Extra-large G proteins regulate disease resistance by directly coupling to immune receptors in Nicotiana benthamiana

Abstract

Heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are key regulators of eukaryotic intracellular signaling. Extra-large G (XLG) proteins are a subfamily of plant-specific Gα proteins interacting with plasma membrane-localized receptors to regulate multiple biological processes. The Nicotiana benthamiana genome encodes seven XLG proteins, NbXLG1–7, whose functions in disease resistance and underlying mechanisms are unknown. In this study, we silenced all the seven genes and found that disease susceptibility was enhanced when both NbXLG3 and NbXLG5 or NbXLG4 was silenced. Then, we generated N. benthamiana xlg3xlg5 double- and xlg4 single-mutant lines using the CRISPR-Cas9 approach. All the mutants showed reduced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, the fungal pathogen Sclerotinia sclerotiorum, and a series of oomycete pathogens, including Phytophthora capsici, Phytophthora infestans, and Phytophthora parasitica. We further demonstrated that NbXLG3/4/5 positively regulated microbial pattern-induced reactive oxygen species burst and defense gene expression by directly coupling to the tested plant immune receptors. In addition, we examined the role of NbXLG3/4/5 in abiotic stress tolerance and observed that NbXLG3 and NbXLG5 negatively regulated plant resistance to high-salt, mannitol, and PEG. Our study demonstrates the possible role of NbXLG3/4/5 in response to biotic and abiotic stresses and provides insights for the improvement of plant resistance to environmental changes.

Background

The heterotrimeric G protein complex, composed of α, β, and γ subunits, is one of the most important signal transducers in eukaryotic cells (Pandey 2019). G proteins are key regulators of extracellular signal transduction. In animals and fungi, the Gα subunit is directly coupled to seven-transmembrane G protein coupled receptors (GPCRs) that perceive extracellular signals through the ectodomains. GPCRs transmit these extracellular signals to Gαs, causing Gαs to exchange GDP for GTP, resulting in the activation of G protein heterotrimers. An activated Gα separates from Gβγ and they each function on their downstream targets (also known as G protein effectors) to transduce and amplify signals (Oldham and Hamm 2008). In plants, there is mounting evidence that G proteins are directly coupled to single-transmembrane receptor-like proteins (RLPs) and receptor kinases (RKs) to transduce extracellular signals to downstream effectors (Bommert et al. 2013; Choudhury and Pandey 2015; Liang et al. 2016; Yu et al. 2018; Zhao et al. 2022). Animals have a much larger number of G protein subunits that can form multiple heterotrimer combinations. For example, humans have 23 Gαs, 5 Gβs, and 12 Gγs. In contrast, plants have fewer G protein subunits. Arabidopsis and rice have only one canonical Gα subunit, three extra-large G (XLG) proteins, one Gβ subunit, and three and five Gγ subunits, respectively (Stateczny et al. 2016). XLG proteins are widespread in all higher plants and contain a C-terminal Gα-like domain and an N-terminal extension (Lee and Assmann 1999).

Plant G proteins are essential for many biological processes. Arabidopsis G proteins have been reported to regulate growth and development and respond to multiple hormones, abiotic stresses, and plant immune responses (Urano et al. 2016; Pandey 2019). Mutation of the Gα or Gβ genes in monocots (rice and maize) leads to a dwarf-like or lethal phenotype (Fujisawa et al. 1999; Ueguchi-Tanaka et al. 2000; Utsunomiya et al. 2012; Bommert et al. 2013). Dense and erect panicle 1 (DEP1), a rice Gγ protein, regulates erectness, panicle branching, and nitrogen assimilation (Huang et al. 2009; Sun et al. 2014). Rice Gγ protein grain size 3 (GS3) has been identified as the major allele controlling rice grain size (Fan et al. 2006). Another Gγ protein, RGG2, has been reported to be a negative regulator of grain size and yield (Miao et al. 2019). CT2, a Gα protein in maize, interacts with the CLAVATA receptor and regulates shoot apical meristem development (Bommert et al. 2013).

