A G-type lectin receptor-like kinase in Nicotiana benthamiana enhances resistance to the fungal pathogen Sclerotinia sclerotiorum by complexing with CERK1/LYK4

Abstract

Fungal pathogens are among the main destructive microorganisms for crops and ecosystems worldwide, causing substantial agricultural and economic losses. Plant cell surface-localized lysin motif (LysM)-containing receptor-like kinases (RLKs) or receptor-like proteins (RLPs) enhance plant resistance to fungal pathogens via sensing chitin, which is a conserved component of the fungal cell wall. Other types of RLKs also regulate chitin signaling via distinct mechanisms in plants. In this study, we identified a G-type lectin RLK, NbERK1, which positively regulated chitin signaling and resistance to the fungal pathogen Sclerotinia sclerotiorum in the model plant Nicotiana benthamiana. In addition, the LysM-RLK NbCERK1/NbLYK4 was shown to mediate plant resistance to S. sclerotiorum positively. Further, the association of chitin-induced NbCERK1-NbLYK4 was found to be essential for chitin perception and signaling. Importantly, NbERK1 was associated with NbCERK1/NbLYK4 and positively regulated chitin-induced NbCERK1-NbLYK4 association. Moreover, chitin could induce the dissociation of NbERK1 from the NbCERK1-NbLYK4 complex. Also, the kinase activity of NbERK1 was likely essential for this dissociation and plant resistance-enhancing activity of NbERK1. Together, these results suggest that NbERK1 is a novel component of the chitin receptor complex and enhances plant resistance to fungal pathogens via regulating chitin signaling.

Background

Plants rely on two layers of the immune system to resist pathogen infection: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and Dangl 2006). Plant surface-localized pattern recognition receptors (PRRs) activate the downstream signaling pathway involved in PTI response after recognizing the conserved pathogen-associated molecular patterns (PAMPs), such as bacterial flg22, fungal chitin, and oomycete necrosis and ethylene-inducing peptide 1-like proteins (NLPs) (Chinchilla et al. 2006; Monaghan and Zipfel 2012; Oome et al. 2014; Shinya et al. 2015). PTI is a key step in priming the plant immune system, and it can successfully counter the invasion of most microorganisms. PRRs are classified into receptor-like kinases (RLKs) and receptor-like proteins (RLPs) (Couto and Zipfel 2016). RLKs contain an extracellular domain (ECD) potentially involved in ligand perception, a transmembrane (TM) domain, and a cytoplasmic kinase domain (CD), while RLPs contain an ECD and TM domains, lacking CD (Shiu and Bleecker 2003; Jamieson et al. 2018). Plant RLKs and RLPs can be classified into various types according to their ECD, such as the leucine-rich repeat (LRR) type, lectin motif type, lysine motif (LysM) type, malectin-like domain type, and epidermal growth factor (EGF)-like motif type (Couto and Zipfel 2016; Tang et al. 2017; Saijo et al. 2018).

Fungi are the primary destructive pathogens of terrestrial plants and cause severe damage to crops on a global scale (Fisher et al. 2018). Chitin is an insoluble polymer of β-1,4-linked N-acetylglucosamine, a highly conserved structural component of fungal cell walls. It is the best-known PAMP in fungi (Shinya et al. 2015; Bressendorff et al. 2016; Kawasaki et al. 2017). During the fungal invasion, chitin is perceived by LysM-containing RLKs (LysM-RLKs) or RLPs (LysM-RLPs), and subsequently, the immune responses are initiated, ultimately leading to plant resistance to fungi (Tanaka et al. 2013; Gong et al. 2020). In Arabidopsis, the LysM-RLKs family has five members (AtCERK1, AtLYK2, AtLYK3, AtLYK4, and AtLYK5) involved in chitin perception and signaling. AtLYK5 is the major chitin receptor, while AtCERK1 is an indispensable chitin coreceptor (Gong et al. 2020). AtLYK5 binds to chitin with a higher affinity than AtCERK1 does. Interestingly, chitin induces the association between AtLYK5 and AtCERK1 (Cao et al. 2014; Xue et al. 2019; Wang et al. 2020). AtLYK4, a minor chitin receptor, bears a certain affinity to chitin and associates with both AtLYK5 and AtCERK1 (Cao et al. 2014). Notably, only AtCERK1 is an active kinase, and chitin-induced phosphorylation and activation of AtCERK1 are essential for chitin signaling (Miya et al. 2007; Cao et al. 2014). In rice, LysM-RLP OsCEBiP is the major chitin receptor with a high affinity to chitin (Kaku et al. 2006; Hayafune et al. 2014). OsCERK1 (ortholog of AtCERK1) is a chitin coreceptor but lacks chitin binding ability. It associates with OsCEBiP upon chitin perception (Shimizu et al. 2010). LysM-RLPs, OsLYP4, and OsLYP6, are minor chitin receptors and bear moderate affinity to chitin (Liu et al. 2012). GhLYK5 is the major chitin receptor in cotton, while GhCERK1 is a chitin coreceptor. Wall-associated kinase GhWAK7A phosphorylates GhLYK5 and regulates the chitin-induced association of GhCERK1 and GhLYK5 (Wang et al. 2020). Nicotiana benthamiana is a model plant for studying plant–pathogen interactions. Its CERK1 (NbCERK1) is reported to be a putative chitin receptor and positively regulates chitin signaling (Segonzac et al. 2011). However, other NbLYKs that regulate chitin signaling in N. benthamiana are unknown.

