. 5a).
(a) Mean (±SE) relative A. alternata-induced scopoletin and scopolin levels and NaF6’H1 transcripts in five replicate 0 leaves of WT and irWRKY3 plants. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: *P<0.05, ***P<0.005).
(b) Schematic diagram of the promoter of NaF6’H1. Black triangles indicated the W-box motifs. Short orange lines indicated DNA probes used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(c) EMSA showed that His-NaWRKY3 could bind to the promoter of NaF6’H1 directly. 50- and 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(d) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by real time PCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control, and asterisks indicated levels of significant difference between negative controls and experimental group (Student’s t-test: *P<0.05).
(e) Schematic diagram of constructs for effectors and reporters (upper panel), and mean (±SE) relative NaF6’H1pro::LUC transient transcription activity activated by NaWRKY3 in N. benthamiana plants (lower panel). The Agrobacterium with NaF6’H1pro::LUC reporter was co-injected with those with indicated effector into leaves of N. benthamiana. After 36 h, values represented the NaF6’H1pro::LUC activity relative to the internal control (35S::REN activity) were obtained. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; *P<0.05).
EMSA results indicated that NaWRKY3 could specifically bind to two probes designed from the W-boxes in the promoter region of NaF6’H1 (Fig. 5b and 5c). This binding of NaWRKY3 to the promoter of NaF6’H1 in vivo was further verified by ChIP-qPCR assay (Fig. 5d). Dual-LUC assay results showed that NaWRKY3 could significantly induced the LUC reporter expression which was driven by the promoter of NaF6’H1 (Fig. 5e).
Our data demonstrates that NaWRKY3 is required for A. alternata-induced scopoletin accumulation by directly binding to NaF6’H1 promoter and activating its expression.
Regulation of NaRboh D-dependent defensive responses by NaWRKY3
In N. attenuata, NaRboh D had been reported to be involved in the ROS burst after insect herbivore attack (Wu et al., 2013). Silencing NaRboh D (Fig. 6a) also strongly reduced ROS production after A. alternata inoculation at 12 hpi (Supplemental Fig. 6). Interestingly, A. alternata infection usually leads to stomata closure, which is essential for plant resistance to this fungus (Sun et al., 2014). However, A. alternata-induced stomata closure was abolished in NaRboh D-silenced irRboh D plants (Fig. 6a). Consistently, irRboh D plants were highly susceptible to A. alternata (Fig. 6b).
(a) Mean (±SE) relative A. alternata-induced NaRboh D transcripts (left panel) and stomatal conductance (right panel) as measured in five replicate 0 leaves at 1 dpi. Asterisks indicated levels of significant difference between WT and irRboh D samples (Student’s t-test: ***P<0.005).
(b) Mean (±SE) diameter of necrotic lesions of 20 inoculation sites on 0 leaves from WT and irRboh D plants. The lesion area analyzes at 4 dpi. Asterisks indicated significant difference from the WT and irRboh D plants according to Student’s t-test at ***P < 0.005.
(c) Mean (±SE) relative A. alternata-induced NaRboh D transcripts as measured by qPCR in five replicate leaves of WT and irWRKY3 plants at 1 and 3 dpi. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: *P<0.05, **P<0.01).
(d) Schematic diagram of the promoter of NaRboh D. Black triangles indicated the W-box motifs. Short orange lines indicated DNA probes used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(e) EMSA showed that His-NaWRKY3 could bind to the promoter of NaRboh D directly. 50- and 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(f) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by real time PCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control, and asterisks indicated levels of significant difference between negative controls and experimental group (Student’s t-test: **P<0.01).
(g) Schematic diagram of constructs for effectors and reporters (left panel), and mean (±SE) relative NaRboh Dpro::LUC transient transcription activity activated by NaWRKY3 in N. benthamiana plants (right panel). The Agrobacterium with NaRboh Dpro::LUC reporter was co-injected with those with indicated d effector into leaves of N. benthamiana. After 36 h, values represented the NaRboh Dpro::LUC activity relative to the internal control (35S::REN activity) were obtained. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; **P<0.01).