Recently, XLG proteins have been considered plant-specific Gα subunits, which greatly increase the functional diversity of plant G proteins. Arabidopsis XLG proteins are required to respond to hormonal and abiotic stresses (Ding et al. 2008; Pandey et al. 2008). The rice genome encodes three XLG proteins required for modeling yield-related traits, including plant height, panicle length, tiller number, and 1000-grain weight (Zhao et al. 2022). Maize XLGs have been reported to regulate shoot apical meristem development, and mutations in all three ZmXLGs are lethal (Wu et al. 2018). XLGs also play important roles in plant resistance to phytopathogens and in regulating immune signals (Zhu et al. 2009; Liu et al. 2013; Maruta et al. 2015; Liang et al. 2016, 2018; Ma et al. 2022; Wang et al. 2022; Zhao et al. 2022). Plant cells can recognize conserved microbial features, termed microbial patterns, using surface-localized pattern-recognition receptors (PRRs) to sense the invasion of microbial pathogens. Perception of microbial patterns by PRRs causes the activation of pattern-triggered immunity (PTI), which includes the transient influx of calcium, burst of reactive oxygen species (ROS), activation of MAP kinases, and transcriptional reprogramming (DeFalco and Zipfel 2021). The Arabidopsis receptor-like kinase (RLK) protein FLS2 recognizes bacterial flagellin (or flg22 epitope) in the presence of the co-receptor BAK1 (Chinchilla et al. 2006, 2007). The Arabidopsis RLK proteins LYK4/5 and CERK1 form a complex that recognizes fungal cell wall-derived chitin (Cao et al. 2014). We have previously shown that XLG2 and XLG3 are directly coupled to the FLS2 receptor to regulate flg22-induced immune signaling (Liang et al. 2016). Zhao et al. (2022) showed that rice XLG proteins regulate microbial pattern-induced immune activation and play different roles in plant resistance to bacterial and fungal infections (Zhao et al. 2022).

In the present study, we showed that the N. benthamiana genome encodes seven XLG proteins (NbXLGs), of which NbXLG3, NbXLG4, and NbXLG5 are required for plant resistance against bacterial and fungal pathogens. We further demonstrated that these three NbXLGs contribute to plant oomycete pathogen resistance. In addition, NbXLG3, NbXLG4, and NbXLG5 were found to regulate microbial pattern-induced immunity by interacting with PRRs. We also showed that NbXLG3 and NbXLG5, but not NbXLG4, negatively regulate plant resistance to abiotic stresses. Overall, our study revealed the biological functions of NbXLG proteins and how they can be used to potentially improve plant resistance to biotic and abiotic stresses.

Results

Identification of the NbXLGs involved in plant immunity

XLG proteins have been reported to regulate plant immunity, growth, and development in the model plant Arabidopsis. However, the functions of XLG proteins in Solanaceae plants have not been studied. We showed that there are 5–7 XLGs in N. benthamiana, Solanum lycopersicum, and Solanum tuberosum (Additional file 1: Figure S1 and Additional file 2: Table S1). Although most XLGs are grouped with Arabidopsis XLGs, a clade of Solanaceae XLGs cannot be grouped with Arabidopsis XLGs (Additional file 1: Figure S1). The N. benthamiana genome encodes one canonical Gα (NbGα) and seven XLG proteins, NbXLG1 (Niben101Scf00372g05021), NbXLG2 (Niben101Scf04286g01030), NbXLG3 (Niben101Scf01202g02006), NbXLG4 (Niben101Scf05674g05014), NbXLG5 (Niben101Scf06100g02001), NbXLG6 (Niben101Scf04383g01013), and NbXLG7 (Niben101Scf01249g03025) (Fig. 1a and Additional file 2: Table S1).