The lectin RLKs (LecRLKs) are named due to the presence of the lectin/lectin-like extracellular domain, which can bind to fungal and bacterial cell wall components (Vaid et al. 2013; Sun et al. 2020). Based on the different extracellular lectin domains, LecRLKs can be classified into L-type, G-type, and C-type (Sun et al. 2020). Compared with L-type lectin RLKs, the involvement of G-type lectin RLKs is less investigated in plant disease resistance. In rice, the G-type LecRLK, Pi-d2, was reported to provide resistance against the fungal pathogen Magnaporthe grisea (Chen et al. 2006). OsLecRK could activate multiple immune signaling pathways to confer plant defenses against M. grisea and bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) (Cheng et al. 2013). SDS2 positively regulated plant resistance to M. grisea by interacting with the receptor-like cytoplasmic kinases OsRLCK118/176 (Fan et al. 2018). In N. benthamiana, NbLRK1 positively regulated plant resistance to Phytophthora infestans via interacting with P. infestans elicitin INF1 and mediating INF1-induced cell death (Kanzaki et al. 2008). Nicotiana tobacum Nt-Sd-RLK modulated plant disease resistance by regulating bacterial lipopolysaccharide (LPS)-induced immune responses (Sanabria et al. 2012). In Arabidopsis, LORE was a direct receptor of bacterial LPS, and LPS could induce LORE auto-phosphorylation. LORE enhanced plant resistance to Pseudomonas syringae via the perception of LPS (Ranf et al. 2015). N. benthamiana ERK1 positively regulated plant resistance to Phytophthora spp. by mediating PTI responses induced via Phytophthora expansin-like protein PcEXLX1 (Pi et al. 2022). However, the function and mechanism of ERK1 in regulating plant resistance to other pathogens remain unclear.

In this study, we found that NbERK1, NbCERK1, and NbLYK4 were involved in plant resistance to the fungal pathogen Sclerotinia sclerotiorum and mediated chitin-induced immune responses in N. benthamiana using virus-induced gene silencing (VIGS) assays. NbERK1 complexed with NbCERK1/NbLYK4 and regulated chitin-induced NbCERK1-NbLYK4 association. Furthermore, the predicted kinase catalytic active site (D644) was found to be essential for NbERK1 dissociation from NbCERK1-NbLYK4 complex upon chitin perception and NbERK1-enhanced plant resistance. Our results showed that a G-type lectin RLK, NbERK1, enhances plant resistance to this fungal pathogen via regulating chitin signaling.

Results

NbERK1 regulates plant resistance to S. sclerotiorum and chitin-induced immune responses in N. benthamiana

Recently, we demonstrated that the G-type lectin RLK, NbERK1, positively regulates plant resistance to the oomycete pathogen Phytophthora capsici by mediating the perception of the apoplastic expansin-like protein PcEXLX1 (Pi et al. 2022). The extracellular domains of LecRLKs are similar to lectin proteins, which can bind to fungal and bacterial cell wall components (Vaid et al. 2013). These observations prompted us to examine the involvement of NbERK1 in plant resistance against fungal and bacterial pathogens.

To determine whether NbERK1 is involved in plant resistance against the fungal pathogen S. sclerotiorum, two gene silencing constructs (TRV:NbERK1 and TRV:NbERK1-1) were generated targeting different regions of NbERK1 (Niben101Scf05948g04005.1). Three weeks after agroinfiltration, RT-qPCR analysis confirmed that NbERK1 was effectively silenced compared with TRV:GFP-treated plants (Additional file 1: Figure S1). The agroinfiltrated plants were inoculated with S. sclerotiorum, which revealed that plants treated with TRV:NbERK1 and TRV:NbERK1-1 exhibited significantly larger lesions than the TRV:GFP-treated plants (Fig. 1a, b). This result indicated that NbERK1 should be involved in plant resistance to S. sclerotiorum.

Fig. 1

NbERK1 regulates plant resistance to Sclerotinia sclerotiorum and chitin-induced immune responses in Nicotiana benthamiana. a Representative leaves showing lesions of N. benthamiana (pre-treated with TRV:GFP, TRV:NbERK1, or TRV:NbERK1-1) caused by S. sclerotiorum. Photos were captured 24 h post-inoculation (hpi). Bars, 1 cm. b Lesion areas caused by S. sclerotiorum on TRV-treated plants. Six leaves were counted in each replicate. The experiment was repeated three times. Error bars indicate ± SD (Student’s t-test, **P < 0.01). c The expression of NbERK1 is induced by chitin treatment in N. benthamiana. N. benthamiana leaf discs were incubated in a 96-well plate with 200 μL water overnight and treated with 200 μg/mL chitin. The expression of NbERK1 was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Error bars represent fold changes (± SD). d MAPK activation of N. benthamiana (pre-treated with TRV:GFP, TRV:NbERK1, or TRV:NbERK1-1) treated with 200 μg/mL chitin. α-pTEpY antibody was used to detect MAPK activation. Protein loading was determined by Ponceau S staining. e ROS production of N. benthamiana (pre-treated with TRV:GFP, TRV:NbERK1, or TRV:NbERK1-1) treated with 200 μg/mL chitin. f Relative expression of pattern-triggered immunity (PTI) marker genes (PTI5 and Acre31) in N. benthamiana (pre-treated with TRV:GFP, TRV:NbERK1, or TRV:NbERK1-1) induced by 200 μg/mL chitin at 3 hpi. Error bars represent fold changes ± SD (Student's t-test, **P < 0.01; ***P < 0.001)