We found that A. alternata-induced NaRboh D transcripts were strongly decreased in irWRKY3 plants during transcriptome analysis. qPCR results confirmed that silencing NaRboh D greatly impaired A. alternata-induced NaRboh D expression (Fig. 6c). EMSA and ChIP-qPCR results showed that NaWRKY3 could specifically bind to the promoter of NaRboh D in vitro and in vivo (Fig. 6d-f). Dual-LUC experiments further showed that NaWRKY3 activated LUC expression driven by NaRboh D promoter (Fig. 6g).
Thus, our data suggests that NaWRKY3 positively regulates NaRboh D expression through binding to its promoter to activate its expression. Meanwhile, NaRboh D is involved in the resistance to A. alternata via ROS and stomata closure.
NaWRKY3 is required for the expression of NaBBL28 after A. alternata inoculation
Berberine bridge like (BBL) genes are believed to encode enzymes involved in specialized metabolism such as alkaloids biosynthesis in Nicotiana. There were several BBL genes found to be induced by A. alternata in WT plants (Supplemental Fig. 4a and 4b, Fig. 7a). Among them, NaBBL28 (XM_019378340.1) transcripts were strongly increased in response to A. alternata, while in irWRKY3 plants the NaBBL28 transcripts were dramatically decreased (Fig. 7a). Furthermore, plants with silenced NaBBL28 via VIGS were highly susceptible to A. alternata (Supplemental data 4c and 4d).
(a) Mean (±SE) relative A. alternata-induced NaBBL28 transcripts as measured by qPCR in five replicate leaves of WT and irWRKY3 plants at 1 and 3 dpi. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: **P<0.01).
(b) Schematic diagram of the promoter of NaBBL28. Black triangles indicated the W-box motifs. Short orange lines indicated DNA probes used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(c) EMSA showed that His-NaWRKY3 could bind to the promoter of NaBBL28 directly. 50- and 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(d) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by real time PCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control. Asterisks indicated the levels of significant differences between WT and irWRKY3 plants with the same treatments (Student’s t-test: *P<0.05).
(e) Schematic diagram of constructs for effectors and reporters (left panel), and mean (±SE) relative NaBBL28pro::LUC transient transcription activity activated by NaWRKY3 in N. benthamiana plants (right panel). The Agrobacterium with NaBBL28pro::LUC reporter was co-injected with those with indicated effector into leaves of N. benthamiana. After 36 h, values represented the NaBBL28pro::LUC activity relative to the internal control (35S::REN activity) were obtained. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; ***P<0.005).
(f) Mean (±SE) relative A. alternata-induced NaBBL28 transcripts as measured by qPCR in five replicate 0 leaves of WT and irBBL28 (irBBL28-2, irBBL28-3, irBBL28-4) plants at 1 dpi. Asterisks indicated the levels of significant differences between WT and irBBL28 plants with the same treatments (Student’s t-test: **P<0.01, ***P<0.005).
(g) HGL-DTGs biosynthetic pathway was used for indication of reduced HGL-DTGs. Red downward pointing arrows indicated the levels of HGL-DTGs significant decreased in irBBL28 leaves, short red lines indicated the levels of Lyciumoside I were not altered between the WT and irBBL28 plants.
(h) Mean (±SE) diameter of necrotic lesions of 20 inoculation sites on 0 leaves from WT and irBBL28 plants. The lesion area analyzes at 4 dpi. Asterisks indicated significant difference from the WT and irBBL28 plants according to Student’s t-test at ***P < 0.005.
(i) Phylogenetic analysis of NaBBL28 and other BBL proteins. BBLs which have been functionally characterized as oligosaccharide oxidase and alkaloid biosynthesis enzymes in Arabidopsis and Nicotiana were used for alignment. NaBBL28 was indicated with the red arrow.
EMSA showed that NaWRKY3 could bind to two W-boxes in the promoter region of NaBBL28 in vitro, and this binding was further verified by ChIP-qPCR assay (Fig. 7b-d). Dual-LUC assay was employed to show that NaWRKY3 could significantly induce the LUC activity driven by the promoters of NaBBL28 (Fig. 7e).