Fig. 1

Analyzation of XLG proteins in Nicotiana benthamiana. a Phylogenetic analysis of Gα proteins from N. benthamiana and Arabidopsis. b Silencing of NbXLG3,5 or NbXLG4 leads to enhanced susceptibility to Sclerotinia sclerotiorum. The indicated NbXLG genes were silenced by virus-induced gene silencing (VIGS). The plants were inoculated with S. sclerotiorum and photographed under UV light at 24 h post-inoculation (hpi). The lesion areas were measured and calculated (mean ± SD, n ≥ 6, student’s t test, **significant difference at P < 0.01). Scale bar, 1 cm. c Silencing of NbXLG3,5 or NbXLG4 leads to compromised flg22-induced ROS production. The indicated NbXLG-silenced plants were subjected to flg22-induced ROS examination (mean ± SD, n ≥ 6, student’s t test, **significant difference at P < 0.01). RLU, relative luminescence units. d Flg22 and chitin induce protein band shift of NbXLG3, NbXLG4, and NbXLG5 in the N-terminus. The N-terminal ~ 200 amino acid of NbXLGs were transiently expressed in N. benthamiana for 2 days, infiltrated with 1 μM flg22, or 200 μg/mL chitin for 10 min, and protein band shift was examined by anti-HA immunoblots

To determine the role of NbXLGs in plant immunity, we silenced NbXLG genes using virus-induced gene silencing (VIGS). Based on the phylogenetic tree, we constructed four VIGS vectors targeting NbXLG1,6, NbXLG2,7, NbXLG3,5, and NbXLG4. We then challenged the plants with S. sclerotiorum and examined lesion development one day later. NbXLG3,5- and NbXLG4-silenced plants exhibited significantly enhanced susceptibility to S. sclerotiorum (Fig. 1b). We then examined flg22-induced ROS burst, a typical assay for measuring microbial pattern-induced immunity, in NbXLG-silenced plants. The results showed that ROS production was slightly reduced in NbXLG1,6- or NbXLG2,7-silenced plants but severely reduced in NbXLG3,5- or NbXLG4-silenced plants (Fig. 1c). Quantitative real-time PCR (qPCR) analysis showed that all the target NbXLG genes were successfully silenced via VIGS (Additional file 1: Figure S2a, b). We previously showed that immune-related Arabidopsis XLGs are phosphorylated upon microbial pattern treatment at the N-terminus (Liang et al. 2016; Ma et al. 2022). Thus, we transiently expressed the N-terminus of approximately 200 amino acids of NbXLGs in N. benthamiana and examined the flg22-induced band shift by western blotting to detect protein phosphorylation. Consistent with the S. sclerotiorum infection and flg22-induced ROS assays, the N-terminus of NbXLG3, NbXLG4, and NbXLG5 showed a prominent band shift in the SDS-PAGE gel upon treatment with bacterial pattern flg22 or fungal pattern chitin (Fig. 1d). These results indicate that NbXLG3, NbXLG4, and NbXLG5 are involved in plant immunity.

Construction of Nbxlg3,5 and Nbxlg4 knockout mutants

To further analyze the roles of NbXLG3, NbXLG4, and NbXLG5 in plant immunity, we generated Nbxlg knockout lines using the CRISPR-Cas9 approach. NbXLG3 and NbXLG5 showed high sequence identities and similarities. Thus, we designed two guide RNAs (gRNAs) targeting NbXLG3 and NbXLG5 and cloned them into the pHEE401E vector (Wang et al. 2015) (Fig. 2a). In addition, we designed two gRNAs targeting the N-terminus of NbXLG4 (Fig. 2a). After screening of transgenic lines, we obtained two mutant alleles for each gene. We generated a 61 bp deletion in NbXLG3 and a 1 bp deletion in NbXLG5 (Nbxlg3,5-L1), a 1 bp insertion in NbXLG3 and a 2 bp deletion in NbXLG5 (Nbxlg3,5-L2), a 4 bp deletion in NbXLG4 (Nbxlg4-L1), and a 33 bp deletion in NbXLG4 (Nbxlg4-L2) (Fig. 2a). PCR and sequencing confirmed the gene editing results (Additional file 1: Figure S3). As shown in Fig. 2b, none of the Nbxlg knockout mutants showed visible severe growth and development defects (Fig. 2b). Therefore, we selected two independent homozygous lines without Cas9 expression (Cas9-free) for each transgene for further studies (Additional file 1: Figure S4a–d).