Like other PAMPs, chitin can induce some well-characterized immune responses, including the reactive oxygen species (ROS) burst, expression of PTI marker genes, and activation of mitogen-activated protein kinase (MAPK) (Heese et al. 2007; Lloyd et al. 2014; Yu et al. 2017). To determine whether NbERK1 is required for chitin-induced immune responses, we first examined the expression pattern of NbERK1 upon chitin treatment in N. benthamiana. The RT-qPCR analysis showed that NbERK1 was induced at 1 h post chitin treatment (Fig. 1c). We then measured chitin-induced immune responses in plants treated with TRV:GFP, TRV:NbERK1, or TRV:NbERK1-1. Chitin-induced MAPK activation was compromised in plants treated with TRV:NbERK1 or TRV:NbERK1-1 at 5 and 15 min compared with TRV:GFP-treated plants (Fig. 1d). Chitin-induced ROS production was also weakened in TRV:NbERK1- and TRV:NbERK1-1-treated plants compared with the control (Fig. 1e). Consistently, the relative expression of chitin-induced PTI marker genes (PTI5, Acre31, WRKY7, and WRKY8) was also reduced in TRV:NbERK1- and TRV:NbERK1-1-treated plants (Fig. 1f and Additional file 1: Figure S2). Collectively, these findings indicated that NbERK1 regulates chitin-induced immune responses in N. benthamiana.

NbERK1 is dispensable for plant resistance to Pst DC3000 mutant ΔhopQ1 and flg22-induced immune responses

To determine whether NbERK1 regulates plant resistance against any bacterial pathogen, we inoculated TRV:GFP- and TRV:NbERK1-treated plants with Pseudomonas syringae pv. tomato (Pst) DC3000 mutant ΔhopQ1 (Wei et al. 2007). The result revealed that the bacterial number showed no significant difference in the GFP- and NbERK1-silenced plants (Additional file 1: Figure S3). Flagelin 22 (flg22), a 22-amino-acid peptide, is the best-studied bacterial PAMP (Felix et al. 1999). The RT-qPCR analysis demonstrated that flg22 did not induce NbERK1 expression in the early stage (Additional file 1: Figure S3). To verify whether NbERK1 was involved in flg22-induced immune responses, ROS production and the relative expression of PTI marker genes (PTI5 and Acre31) were measured in the GFP- and NbERK1-silenced plants. Silencing of NbERK1 did not affect flg22-induced ROS production (Additional file 1: Figure S3) and the upregulation of PTI5 and Acre31 (Additional file 1: Figure S3) compared with control plants treated with TRV:GFP. The above results suggested that NbERK1 is dispensable for plant resistance to Pst DC3000 mutant ΔhopQ1 and flg22-induced immune responses.

NbCERK1 and NbLYK4 are essential for chitin perception in N. benthamiana

To determine which LYK gene regulates chitin perception in N. benthamiana, we first constructed a phylogenetic tree of all LYK protein family members in Arabidopsis and N. benthamiana (Additional file 1: Figure S4). Next, the TRV VIGS constructs were generated to silence NbCERK1, NbLYK2, NbLYK3, NbLYK4, and NbLYK5. The RT-qPCR analyses confirmed that these genes were silenced effectively in TRV-treated plants except NbLYK2, and the expression of the other four NbLYK homologs was not affected in the corresponding silenced plants (Additional file 1: Figure S5).

The silenced plants were inoculated with S. sclerotiorum, revealing that NbCERK1- and NbLYK4-silenced plants exhibited significantly larger lesions than GFP-silenced plants (Fig. 2a, b). To further examine the functions of these NbLYKs in regulating S. sclerotiorum resistance, GFP, NbCERK1, NbLYK2, NbLYK4, and NbLYK5 each were transiently expressed in N. benthamiana (NbLYK3 was not successfully cloned). Subsequently, S. sclerotiorum was inoculated at the agroinfiltration sites after 24 h. The results showed that only the transient expression of NbCERK1 enhanced the resistance of plant to S. sclerotiorum (Additional file 1: Figure S6).

Fig. 2

NbCERK1 and NbLYK4 are essential for plant resistance to Sclerotinia sclerotiorum and chitin perception. a Representative leaves showing lesions of Nicotiana benthamiana (pre-treated with TRV:GFP, TRV:NbCERK1, TRV:NbLYK2, TRV:NbLYK3, TRV:NbLYK4, or TRV:NbLYK5) caused by S. sclerotiorum. Photos were captured at 24 h post-inoculation (hpi). Bars, 1 cm. b Lesion areas caused by S. sclerotiorum on TRV-treated plants. Six leaves were counted in each replicate. The experiment was repeated three times. Error bars indicate ± SD (Student’s t-test, **P < 0.01; ***P < 0.001). c ROS production of N. benthamiana (pre-treated with TRV:GFP, TRV:NbCERK1, TRV:NbLYK2, TRV:NbLYK3, TRV:NbLYK4, or TRV:NbLYK5) treated with 200 μg/mL chitin. d The total RLU was the sum of the RLUs for the first 30 min. Error bars indicate ± SD (Student’s t-test, ***P < 0.001). e MAPK activation of N. benthamiana (pre-treated with TRV:GFP, TRV:NbCERK1, or TRV:NbLYK4) treated with 200 μg/mL chitin. α-pTEpY antibody was used to detect the MAPK activation. Protein loading was determined by Ponceau S staining. f Relative expression of PTI marker genes (PTI5 and Acre31) in N. benthamiana (pre-treated with TRV:GFP, TRV:NbCERK1, or TRV:NbLYK4) induced by 200 μg/mL chitin at 3 hpi. Error bars represent fold changes ± SD (Student's t-test, **P < 0.01). g NbLYK4 associates with NbCERK1 upon chitin treatment in N. benthamiana. GFP-tagged NbLYK4 and HA-tagged NbCERK1 were co-expressed in N. benthamiana for 36–48 h. Samples were treated with 200 μg/mL chitin for 15 min before harvesting. Protein associations were detected by Co-IP assays