To further investigate the role of NaBBL28 in N. attenuata resistance to A. alternata, stable NaBBL28 RNAi plants were obtained (Fig. 7f). qPCR showed NaBBL28 expression was successfully silenced in three NaBBL28 RNAi lines, irBBL28-2, irBBL28-3 and irBBL28-4 after A. alternata inoculation at 1 dpi (Fig. 7f). All three RNAi lines were highly susceptible to A. alternata (Fig. 7h), which was consistent with the phenotype observed in VIGS plants (Supplemental Fig. 4c and 4d).
NaBBL28 had a FAD (flavin adenine dinucleotide)-binding domain that was known feature of BBE-like proteins, belonged to FAD/FMN-containing dehydrogenase family with a N-terminal signal peptide (Supplemental Fig. 4e and 4f). A phylogenetic analysis including functional characterized BBLs (Carter and Thornburg, 2004; Benedetti et al., 2018; Locci et al., 2019) showed a closer relationship of NaBBL28 to oxidize glucose/oxidize cellulose rather than alkaloid-related BBLs (Fig. 7g), suggesting that NaBBL28 might act as a oligosaccharide oxidase or dehydrogenase.
N. attenuata plants accumulated large quantities of defensive 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). We thus investigated HGL-DTGs in NaBBL28 RNAi lines by HPLC-MS/MS. Silencing NaBBL28 resulted several HGL-DTGs decreased significantly. A marked reduction in Nicotianoside III, Attenoside, Nicotianosides I, Nicotianoside II, Nicotianoside VII were observed in NaBBL28-silenced plants, whereas little effect was found in their precursor Lyciumoside I (Supplemental Fig. 5). The results revealed that NaBBL28 might be important to hydroxylate the aglycones especially the rhamnose at C-3 of Lyciumoside I to form Nicotianoside III, and Attenoside.
Taken together, our results reveal that NaWRKY3 positively regulates NaBBL28, which is required for A. alternata resistance likely by acting as a rhamnose hydroxylase of HGL-DTGs.
NaWRKY3 negative regulates its own expression after A. alternata inoculation
To further characterize the role of NaWRKY3 in A. alternata resistance, we generated transgenic N. attenuata plants with constitutively over-expressed NaWRKY3 in two versions (Supplemental Fig. 1c). Under the control of 35S promoter, NaWRKY3 ORF with 5’-UTR (OV3C1) and genomic DNA of NaWRKY3 (OV3G1) were fused with eGFP and transformed into N. attenuata plants. qPCR and Western Blot showed that the NaWRKY3-eGFP transcripts and proteins were successfully expressed in both versions of transgenic plants (Supplemental Fig. 1b and 1c). However, we could not detect the higher levels of total NaWRKY3 transcripts in all over-expressed lines (Supplemental Fig. 1c).
Both EMSA and ChIP-qPCR indicated that NaWRKY3 could specifically bind to its own promoter (Fig. 8a-c). Furthermore, NaWRKY3 could significantly repress the LUC activity driven by its own promoter in dual-LUC assay, and this suppression could not be relieved even at A. alternata infected condition (Fig. 8d).
(a) Schematic diagram of the promoter of NaWRKY3. Black triangle indicated the W-box motif. Short orange line indicated DNA probes used for EMSA, and green arrows indicated primers used for ChIP-qPCR assays. The translational start site (ATG) was shown at position +1.
(b) EMSA showed that His-NaWRKY3 could bind to the promoter of NaWRKY3 directly. 50- and 200-fold excess of unlabeled probes were used for competition. Biotin-Probe-m was the probe with mutated W-box motif.
(c) Mean (±SE) fold enrichment on the NaWRKY3-eGFP plants in ChIP-qPCR. DNA from NaWRKY3-eGFP over-expressed plants at 1 dpi was immune-precipitated by GFP or IgG antibody, and was further analyzed by real time PCR with indicated primers. The immune-precipitated DNA from IgG antibody was served as control. Asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; *P<0.05).
(d) Schematic diagram of constructs for effectors and reporters (left panel), and mean (±SE) relative NaWRKY3pro::LUC transient transcription activity activated by NaWRKY3 in N. benthamiana plants (right panel). The Agrobacterium with NaWRKY3pro::LUC reporter was co-injected with those with indicated effector into leaves of N. benthamiana. AA1 indicated A. alternata inoculation 1 dpi, asterisks indicated significant differences between control and Ov-NaWRKY3 samples (Student’s t-test; n=4; *P<0.05).