Fig. 2

Generation of Nbxlg3,5- or Nbxlg4-knockout mutants. a Knocking out of NbXLG3,5 or NbXLG4 via CRISPR/Cas9 approach. In the schematic map, the exons, introns, and UTRs are indicated as yellow boxes, lines, and bule boxes, respectively. The gRNA sequences and gene editing results are listed in the diagram. The DNA-sequencing chromatograms are shown in Additional file 1: Figure S3. b Morphologic phenotypes of 20- and 40- days-old NbXLG3,5 and Nbxlg4 mutant plants. Bar, 1 cm

NbXLG3/4/5 are required for plant resistance to bacterial and fungal pathogens

To evaluate the function of NbXLG3/5 and NbXLG4 in plant resistance, we examined plant resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and the fungal pathogen S. sclerotiorum. All Nbxlg3,5 (Nbxlg3,5-L1 and L2) and Nbxlg4 (Nbxlg4-L1 and L2) mutant lines showed significantly reduced resistance to Pst DC3000 (Fig. 3a). We then introduced Pst DC3000 hrcC⁻, a mutant strain defective in secreting virulence effectors and used to measure microbial pattern-induced immune responses. All Nbxlg3,5 and Nbxlg4 mutants showed enhanced susceptibility to Pst hrcC⁻ infection (Fig. 3b), suggesting a positive role for NbXLG3/5 and NbXLG4 in microbial pattern-triggered immunity. Next, we infected Nbxlg mutants with the fungal pathogen S. sclerotiorum. We found that Nbxlg3,5 and Nbxlg4 mutants exhibited much larger lesions than the wild-type (WT) plants (Fig. 3c), consistent with the result of S. sclerotiorum infection assay in NbXLG-silenced plants (Fig. 1b). Our results show that NbXLG3/5 and NbXLG4 positively regulate plant resistance against bacterial and fungal pathogens.

Fig. 3

Nbxlg3,5 and Nbxlg4 mutant plants have reduced resistance to bacterial and fungal pathogens. a, b Nbxlg3,5 and Nbxlg4 mutants showed enhanced disease susceptibility to Pst DC3000 (a) and Pst DC3000 hrcC⁻ (b). The plants were infiltrated with the indicated Pst strains, and in planta bacterial titers were determined at 3 days post-inoculation (dpi) (mean ± SD, n ≥ 6, student’s t-test, **significant difference at P < 0.01). c Nbxlg3,5 and Nbxlg4 mutants showed enhanced disease susceptibility to Sclerotinia sclerotiorum. The leaves were inoculated with S. sclerotiorum and photographed at 1 dpi, and the lesion areas were measured (mean ± SD, n ≥ 8, student’s t test, **significant difference at P < 0.01). Scale bar, 1 cm

NbXLG3/4/5 are required for plant resistance to oomycete pathogens

We next investigated the role of NbXLG proteins in plant resistance to oomycete pathogens, which has not been studied. We inoculated the WT, Nbxlg3,5, and Nbxlg4 plants with oomycete pathogens, including P. capsici, P. infestans, and P. parasitica. Knocking out NbXLG3,5 or NbXLG4 substantially reduced plant resistance to P. capsici (Fig. 4a). In addition, the mutant lines developed a much larger lesion than the WT plants upon P. capsici infection (Fig. 4a). Similarly, all Nbxlg3,5 and Nbxlg4 mutant lines showed enhanced susceptibility to P. infestans and P. parasitica (Fig. 4b, c). The lesions in the Nbxlg3,5 and Nbxlg4 mutant lines were much larger than those in the WT plants upon P. infestans or P. parasitica infection (Fig. 4b, c). Altogether, our findings revealed that NbXLG3, NbXLG4, and NbXLG5 play pivotal roles in plant resistance to oomycete pathogens.

Fig. 4

Nbxlg3,5 and Nbxlg4 mutant plants have reduced resistance to oomycete pathogens. The WT, Nbxlg3,5 and Nbxlg4 plants were inoculated with Phytophthora capsici (a), Phytophthora infestans (b), or Phytophthora parasitica (c). Photographs were taken under UV light and lesion areas were measured at 36–48 hpi, 7 dpi, and 60 hpi for P. capsici, P. infestans, and P. parasitica, respectively (mean ± SD, n ≥ 8, student’s t test, **significant difference at P < 0.01). Scale bar, 1 cm

NbXLGs positively regulate microbial patterns-induced immunity by coupling to plant immune receptors