Chitin-induced ROS production was measured to determine which NbLYK-silenced plants showed altered responses to chitin treatment. The results showed that chitin-induced ROS production was remarkably compromised in NbCERK1- and NbLYK4-silenced plants and partially reduced in NbLYK3- and NbLYK5-silenced plants compared to TRV:GFP-treated control plants (Fig. 2c, d). These findings raised our interest in further exploring the functions of NbCERK1 and NbLYK4. Consistent with the above results, chitin-induced MAPK activation was compromised at 5, 15, and 30 min in NbCERK1- and NbLYK4-silenced plants compared with TRV:GFP-treated plants (Fig. 2e). In addition, the expression of chitin-induced PTI marker genes (PTI5 and Acre31) was significantly reduced in NbCERK1- and NbLYK4-silenced plants (Fig. 2f). Since NbLYK2 was not effectively silenced in N. benthamiana, it is uncertain whether it regulates plant disease resistance and chitin signaling.

In Arabidopsis, the association of AtCERK1 and AtLYK5 induced by chitin is essential for triggering chitin-induced immune signaling (Cao et al. 2014; Erwig et al. 2017). Previously, the association of AtLYK4 with AtCERK1 was found to be chitin-independent (Cao et al. 2014). To determine whether chitin could induce the association of NbCERK1 with NbLYK4, GFP-tagged NbLYK4 (NbD044705.1) was co-expressed with HA-tagged NbCERK1 (NbD008475.1) in N. benthamiana for co-immunoprecipitation (Co-IP) assay, which showed that NbLYK4 was associated with NbCERK1 upon chitin treatment (Fig. 2g). Collectively, NbCERK1 and NbLYK4 are essential for chitin perception and plant resistance to the fungal pathogen.

NbERK1 associates with NbCERK1 and NbLYK4

NbERK1 regulates chitin-induced immune responses, and NbCERK1-NbLYK4 complex is essential for chitin perception. Therefore, it is imperative to explore the potential protein–protein interactions between NbERK1 and NbCERK1/NbLYK4. For the Co-IP assay, the NbERK1-HA was co-expressed with NbCERK1-GFP in N. benthamiana. NbERK1 was immunoprecipitated with NbCERK1 irrespective of the chitin treatment (Fig. 3a). However, the association between NbERK1 and NbCERK1 was remarkably weakened upon chitin treatment (Fig. 3a). The same association was also observed between NbERK1 and NbLYK4 by Co-IP (Fig. 3b).

Fig. 3

NbERK1 associates with NbCERK1 and NbLYK4. a NbERK1 associates with NbCERK1 in Nicotiana benthamiana. GFP-tagged NbCERK1 and HA-tagged NbERK1 were co-expressed in N. benthamiana for 36–48 h. Samples were treated with 200 μg/mL chitin for 15 min before harvesting. Protein associations were detected by Co-IP assays. b NbERK1 associates with NbLYK4 in N. benthamiana. GFP-tagged NbLYK4 and HA-tagged NbERK1 were co-expressed in N. benthamiana for 36–48 h. Samples were treated with 200 μg/mL chitin for 15 min before harvesting. Protein associations were detected by Co-IP assays. c NbERK1 associates with NbLYK4 by a split-luciferase assay. NbERK1-Nluc was co-expressed with the indicated Cluc constructs in N. benthamiana. The intensity of protein interaction was shown by the relative luminescence unit (RLU), and protein expression was shown by immunoblotting (mean ± SD, n ≥ 6). d NbERK1 associates with NbLYK4 in the BiFC assay. NbERK1 and NbLYK4 were fused to the N terminus or C terminus of the YFP fragment, respectively. YFP fluorescence was detected by confocal microscopy. Bars, 25 μm. e NbERK1 associates with NbCERK1 in the yeast two-hybrid assay. Yeast cells with corresponding vectors were grown on SD-Leu/-Trp or SD-Ade/-His/-Leu/-Trp medium. AD and BD represent empty plasmids. ECD, extracellular domain; CD, cytosolic domain

To further verify the association of NbERK1 with NbCERK1 and NbLYK4, a luciferase complementation assay was performed in N. benthamiana. We observed that NbERK1 was associated with NbLYK4, but not with NbCERK1 or EV, and all proteins were normally expressed (Fig. 3c). These results demonstrated that NbERK1 was associated with NbLYK4 in vivo. Similarly, in the bimolecular fluorescence complementation (BiFC) assay, the YFP signal was detected only in N. benthamiana leaves expressing NbERK1-nYFP and cYFP-NbLYK4 (Fig. 3d), but not in leaves expressing NbERK1-nYFP and cYFP-NbCERK1 (Additional file 1: Figure S7). These results indicated that NbERK1 was associated with NbLYK4, but not with NbCERK1 in the BiFC assay. We speculate that the association of NbERK1 and NbCERK1 might be via their extra-cellular domain (ECD). Therefore, we performed a yeast two-hybrid assay using pGADT7/pGBKT7 plasmids containing ECDs or CDs, which showed that NbERK1 was indeed associated with NbCERK1 via ECD (Fig. 3e). Collectively, these data suggest that NbERK1 forms complexes with NbCERK1 and NbLYK4, and it is released from the NbCERK1-NbLYK4 complex upon chitin perception.