Thus, we find that NaWRKY3 could bind to its own promoter but acts as a transcriptional repressor.
Discussion
NaWRKY3 regulates phytoalexins and upstream signals
Many WRKYs have been reported to be involved in plant resistance to pathogens, including AtWRKY33, AtWRKY8, AtWRKY57, AtWRKY46, AtWRKY70, and AtWRKY53 in Arabidopsis (Birkenbihl et al., 2012; Chen et al., 2018; Jiang and Yu, 2016; Hu et al., 2012). AtWRKY46 coordinates with AtWRKY70 and AtWRKY53 in basal resistance against P. syringae partially involved in SA-signaling pathway (Hu et al., 2012). AtWRKY8 is a negative regulator of basal resistance to P. syringae due to decrease expression of PR1and positive regulator to B. cinerea by elevating PDF1.2 transcripts (Chen et al., 2010; Chen et al., 2018). NbWRKY7, 8, 9 and 11 played redundant roles in immunity to P. infestans (Adachi et al., 2015). A. alternata is notorious necrotrophic fungal pathogen which causes severe loss in Nicotiana species. However, to date no WRKYs has been identified to be involved in defense responses to this pathogen.
Previously, we have shown that NaF6’H1 is the key enzyme gene for the biosynthesis of phytoalexins scopoletin and scopolin, which serves as the first line of defense to A. alternata in N. attenuata (Sun et al., 2014). Analysis of NaF6’H1 promoter revealed that its expression was likely regulated by some WRKYs as several W-boxes occurred in the promoter region. Indeed, several WRKYs were found to be highly induced by A. alternata during transcriptome analysis (Song et al., 2019). Among them, NaWRKY3 was one of the WRKYs elicited significantly by A. alternata not only during transcriptome analysis but also verified by real time PCR (Fig. 1). Importantly, silencing NaWRKY3 resulted plants highly susceptible to A. alternata (Fig. 1). Further investigation revealed that NaWRKY3 was required for A. alternata-induced scopoletin and scopolin accumulation by directly binding to NaF6’H1 promoter and activating its expression, which were proved by EMSA, ChIP-qPCR, dual-LUC assay and analysis of RNAi plants (Fig. 5). Thus, NaWRKY3 was the first WRKY identified in Nicotiana species to regulate plant resistance to A. alternata by regulation of biosynthesis of phytoalexins, scopoletin and scopolin.
Plant hormones JA and ethylene are usually associated with plant resistance to necrotrophs (Glazebrook, 2005). N. attenuata plants activate both JA and ethylene signaling after A. alternata attack, which subsequently regulate scopoletin and scopolin biosynthesis to defend against this fungus (Sun et al., 2014; Sun et al., 2017). Here we reported that NaWRKY3 not only regulated phytoalexin biosynthesis directly through NaF6’H1 (Fig. 5), but also controlled A. alternata-induced levels of JA and ethylene through binding and activating NaLOX3, NaACS1 and NaACO1 promoter (Fig. 2, Fig. 4).
Rboh D is an NADPH oxidase, which is crucial for ROS production after pathogens infection, and is essential for plant resistance to pathogens, AtWRKY55 activates the expression of AtRboh D positively regulates defense against P. syringae (Wang et al., 2020). In this study, we found that NaRboh D was involved in the resistance to A. alternata via ROS and stomata closure (Fig. 6 and Supplemental Fig. 6). Interestingly, NaWRKY3 positively regulated NaRboh D expression through binding to its promoter to activate its expression (Fig. 6).
Thus, we propose that NaWRKY3 is a regulator of defense networks in N. attenuata to A. alternata; it not only regulates the direct defense chemicals scopoletin and scopolin, but also controls upstream signals including JA, ethylene and NaRboh D-based ROS.
NaWRKY3 is a functional homolog of AtWRKY33, but with different working mechanism
In Arabidopsis, AtWRKY33 is proposed as a key regulator of hormonal and metabolic responses in plant resistance to B. cinerea (Birkenbihl et al., 2012; Zhou et al., 2022). Our NaWRKY3 shared 46.44% amino acid sequence identity to AtWRKY33, and was the most closed AtWRKY33 homolog in N. attenuata (Supplemental Fig. 7).