Pattern-triggered immunity (PTI) confers broad resistance to most microbes. Considering NbXLG3/5 and NbXLG4 are required for plant resistance to multiple pathogens, we deduced that NbXLG3/5 and NbXLG4 are required for PTI. Microbial pattern-triggered ROS burst is a specific assay for examining PTI activation, we thus treated N. benthamiana plants with bacteria-derived flg22 or fungal-derived chitin and examined ROS production. All Nbxlg3,5 and Nbxlg4 mutant lines displayed significantly reduced flg22- and chitin-induced ROS bursts compared to the WT plants (Fig. 5a, b). We next checked defense gene expression in different Nbxlg mutants upon flg22 or chitin treatment. We examined the expression of ACRE31 and PTI5, and found they were highly induced by flg22 and chitin at 3 h after treatment (Fig. 5c, d). However, the Nbxlg3,5 and Nbxlg4 mutant lines showed a significantly compromised expression of ACRE31 and PTI5 upon flg22 or chitin treatment compared with the WT plants (Fig. 5c, d). The microbial pattern-induced phosphorylation of MAPKs is another typical assay for examining PTI. Therefore, we examined flg22- or chitin-induced activation of MAPKs in Nbxlg3,5 and Nbxlg4 mutants using anti-p-ERK immunoblots, and found that flg22- or chitin-induced activation of MAPKs were not affected by mutations in NbXLG3/5 or NbXLG4 (Additional file 1: Figure S5a, b).

Fig. 5

NbXLG3, NbXLG4, and NbXLG5 regulate flg22- or chitin-induced ROS and defence gene expression by coupling to plant immune receptors. a, b The Nbxlg3,5 and Nbxlg4 mutant lines showed significantly reduced ROS burst upon flg22 (a) or chitin (b) treatment. The indicated plants were examined for flg22- or chitin-induced ROS burst and the peak RLU value were recorded (mean ± SD, n ≥ 6, student’s t test, **significant difference at P < 0.01). c, d Flg22- or chitin- induced expression of defence marker gene ACRE31 and PTI5 are compromised in Nbxlg3,5 and Nbxlg4 mutant lines (Mean ± SD; student’s t test, **significant difference at P < 0.01.). e, f NbXLG3/5 and NbXLG4 interact with NbFLS2 (e) and NbCERK1(f). The indicated constructs were transiently expressed in Nicotiana benthamiana and protein interactions were examined by luciferase complementation image (LCI) assays (mean ± SD; n ≥ 6, student’s t test, **significant difference at P < 0.01). In a, b, e, and f, RLU indicates relative luminescence units

To confirm that compromised PTI activation was caused by mutations in NbXLG3/5 or NbXLG4, we transiently expressed NbXLG3 and NbXLG4 in Nbxlg3,5 and Nbxlg4 mutants, respectively, and examined flg22-induced ROS production. We noticed that transient expression of NbXLG3 could fully restore the reduced ROS levels in Nbxlg3,5 (Additional file 1: Figure S6a). Similarly, NbXLG4 expression restored the Nbxlg4 defect in flg22-induced ROS (Additional file 1: Figure S6b). These results confirm the role of NbXLG3/5 and NbXLG4 in plant immunity. Notably, the expression of the Arabidopsis XLG1, XLG2 or XLG3 cannot restore the reduced flg22-induced ROS burst caused by the mutation of NbXLG3/5 and NbXLG4 (Additional file 1: Figure S6a–d).

We have previously shown that AtXLGs form complexes with Gβγ dimers and are directly coupled to the plant immune receptor complex. Luciferase complementation image (LCI) assays showed that NbXLG3/5 and NbXLG4 interacted with NbGβ (Additional file 1: Figure S7a). Co-IP assays showed that NbXLG3 and NbXLG4 interacted with NbGβ (Additional file 1: Figure S7b). These results indicate that NbXLGs can form heterotrimers with Gβγ dimers. Next, we showed that NbXLG3, NbXLG4, and NbXLG5 interacted with the NbFLS2 and NbCERK1 receptors by LCI assays (Fig. 5e, f). We further confirmed NbXLG3-NbFLS2 and NbXLG4-NbFLS2 interactions using Co-IP assays (Additional file 1: Figure S7c). Collectively, we demonstrated that NbXLG3, NbXLG5, and NbXLG4, coupled with immune receptors to regulate pattern-triggered immunity.