NbERK1 is important for the chitin-triggered association of NbCERK1 and NbLYK4

We further investigated the potential role of NbERK1 in the chitin-triggered association of NbCERK1 and NbLYK4. NbCERK1-HA was co-expressed with NbLYK4-GFP in TRV:GFP- and TRV:NbERK1-treated plants. Silencing of NbERK1 weakened the chitin-induced association of NbCERK1-NbLYK4 (Fig. 4a), suggesting that NbERK1 is important for the chitin-induced formation of NbCERK1-NbLYK4 complex. To determine the dynamic of NbERK1/NbCERK1/NbLYK4 complex upon chitin induction, NbERK1-HA was co-expressed with NbCERK1-Flag and NbLYK4-GFP in N. benthamiana. The result showed that the overexpression of NbERK1 also weakened the chitin-induced NbCERK1-NbLYK4 association (Additional file 1: Figure S8). This result suggests that NbERK1 is possibly also required to promote NbCERK1-NbLYK4 dissociation after the activation of chitin-induced immune responses.

Fig. 4

NbERK1 is important for the chitin-triggered association of NbCERK1 and NbLYK4. a Silencing of NbERK1 reduces the chitin-triggered association of NbCERK1 and NbLYK4 in Nicotiana benthamiana. GFP-tagged NbLYK4 and HA-tagged NbCERK1 were co-expressed in N. benthamiana (pre-treated with TRV:GFP or TRV:NbERK1) for 36–48 h. Samples were treated with 200 μg/mL chitin for 15 min before harvesting. Protein associations were detected by Co-IP assays. b Chitin does not induce dissociation of NbERK1-D644N from NbCERK1. GFP-tagged NbCERK1 was co-expressed with HA-tagged NbERK1-D644N or NbERK1 in N. benthamiana for 36–48 h. Samples were treated with 200 μg/mL chitin for 15 min before harvesting. Protein associations were detected by Co-IP assays. c Chitin does not induce dissociation of NbERK1-D644N from NbLYK4. GFP-tagged NbLYK4 was co-expressed with HA-tagged NbERK1-D644N or NbERK1 in N. benthamiana for 36–48 h. Samples were treated with 200 μg/mL chitin for 15 min before harvesting. Protein associations were detected by Co-IP assays

To further clarify the role of NbERK1 kinase activity in chitin-induced NbCERK1-NbLYK4 complex formation, we first examined the kinase activity of NbERK1 using a prokaryotic expression system. However, the CD of NbERK1 was found to be toxic when expressed in Escherichia coli, which prevents recombinant protein production. Therefore, we generated the potential NbERK1 kinase-inactive mutant (NbERK1-D644N) by mutating the Asp residue at position 644 (D644). D644 is an essential residue in the catalytic loop, which was identified by sequence comparison with Arabidopsis LecRK-IX.1 and LecRK-IX.2 (Wang et al. 2015). Next, we determined the importance of NbERK1 kinase activity for NbERK1 dissociation from NbCERK1-NbLYK4 complex upon chitin perception. For the Co-IP assay, NbERK1-D644N-HA or NbERK1-HA was co-expressed with NbCERK1-GFP in N. benthamiana. We observed no change in the association between NbERK1-D644N and NbCERK1 upon chitin treatment (Fig. 4b). Similarly, we found no change in the association between NbERK1-D644N and NbLYK4 upon chitin treatment (Fig. 4c), suggesting that the kinase catalytic active site (D644) of NbERK1 is important for NbERK1 dissociation from NbCERK1-NbLYK4 complex upon chitin perception.

The kinase catalytic active site (D644) of NbERK1 is essential for plant resistance-enhancing activity and its protein stability

Programmed cell death (PCD) is critical in plant immunity (Fan et al. 2018). Activation of cell-surface receptors may lead to plant cell death (Gao et al. 2009). To determine whether NbERK1 has cell death-inducing activity, NbERK1-HA was transiently expressed in N. benthamiana. We observed NbERK1-induced cell death in N. benthamiana at 3 days post-infiltration (Fig. 5a). To determine the importance of the kinase catalytic active site for NbERK1-induced cell death activity, NbERK1-D644N-HA was also transiently expressed in N. benthamiana. The kinase-null mutant (NbERK1-D644N-HA) expression failed to induce cell death in N. benthamiana, which was confirmed by quantifying ion leakage (Fig. 5a, b). Immunoblotting analysis confirmed protein expression (Fig. 5c). These results indicated that the NbERK1 kinase catalytic active site (D644) is essential for the cell death-inducing activity of NbERK1.