To some extent, AtWRKY33 and NaWRKY3 act in a similar way in defense responses but with different target genes. AtWRKY33 was involved in B. cinerea-induced ethylene produced by regulation of AtACS2 and AtACS6 expression through directly binding to the promoter of those genes (Li et al., 2012). Similarly, NaWRKY3 also acted as a positive regulator of A. alternata-induced ethylene, it could directly bind to NaACS1 and NaACO1 promoter and activate their expression (Fig. 4). AtWRKY33 bound to PAD3 promoter and positively regulated phytoalexin camalexin biosynthesis (Zhou et al., 2022). Scopoletin and scopolin were phytoalexins produced in N. attenuata which were required for A. alternata resistance (Sun et al., 2014; Sun et al., 2017). A. alternata-induced NaF6’H1 expression was strongly reduced in NaWRKY3-silenced plants, and NaF6’H1 was regulated by NaWRKY3 by directly binding to its promoter and activating its expression (Fig. 5).
More interestingly, some of the target genes or pathways are regulated by AtWRKY33 and NaWRKY3 in opposite ways. AtRboh D expression was elevated in Arabidopsis upon B. cinerea infection, but its levels in wrky33 mutants were similar to those of WT plants (Birkenbihl et al., 2012). However, A. alternata-induced NaRboh D expression was greatly impaired in NaWRKY3-silenced plants and we demonstrated that NaWRKY3 positively regulated NaRboh D through binding to its promoter to activate its expression after inoculation of A. alternata (Fig. 6). At 1 dpi, wrky33 mutants accumulated more B. cinerea-induced JA levels than WT, which might be due to AtWRKY33 negatively regulated SA signaling in Arabidopsis (Birkenbihl et al., 2012). But in N. attenauta, A. alternata-induced JA levels were significantly reduced in NaWRKY3-silenced plants, and we found that JA biosynthetic gene NaLOX3 was the direct target of NaWRKY3 (Fig. 2).
Thus, we propose that NaWRKY3 is a functional homolog of AtWRKY33. Although NaWRKY3 and AtWRKY33 are both required for plant resistance to necrotrophic fungal pathogens and involved in phytohormone signaling and phytoalexin biosynthesis, their detailed mode of action is quite different.
NaWRKY3 regulates NaBBL28, a gene required for N. attenuata resistance to A. alternata and related to HGL-DTGs oligosaccharide hydroxylation
The BBL genes are believed to catalyze the oxidative cyclization in specialized metabolism biosynthesis. Overexpressing of BBL genes OGOX1 and CELLOX in A. thaliana increased plant resistance to B. cinerea, because oxidized oligogalacturonides (OGs) and cellodextrins (CDs) are inefficient carbon source that cannot be utilized by B. cinerea (Benedetti et al., 2018; Locci et al., 2019). However, whether any BBL genes are involved in defense responses to A. alternata is unknown. In our study, we found that NaBBL28 gene was strongly up-regulated in response to A. alternata inoculation at both 1 and 3 dpi in 0 leaves (Fig. 7), and bigger lesions were observed in stable NaBBL28 RNAi plants and NaBBL28 VIGS plants (Fig. 7h, Supplemental Fig. 4d). These findings suggest that NaBBL28 is required for the resistance of N. attenuata to A. alternata.
Currently, there is not report how BBLs are regulated. In our study, we found several W-boxes in the promoter of NaBBL28 and strongly decreased transcripts of NaBBL28 in irWRKY3 plants (Fig. 7). Further EMSA, ChIP-qPCR and dual-LUC assay demonstrated NaWRKY3 directly up-regulated the expression of NaBBL28 by binding and activating its promoter (Fig. 7b-e).