NbXLG3/5 negatively regulates plant abiotic stresses

Arabidopsis XLG proteins have been reported to play a positive role in response to abiotic stresses (osmotic and salt stresses) and hormones (ABA and ET) (Ding et al. 2008; Urano et al. 2016). Therefore, we analyzed the roles of NbXLG3, NbXLG4, and NbXLG5 in high-salt or osmotic stress tolerance and examined the root length under different concentrations of NaCl, mannitol, and polyethylene glycol (PEG). Nbxlg3,5-L1 and Nbxlg3,5-L2 mutant lines showed significantly enhanced resistance to 200 mM NaCl but normal resistance to 250 mM NaCl (Fig. 6a and Additional file 1: Figure S8). Compared to the WT, the Nbxlg3,5-L1 and Nbxlg3,5-L2 mutants showed enhanced resistance to mannitol (300 and 400 mM) and PEG (2.5% and 5%) (Fig. 6a and Additional file 1: Figure S8), suggesting a negative role of NbXLG3/5 in osmotic stress tolerance. However, the Nbxlg4-L1 and Nbxlg4-L2 mutant lines showed normal resistance to NaCl, mannitol, and PEG stresses, indicating that NbXLG4 is not required for salt or osmotic stress (Fig. 6b and Additional file 1: Figure S9). Altogether, we demonstrated that NbXLG3 and NbXLG5 play a negative role in plant response to abiotic stresses.

Fig. 6

Nbxlg3,5 mutant plants have enhanced resistance to abiotic stresses. a Nbxlg3,5 mutant plants showed significantly enhanced resistance to salt and osmotic stresses. Nicotiana benthamiana seedlings were vertically grown on 1/2 MS medium supplemented with different concentrations of NaCl, mannitol, or PEG for 7 days. The seedlings were photographed and root length was measured. The related statistical data was shown in Additional file 1: Figure S8. b Nbxlg4 mutant plants showed normal resistance to salt and osmotic stresses. N. benthamiana seedlings were vertically grown on 1/2 MS medium supplemented with different concentrations of NaCl, mannitol, or PEG for 7 days. The seedlings were photographed and root length was measured. The related statistical data was shown in Additional file 1: Figure S9

Discussion

Heterotrimeric G proteins have been well studied regarding their functions, regulatory mechanisms, and structures in animals and fungi. The animal working model for G proteins has been established for years and is considered the most well-understood pathway. Over the last decade, plant heterotrimeric G proteins have been extensively studied, and the similarities and differences between plant and animal G proteins are actively being discovered. The plant genome encodes several G protein subunits and the theoretical number of heterotrimer combinations is limited. However, the discovery of XLG proteins, a subfamily of plant-specific Gα proteins, has greatly increased the number of heterotrimers and the functional diversity of plant G proteins. To date, most studies on XLG proteins have been conducted in Arabidopsis. However, XLG proteins in Solanaceae plants, such as N. benthamiana, have not been studied.

Bioinformatics analysis revealed that Solanaceae plants have more XLG proteins than Arabidopsis plants. There are five XLGs in tomato and potato plants, while seven XLGs are present in N. benthamiana (Additional file 1: Figure S1). We noticed that a subfamily of XLG proteins in Solanaceae plants did not cluster with AtXLGs, suggesting that they might possess specific functions that differ from those of AtXLGs. We silenced NbXLGs via VIGS and showed that NbXLG3/5 and NbXLG4 contribute to S. sclerotiorum resistance and flg22-induced ROS production (Fig. 1b, c). These results suggest a role for NbXLG3/5 and NbXLG4 in plant immunity. We previously reported that AtXLG2 and AtXLG3 are phosphorylated at the N-terminus following flg22 treatment (Liang et al. 2016). In this study, we showed that flg22 and chitin induced N-terminal phosphorylation of NbXLG3, NbXLG4, and NbXLG5 (Fig. 1d), further supporting their role in plant immunity. The specific phosphosites of these three NbXLGs and their functions remain unknown. It would be interesting to study whether the phosphosites of XLGs are conserved in Arabidopsis and N. benthamiana.