Fig. 5

The kinase catalytic active site of NbERK1 is essential for cell death-inducing/plant resistance-enhancing activity and protein stability. a The kinase catalytic active site of NbERK1 affects its cell death-inducing activity. Transient expression of NbERK1 and NbERK1-D644N in Nicotiana benthamiana by agroinfiltration. INF1 and green fluorescent protein (GFP) were used as controls. Photos were captured under UV light. b Cell death was quantified by measuring electrolyte leakage. Error bars indicate ± SD (Student’s t-test, ***P < 0.001). c Immunoblotting analysis of NbERK1 and NbERK1-D644N expressed in N. benthamiana. d, e The kinase catalytic active site of NbERK1 affects its plant resistance-enhancing activity. Representative leaves showing lesions of N. benthamiana caused by Sclerotinia sclerotiorum. Leaves were infiltrated by Agrobacterium tumefaciens strains harboring GFP, NbERK1, or NbERK1-D644N 24 h before S. sclerotiorum inoculation. Photos were captured 24 hpi. Lesion areas were counted by ImageJ. Error bars indicate ± SD (Student’s t-test, *P < 0.05). f The kinase catalytic active site of NbERK1 affects protein stability. NbERK1-HA and NbERK1-D644N-HA were transiently expressed in N. benthamiana for 2 d. Leaves were treated with 50 μM CHX for 0, 2, and 4 h before harvesting. Protein abundances were detected by immunoblotting using anti-HA antibodies

Next, GFP and NbERK1 were transiently expressed in N. benthamiana to determine the plant resistance-enhancing activity of NbERK1. Subsequently, S. sclerotiorum was inoculated at the agroinfiltration sites after 24 h. We found that pretreatment of plants with NbERK1 exhibited significantly enhanced resistance to S. sclerotiorum compared with the control GFP (Fig. 5d). NbERK1-D644N-HA was also transiently expressed in N. benthamiana to determine its function in enhancing plant resistance. As expected, the expression of NbERK1-D644N-HA did not affect plant resistance compared with GFP (Fig. 5e). Collectively, these findings indicate that the kinase catalytic active site (D644) of NbERK1 is essential for plant resistance-enhancing activity.

The protein abundance of kinase-null mutant (NbERK1-D644N-HA) in Fig. 5c was significantly less than that of NbERK1, which led us to explore the importance of the NbERK1 kinase catalytic active site for the NbERK1 protein stability. NbERK1-HA and NbERK1-D644N-HA were transiently expressed in N. benthamiana for 2 d, and leaves were treated with 50 μM cycloheximide (CHX, a protein synthesis inhibitor) for 0, 2, and 4 h before harvesting. We found that the protein abundance of NbERK1-D644N-HA was less than NbERK1 before and after CHX treatment (Fig. 5f). These results suggest that the kinase catalytic active site (D644) of NbERK1 is also essential for its protein stability.

Discussion

Plant pathogenic fungi often cause severe plant diseases leading to significant economic losses. S. sclerotiorum is an important fungus with a broad host range, which can cause yield reduction in many crops, such as soybeans (Bolton et al. 2006). Chitin is a fungal cell wall component that can activate PTI response, including ROS burst, expression of marker genes, and activation of MAPK (Heese et al. 2007; Lloyd et al. 2014; Yu et al. 2017). Previously, we found that NbERK1 positively regulated plant resistance to the oomycete pathogen P. capsici by mediating the perception of the apoplastic expansin-like protein PcEXLX1. In this study, we showed that NbERK1 enhances plant resistance to the fungal pathogen S. sclerotiorum by regulating chitin signaling, though it is not involved in plant resistance to the bacterial pathogen Pst DC3000 mutant ΔhopQ1. NbERK1 was associated with chitin sensory receptors NbCERK1-NbLYK4 and was found to be significant for chitin-induced NbCERK1 and NbLYK4 association. The results further indicated that the kinase catalytic active site (D644) of NbERK1 was essential for NbERK1 dissociation from NbCERK1-NbLYK4 complex upon chitin perception and enhancing plant resistance through NbERK1 in N. benthamiana.

LysM-RLKs function in chitin perception, signaling, and plant resistance to fungal pathogens (Kawasaki et al. 2017). The major chitin receptor of Arabidopsis and cotton is LYK5 (AtLYK5 and GhLYK5). Chitin-induced AtCERK1-AtLYK5/ GhCERK1-GhLYK5 complex formation is essential for chitin signaling in Arabidopsis and cotton (Wan et al. 2012; Cao et al. 2014; Wang et al. 2020). However, our results showed that N. benthamiana NbLYK5 exerted an insignificant effect on plant resistance to S. sclerotiorum and chitin signaling (Fig. 2). Conversely, silencing NbLYK4 significantly reduced chitin-induced immune responses and plant resistance to S. sclerotiorum (Fig. 2). Importantly, chitin could induce the association of NbLYK4 with NbCERK1 (Fig. 2), which was different from the association of AtLYK4-AtCERK1 (Cao et al. 2014), suggesting that NbLYK4 might be the major chitin receptor of N. benthamiana. These observations indicate that chitin signaling is different among different species.