The substrates of NaBBL28 are still unclear. BBLs acting as glycosides oxidases is proved in PpBBE1, AtBBE13, Nectarin V, OGOX1, OGOX2, OGOX3, OGOX4, CELLOX (Carter and Thornburg, 2004; Benedetti et al., 2018; Toplak et al., 2018; Locci et al., 2019; Scortica et al., 2022). HGL-DTGs are acyclic diterpene glycosides found in Nicotiana species (Heiling et al., 2016). HGL-DTGs have a 17-hydroxygeranyllinalool (HGL) backbone that can be decorated at the C-3 and C-17 positions with glucose and rhamnose by oligosaccharide oxidases and dehydrogenases (Heiling et al., 2016), forms the precursor Lyciumoside I. Sequence alignment indicated that NaBBL28 was more likely an oligosaccharide oxidase or dehydrogenase (Fig. 7i, Supplemental Fig. 4e and 4f). In our study, the levels of precursor Lyciumoside I did not alter in NaBBL28-silenced plants, while Nicotianoside III, Attenoside, Nicotianosides I, Nicotianoside II, and Nicotianoside VII were all significantly decreased in NaBBL28-silenced plants (Fig. 7 and Supplemental Fig. 5), suggesting that NaBBL28 might be a rhamnose dehydrogenase, adding rhamnose to C-3 of Lyciumoside I.
It’s for the first time that a WRKY was identified in the transcriptional regulation of BBL gene. NaWRKY3 positively regulates NaBBL28, which is required for A. alternata resistance. NaBBL28 acts likely as an oligosaccharide dehydrogenase participating in HGL-DTGs hydroxylation at C-3 of the Lyciumoside I aglycones. However, more work is still needed to prove whether Nicotianoside III, Attenoside, Nicotianosides I, Nicotianoside II, and Nicotianoside VII are phytoalexins against A. alternata or not.
The lncRNA L2 required for A. alternata resistance is regulated by NaWRKY3 and influences JA accumulation
Plant lncRNAs have been reported to play important roles pathogen resistance. GblncRNA7 and GblncRNA2 regulate the resistance to V. dahlia in opposite ways (Zhang et al., 2022). LncRNA33732, Sl-lncRNA15492 and Sl-lncRNA39896 are important regulators for tomato resistance to P. infestans (Cui et al., 2019; Jiang et al., 2020; Hong et al., 2022). However, evidence regarding any lncRNAs involved in defense responses to A. alternata is still lacking. The presented data here strongly indicated that N. attenuata plants up-regulated lncRNA L2 as a defense regulator against A. alternata, since its levels were dramatically increased after A. alternata infection (Fig. 3c), and bigger lesions were developed in L2-silenced plants via RNAi or VIGS (Fig. 3 and Supplemental Fig. 3). Further analysis revealed that silencing L2 impaired A. alternata-induced JA and NaF6’H1 expression (Fig. 3). However, the detail mechanism how lncRNA L2 regulates these responses is currently needed more investigation.
NaWRKY3 is a master regulator of defense networks in N. attenuata to A. alternata
In this study, we demonstrate that NaWRKY3 is a master regulator of defense network against A. alternata in N. attenuata. Here is the working model we proposed (Fig. 9). When host plants sense the attack of A. alternata, NaWRKY3 up-regulates 1) JA biosynthesis by regulation of NaLOX3 and lncRNA L2; 2) ethylene biosynthesis by NaACS1 and NaACO1; and 3) the respiratory burst oxidase NaRboh D, which is required for A. alternata-induced ROS and stomal closure. These results suggest that NaWRKY3 regulates up-stream signaling including JA, ethylene and ROS. In addition, NaWRKY3 also regulates downstream specialized metabolites involved in A. alternata resistance. Scopoletin and scopolin biosynthesis is directly activated by NaWRKY3 on NaF6’H1 expression, and NaWRKY3 also controls NaBBL28, which is required for A. alternata resistance and likely involved in hydroxylation of the rhamnose of Lyciumoside I. Finally, as a key transcriptional regulator, the expression of NaWRKY3 is fine-tuned by itself during defense.