To better analyze the functions of NbXLG3/5 and NbXLG4, we constructed two independent mutant lines, Nbxlg3,5 and Nbxlg4, using the CRISPR-Cas9 approach. Notably, Nbxlg4-L2 possesses a 33-bp deletion in NbXLG4 (Fig. 2a), leading to a truncation of amino acids 30–41. However, Nbxlg4-L2 showed immune defect similar to those of Nbxlg4-L1, indicating that these 11 amino acids are important for the function of NbXLG4. Consistent with previous reports that XLG proteins are required for plant resistance against bacterial and fungal pathogens (Maruta et al. 2015; Liang et al. 2016; Urano et al. 2016; Zhao et al. 2022), here we showed that mutations in NbXLG3/5 and NbXLG4 resulted in severely impaired resistance to the bacterial pathogen Pst DC3000 and the fungal pathogen S. sclerotiorum (Fig. 3). To date, the role of XLG proteins in plant resistance to oomycete pathogens has not been investigated. Therefore, we challenged Nbxlg mutants with P. capsici, P. infestans, and P. parasitica and found that NbXLG3/5 and NbXLG4 are essential for plant resistance to these oomycete pathogens (Fig. 4). These findings are the first to show that XLG proteins are required for plant resistance to oomycete pathogens and may help to improve our understanding of the role of XLG proteins in plant immunity.

Next, we examined the functions of NbXLG3/5 and NbXLG4 in microbial pattern-induced immunity. The results showed that these three NbXLGs are required for flg22- or chitin-induced ROS burst and defense gene expression (Fig. 5). Consistent with the roles of XLGs in Arabidopsis and rice, NbXLG3, NbXLG4, and NbXLG5 interacted with NbGβ and were complexed with PRRs (NbFLS2 and NbCERK1) (Additional file 1: Figure S7). This result indicated that the XLG-Gβγ heterotrimers are involved in PRR complexes to regulate plant immune signaling. The role of NbGβ in plant immunity needs to be investigated in future studies. Intriguingly, we observed that transient expression of AtXLGs cannot restore the defect of Nbxlg4 in flg22-induced ROS production and only partially restores the impaired ROS burst in Nbxlg3,5 (Additional file 1: Figure S6).

Moreover, we showed that NbXLG3 and NbXLG5 negatively regulated plant resistance to salt and osmotic stresses. In contrast, AtXLGs are required for plant resistance to osmotic and salt stresses (Ding et al. 2008; Urano et al. 2016). The Nbxlg3,5 mutant lines had significantly enhanced resistance to high-salt, mannitol, and PEG (Fig. 6). However, Nbxlg4 mutant lines showed normal resistance to salt and osmotic stresses (Fig. 6). Thus, NbXLG3 and NbXLG5 can potentially improve plant resistance to biotic and abiotic stresses. Further studies are required to investigate the role of XLG proteins in plant resistance to biotic and abiotic stresses in other Solanaceae plants. It will also be worth studying the effect of XLG proteins on growth, development, and yield-related agronomic traits in Solanaceae plants such as tomatoes and potatoes.

Conclusions

In this study, we generated Nbxlg3,5 and Nbxlg4 knockout mutants and analyzed their immune phenotypes. Nbxlg3,5 and Nbxlg4 mutants showed severe defects in resistance against fungal and bacterial pathogens. We further demonstrated that XLG proteins are required for plant resistance to oomycete pathogens such as P. capsici, P. infestans, and P. parasitica. We revealed that NbXLG3/5 and NbXLG4 are involved in the immune receptor complex to regulate microbial patterns-induced immune responses. In addition, we demonstrated that the Nbxlg3,5 mutant has enhanced resistance to salt and osmotic stresses. NbXLG3 and NbXLG5 potentially play an important role in the coordinated regulation of plant resistance to biotic and abiotic stresses and might be ideal targets for the improvement of plant adaption to environmental changes.