Evidence suggests that additional RLKs in the chitin receptor complex regulate chitin signaling. For instance, the malectin-like domain-containing RLK, IOS1, from Arabidopsis regulate BAK1-dependent and -independent PTI responses. IOS1 positively modulates chitin signaling by associating with AtCERK1, which is BAK1-independent (Yeh et al. 2016). Similarly, Arabidopsis malectin-like RLK FERONIA promotes chitin signaling by associating with AtCERK1 (Stegmann et al. 2017). Here, we found that NbERK1 also regulated chitin-induced immune responses by associating with NbCERK1 (Figs. 1, 3). In cotton, a wall-associated RLK, GhWAK7A, functions in chitin signaling via regulating chitin-induced GhCERK1-GhLYK5 association (Wang et al. 2020). Likewise, our results revealed that NbERK1 positively mediated chitin signaling by regulated chitin-induced NbCERK1-NbLYK4 (chitin sensory receptors) association (Figs. 1, 4). Additionally, the LRR-RLK LIK1 is a negative chitin response regulator by interacting with AtCERK1. The lik1 mutant plants were found to be more susceptible to S. sclerotiorum (Le et al. 2014). However, we observed that NbERK1 is a positive regulator of chitin signaling, and NbERK1-silenced plants were also more susceptible to S. sclerotiorum (Fig. 1). In addition to the above examples of association with chitin sensory receptors, a few RLKs regulate chitin signaling via interacting with RLCK. For example, the G-type lectin RLK SDS2 positively regulates chitin signaling by interacting with OsRLCK118/176 in rice (Fan et al. 2018). In summary, our results highlight a new mechanism by which G-type lectin RLK regulates chitin signaling. Furthermore, it was established as a novel component in the chitin receptor complex.

Overexpression of LecRLKs can induce plant cell death and disease resistance. For example, overexpression of G-type lectin RLK SDS2 from O. sativa induces plant programmed cell death and resistance to M. grisea (Fan et al. 2018). The L-type lectin RLK LecRK-IX.2 induces SA accumulation, which leads to cell death in Arabidopsis (Luo et al. 2017). In addition, overexpression of L-type lectin RLKs LecRK-IX.1 and LecRK-IX.2 from Arabidopsis can enhance plant resistance to Phytophthora and induce plant cell death. Moreover, the cell death-inducing/plant resistance-enhancing activity of LecRK-IX.1 and LecRK-IX.2 rely on their kinase catalytic active site (Wang et al. 2015). Similar to our results, the NbERK1 kinase catalytic active site was also essential for the cell death-inducing/plant resistance-enhancing activity of NbERK1 (Fig. 5). In addition, our results showed that the kinase catalytic active site of NbERK1 was important for NbERK1 dissociation from NbCERK1-NbLYK4 complex upon chitin perception (Fig. 4b, c). These findings suggest that NbERK1 modulates chitin sensory complex NbCERK1-NbLYK4 to enhance plant resistance in a kinase-dependent manner.

Conclusions

In this study, we identified a G-type lectin RLK, NbERK1, which enhances plant resistance to the fungal pathogen S. sclerotiorum via regulating chitin signaling. NbERK1 associates with the chitin receptor complex in N. benthamiana and likely modulates the chitin sensory complex NbCERK1-NbLYK4 to enhance plant resistance in a kinase-dependent manner. Collectively, these results provide a novel regulator of chitin-induced plant immunity.

Methods

Plant material and strain cultures

Nicotiana benthamiana plants used in this study were grown in a glasshouse (25°C, 16 h photoperiod, and 60% relative humidity). Sclerotinia sclerotiorum was grown at 25°C on potato dextrose agar (PDA) medium (boiled extracts from 200 g of fresh potato, 20 g of glucose, and 15 g agar per liter). Escherichia coli strain DH5α was cultured on Luria–Bertani (LB) medium at 37°C and Agrobacterium tumefaciens strain GV3101 was cultured on LB medium at 28°C.

Plasmid construction

For virus-induced gene silencing (VIGS) in N. benthamiana, the gene fragments were amplified from the cDNA of N. benthamiana and ligated into the pTRV2 vector (Liu et al. 2002). For co-immunoprecipitation (Co-IP) in N. benthamiana, constructs were generated using the pCAMBIA1300-GFP, pCAMBIA1300-HA, or pCAMBIA1300-Flag vectors. For transient expression in N. benthamiana, constructs were generated by the pCAMBIA1300-HA or pSuper-HA vectors. For split-luciferase complementation assays in N. benthamiana, constructs were generated by the pCAMBIA1300-Cluc-3 × Flag or pCAMBIA1300-Nluc-HA vectors. For bimolecular fluorescence complementation assays in N. benthamiana, constructs were generated by using the pSuper-HA-cYFP or pSuper-cMyc-nYFP vectors. For the yeast two-hybrid assays, constructs were generated using the pGADT7 or pGBKT7 vectors. Primers used in this study are listed in Additional file 2: Table S1.

Agrobacterium-mediated transient expression and VIGS in N. benthamiana

Plasmids were transformed into A. tumefaciens strain GV3101 by electroporation. A. tumefaciens carrying the plasmid were pelleted and resuspended in infiltration solution (10 mM magnesium chloride (MgCl₂), 10 mM 2-(N-morpholino) ethanesulfonic acid (MES) pH 5.7, and 200 μM acetosyringone). The OD₆₀₀ was adjusted to 0.6 and infiltrated into six-week-old plant leaves.

For VIGS assay, A. tumefaciens cultures harboring pTRV2 constructs were mixed with A. tumefaciens cultures harboring pTRV1 construct in a 1:1 ratio to a final OD₆₀₀ of 0.8 before infiltration into leaves of four-leaf-stage N. benthamiana. The efficiency of gene silencing was determined by RT-qPCR and TRV:GFP was used as a control.

RNA isolation and RT-qPCR

N. benthamiana total RNAs were extracted from leaves using the RNA kit (ZomanBio) and 500 ng of total RNA were used as templates for first-strand cDNA synthesis using the reverse transcription kit (ZomanBio). RT-qPCR was performed on the ABI QuantStudio 6 Flex system (Thermo Fisher) using 2 × HQ SYBR qPCR Mix (Low ROX) (ZomanBio). The primers used for RT-qPCR are listed in Additional file 2: Table S1.