N. attenuata plants increase NaWRKY3 expression after A. alternata infection. The NaWRKY3 protein then binds to the promoters of jasmonic acid (JA) synthetic gene NaLOX3, JA related LncRNA L2, ethylene synthetic gene NaACS1 and NaACO1, respiratory burst oxidase gene NaRboh D, scopoletin and scopolin synthetic gene NaF6’H1 and HGL-DTGs related dehydrogenase NaBBL28, and activates their transcription, thereby regulating a complicate defense network against A. alternata. It includes defense hormones JA and ethylene, NaRboh D-based ROS for stomata closure, scopoletin and scopolin as the direct chemical weapon, LncRNA L2 for JA and NaF6’H1 expression, and NaBBL28 for hydroxylation of the rhamnose of Lyciumoside I to form Nicotianoside III, Attenoside, and other HGL-DTGs. Silencing LncRNA L2, NaBBL28 and NaRboh D separately will lead to plants susceptible to the fungus. Finally, NaWRKY3 fine-tuned its expression by repressing itself.
Materials and Methods
Plant growth and generation of stably transformed plants
Seeds of 33rd inbreeding lines of N. attenuata were used as the wild-type (WT) in all experiments. Stably transformed lines of irWRKY3 (NaWRKY3-deficient; Skibbe et al., 2008), irRboh D (NaRboh D-deficient; Wu et al., 2013) were kindly provided by Prof. Ian T Baldwin.
Stable transgenic plants of irBBL28 (NaBBL28-RNAi), irL2 (L2-RNAi), OV3G1 (over-expression of NaWRKY3 ORF with 5’-UTR), OV3C1 (over-expression of genomic NaWRKY3) were generated in this study as followed. Seed germination and plant growth were conducted as described by Krügel et al. (2002).
Generation of stably transformed plants
For RNAi lines, inverted-repeat orientation of the target gene fragments of lncRNA L2 or NaBBL28 were amplified by primers (Supplementary table) and inserted into the pRESC8. For over-expression line, NaWRKY3 ORF with 5’-UTR (OV3C1) and genomic NaWRKY3 (OV3G1) was infused with eGFP and inserted into pCAMBIA1301 under the control of 35S promoter. These constructed vectors were transformed into N. attenuata WT plants by using Agrobacterium-mediated transformation procedure (Zhao et al., 2021). Single-insertion RNAi lines of irL2 (irL2-1, irL2-3) and irBBL28 (irBBL28-2, irBBL28-3, irBBL28-4) were identified, bred to homozygosity in T2 generation and used in this study.
Total RNA extraction, RT-qPCR analysis and RNA-seq
Total RNA was extracted from a 1.5 ×1.5 cm2 area of leaf lamina with the inoculation site at the center using TRIzol reagent. Around 1 μg of total RNA was used as templates for reverse transcription into cDNA with reverse transcriptase (www.thermofisher.cn). qPCR was performed using SYBR Green mix (BioRad) on a CFX Connect qPCR System (Bio-Rad) and gene-specific primers according to the manufacturer’s instructions. For RT-qPCR analysis, the cDNA was diluted 1.5 times, and 2 μL reaction products were used as template in each reaction. The fold changes in target gene expression were normalized using Actin II (Xu et al., 2018). All primers used in this study are listed in Supplementary table. Five to seven biological replicates were included in assays.
RNA-seq service was provided by oebiotech company (www.oebiotech.com). Total RNA of three biological replicates of WT mock, inoculated WT leaf samples, irWRKY3 mock, inoculated irWRKY3 leaf samples were isolated with TRIzol reagent.
Sequencing was performed at 8 G depth and mapped to the N. attenuata reference genome sequence. The relative abundance of the transcripts was measured with the FPKM (RPKM) method. The differential expression between WT and irWRKY3 with or without A. alternata inoculation samples with a cutoff of 2-fold change.
ChIP-qPCR assay
ChIP assays were performed using an EpiQuik Plant ChIP Kit (www.epigentek.com). Briefly, 7-week-old seedlings over-expressing NaWRKY3-eGFP were harvested with A. alternata at 1 dpi. Chromatin was isolated from 3 g leaf samples and sonicated with a Bioruptor pico-diagenode for 90 min. After coating with anti-GFP (1:1000, Abcam ab290) antibodies, NaWRKY3-eGFP protein/DNA complexes were immunoprecipitated according to the manufacturer’s instructions. The enriched DNA fragments were detected by qPCR using the specific primers listed in Supplementary table. The Actin II promoter was used as a negative control. All ChIP assays were performed with three biological replicates.
Electrophoretic mobility shift assay (EMSA) assays
EMSA experiments were performed using an EMSA/Gel-Shift Kit (Beyotime) according to the manufacturer’s instructions. The NaWRKY3 contained two WRKY domains were cloned into the pET-28a (containing six His tag) vectors. The recombinant His-NaWRKY3 proteins were induced by 3 mM IPTG and expressed in Escherichia coli strain BL21. These recombinant proteins were purified with Ni-NTA agarose. Oligonucleotide probes were labeled by 5’-end biotins, and competitors were unlabeled in the binding assays (sequences are listed in Supplementary table). Equal amounts of His-NaWRKY3 proteins (1 µL) were incubated according to the manufacturer’s instructions in PCR volumes for 20 min at room temperature. The DNA-protein complexes were separated and transferred to a nylon membrane (Thermo Fisher Scientific). After UV cross linking, the biotin signal was detected according to the manufacturer’s instructions.
Dual-LUC assays
Dual-LUC assays were performed in N. benthamiana leaves (Hellens et al., 2005) with pCAMBIA3301-LUC vector system which contained a Renilla luciferase (REN) gene driven by the 35S promoter as an internal control and a firefly luciferase (LUC) gene driven by the promoter of target gene. The target gene promoter fragments (the linear of fragment range from 1.1 kb to 3 kb from ATG site) were cloned and infused into pCAMBIA3301-LUC through the Pst I and Nco I sites, creating NaACS1pro::LUC, NaACO1pro::LUC, NaLOX3pro::LUC, NaF6’H1pro::LUC, NaBBL28pro::LUC, NaRboh Dpro::LUC, L2pro::LUC vectors, independently. Agrobacterium carrying each reporter together with 35S::NaWRKY3-eGFP or 35S::eGFP were co-transformed into N. benthamiana leaves. The LUC activity of plant extracts was analyzed using a microplate reader (Tecan infinite M200 PRO) with commercial LUC reaction reagents following the manufacturer’s instructions (Yeasen Biotechnology). LUC activity was normalized with Renilla luciferase activity (LUC/REN). At least three replicates were performed for each experiment.
Accession Numbers
Sequence data from this article can be found in the GeneBank under accession numbers: NaWRKY3 (XM_019394677.1), NaACS1 (AY426752), NaLOX3 (AY254349), NaACO1 (AY426756), NaF6’H1 (KF771989), NaBBL28 (XM_019378340.1), lncRNA L2 (XR_002068323.1), NaRboh D (XM_019378071.1).
Supplemental Data
Supplementary table
Primers and probes used in this study.
Supplemental Fig. 1 The expression of NaAOS after A. alternata inoculation in WT and irWRKY3 plants, and generation of NaWRKY3-eGFP over-expression plants.
Supplemental Fig. 2 The expression of lncRNAs after A. alternata inoculation in WT and irWRKY3 plants.
Supplemental Fig. 3 lncRNA L2 is required for N. attenuata resistance against A. alternata.
Supplemental Fig. 4 NaBBL28 is required for N. attenuata resistance against A. alternata.
Supplemental Fig. 5 Silencing NaBBL28 decreased HGL-DTGs.
Supplemental Fig. 6 DAB staining of A. alternata-infected WT and irRboh D plants.
Supplemental Fig. 7 Alignment of the Arabidopsis thaliana AtWRKY33 and N. attenuata NaWRKY3 protein sequences.
Author Contributions
Jinsong Wu conceived the project; Jinsong Wu and Zhen Xu designed the experiments and prepared the manuscript. Zhen Xu performed most of experiments and analyzed the data. Shuting Zhang assisted in experiments of JA and ethylene parts and discussed the results.
Conflict of Interest
All authors declare that they have no conflict of interest.
Acknowledgments and Funding
We thank Prof. Ian T. Baldwin (Max-Planck Institute for Chemical Ecology, Jena, Germany) for providing irWRKY3 and irRboh D transgenic N. attenuata seeds and Biological Technology Open Platform of Kunming Institute of Botany for greenhouse and instrument services. This project was supported by the National Natural Science Foundation of China (NSFC Grant No. 31670262) and top-talent recruitment program of Yunnan to Prof. Jinsong Wu.
References