ROS burst assay

N. benthamiana leaf discs (Ø 0.5 cm) were taken using a cork-borer set (Sigma-Aldrich) and incubated in a 96-well plate with 200 μL water for 12 h. To perform the flg22-induced ROS production assay, the water was replaced with the reaction buffer containing 1 μM flg22 (Sangon), 20 μM luminol (Sigma-Aldrich), and 20 μg/mL horseradish peroxidase (Sigma-Aldrich). To perform the chitin-induced ROS production assay, the water was replaced with the reaction buffer containing 200 μg/mL chitin (Sangon), 5 μM L-012 (Waco), and 20 μg/mL horseradish peroxidase (Sigma-Aldrich). Luminescence was measured using the luminometer (Tecan F200).

S. sclerotiorum and bacterial infection assays

For S. sclerotiorum infection assay, it was grown on PDA plates for 3 days at 25°C in the dark. The mycelial plug of S. sclerotiorum was taken using a cork-borer set and then inoculated onto the N. benthamiana leaves (pre-treated with TRV or pre-infiltrated with A. tumefaciens cultures). The inoculated plants were stored in boxes with high humidity for 24 h in the dark. The lesion areas were measured by ImageJ.

For bacterial infection assay, N. benthamiana leaves (pre-treated with TRV) were infiltrated with Pst DC3000 ΔhopQ1. Leaf bacterial number was determined 3 days post inoculation (Zhang et al. 2010).

MAPK activation assay

N. benthamiana leaf discs (Ø 0.5 cm) (pre-treated with TRV) were taken using a cork-borer set and incubated in a 96-well plate with 200 μL ddH₂O for 12 h. To perform the chitin-induced MAPK activation assay, ddH₂O was replaced with the reaction buffer containing 200 μg/mL chitin for 0, 5, 15, and 30 min. For each time point, three leaf discs were collected and total proteins were extracted by RIPA buffer (150 mmol/L sodium chloride (NaCl), 50 mmol/L Tris pH 7.5, 1% Triton X-100, 1% SDS, 1% deoxycholate, 0.5 mmol/L EDTA, 1 × phenylmethanesulfonyl fluoride (PMSF), and 1 × protease inhibitor cocktail). Total proteins were separated by SDS-PAGE gels. MAPK signals were detected by anti-Phospho-p44/p42 MAPK antibody (anti-pTEpY) (Cell Signaling Technology) and stained by Ponceau S for protein loading.

Western blotting and Co-IP assay

To determine the expression of the protein in N. benthamiana leaves, total proteins were extracted using RIPA buffer and then separated by SDS-PAGE gels. Western blotting was detected with corresponding antibodies. For the Co-IP assay, total proteins were extracted using Lysis buffer (150 mmol/L NaCl, 10 mmol/L Tris pH 7.5, 0.5% Triton X-100, 0.5 mmol/L EDTA, 2% (w/v) polyvinylpolypyrrolidone, 10% glycerol, 1 × PMSF, 1 × protease inhibitor cocktail, and 1 × Ps341), incubated with GFP-Nanoab-Agarose (Lablead) for 2 h and washed four times with Wash buffer (10 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 0.5 mmol/L EDTA). Immunoprecipitations were detected using anti-HA (Cwbio), anti-GFP (Proteintech), or anti-FLAG (Proteintech) antibodies.

Chitin treatment method

For the Co-IP and PTI marker gene assays, chitin treatment means injecting chitin solution (200 μg/mL) into the leaves before harvesting. For other assays, chitin treatment means incubating the leaves in chitin solution (200 μg/mL) before harvesting.

Luciferase complementation assay

The indicated Nluc and Cluc constructs were expressed in N. benthamiana by agroinfiltration for 2 days. Leaf discs (Ø 0.5 cm) were collected and into a 96-well plate with 1 mM luciferin (Biovision). The luciferase activity was measured with the luminometer (Tecan F200).

Bimolecular fluorescence complementation assay

The indicated nYFP and cYFP constructs were expressed in N. benthamiana by agroinfiltration for 2 days. YFP signal was detected by confocal microscopy (Leica SP8). The excitation and emission wavelengths for YFP were 514 nm and bright field was used to observe the cell contour of epidermis of N. benthamiana.

Yeast two-hybrid assay

The indicated plasmids were co-transformed into yeast strain AH109. Select positive clones on synthetic defined (SD) medium without Leu and Trp. Protein–protein interactions were tested by transferring transformants to the SD medium without Ade, His, Leu, and Trp.

Electrolyte leakage assay

Five N. benthamiana leaf discs (1.0 cm in diameter) were collected 72 h after agro-infiltration and floated on 5 mL ddH₂O for 3 h at room temperature. The electrolyte leakage value A was measured by the conductivity meter (Mettler Toledo, LE703). Then the leaf discs were boiled for 20 min. Value B was measured with the conductivity meter when the solution was restored to room temperature. Relative electrolyte leakage was calculated by comparing value A and value B.

Protein stability assay in N. benthamiana

To detect the effect of the kinase catalytic active site on protein stability, NbERK1 and NbERK1-D644N were transiently expressed in N. benthamiana for 2 days. Leaves were treated with 50 μM protein synthesis inhibitor cycloheximide (CHX) for 0, 2, and 4 h before harvesting. NbERK1 and NbERK1-D644N abundances were detected by immunoblotting with anti-HA (Cwbio).