Methods

Plant materials and conditions

The N. benthamiana plants used for most of the experiments in this study were soil-grown at 23 °C under a 10-h light/14-h dark photoperiod. The Nbxlg3,5 and Nbxlg4 mutants were generated by CRISPR-Cas9 approach. The N. benthamiana plants used for osmotic stress assays were grown at 23 °C on 1/2 MS medium under a 14-h light/10-h dark photoperiod.

Bioinformatic analyses

Full-length protein sequences of G proteins were used for construction of the phylogenetic trees and the protein sequences were listed in Additional file 2: Table S1. Phylogenetic neighbor-Joining dendrograms were constructed using MEGA 11 software.

Plasmid construction and generation of Nbxlg mutant lines

To perform VIGS assay, a 200–300 bp fragment targeting NbXLG1,6, NbXLG2,7, NbXLG3,5 or NbXLG4 was amplified and cloned into pTRV2 vector. For LCI assay, the coding sequences of the target genes were amplified and cloned into pCAMBIA1300-35S-Cluc-RBS or pCAMBIA1300-35S-HA-Nluc-RBS vector. To perform Co-IP assays, the corresponding genes were amplified and inserted into pCAMBIA1300-35S-FLAG-RBS or pCAMBIA1300-35S-HA-RBS vector. To generate knockout lines of NbXLGs, a pair of guide RNAs targeting the corresponding gene were designed and cloned into pHEE401 vector (Wang et al. 2015). The constructs were then introduced into N. benthamiana plants by Agrobacterium-mediated transformation (Ellis et al. 1987). The primers used in this study are listed in Additional file 3: Table S2.

Virus-induced gene silencing (VIGS)

VIGS was performed as previously described (Liu et al. 2002). A. tumefaciens strains harboring the constructed TRV2 vector or TRV1 vector were resuspended in an infiltration solution (10 mmol/L MgCl₂, 10 mmol/L MES pH5.7, and 200 μM acetosyringone) to a final OD 600 of 1.0. Equal amount of A. tumefaciens with TRV1 or TRV2 was mixed and infiltrated into primary leaves of N. benthamiana during the four‐leaf stage. TRV2:GFP and TRV2:PDS were used as negative and positive controls, respectively. The gene silencing efficiency was examined by qPCR analysis.

Pathogen infection assays

For bacterial inoculation assay, 4- to 5-week-old soil grown N. benthamiana plants were infiltrated with Pst DC3000 or Pst DC3000 hrcC⁻ at a concentration of 1 × 10⁵ CFU/mL. In planta bacterial titers were determined at 3 days post-inoculation (dpi).

For P. capsici infection assay, P. capsici strain LT263 was cultured at 25 °C on V8 agar plates for 2 days. Mycelial plugs were cultured in liquid V8 medium for 3 days, washed with sterilized water, and incubated in water to promote sporangia formation. To release the zoospores, the cultures were incubated at 4 °C for 40 min, followed by at 25 °C for 1 h. Detached N. benthamiana leaves were incubated with 150 zoospores and were kept in plastic boxes with high humidity in the dark. The leaves were photographed under UV light at 36–48 h post-inoculation (hpi). Lesion areas were measured and calculated by Image J software (Yu et al. 2012).

For P. infestans infection assay, the P. infestans strain TDT-88069 was cultured at 20 °C on Rye agar plates. The mycelium was flooded with water and scraped with a glass rod to release sporangia. Leaves were incubated with 350 sporangia and kept in plastic boxes with high humidity in the dark. The leaves were photographed under UV light at 7 dpi and lesion areas were measured by Image J software (Liang et al. 2021).

For S. sclerotiorum and P. parasitica infection assays, N. benthamiana leaves were inoculated with fresh mycelial plugs (5 mm in diameter). The leaves were put in a plastic box with high humidity and were photographed at 24 hpi (S. sclerotiorum) or 60 hpi (P. parasitica), and lesion areas were measured by Image J software (Huang et al. 2019; Nie et al. 2019).

Oxidative burst measurement

N. benthamiana leaf discs were collected and incubated overnight in a 96-well plate containing 200 μL water. The water was replaced with 200 μL reaction buffer containing 20 μM L-012 (Wako Chemical, Tokyo, Japan), 10 μg/mL horseradish peroxidase (Sigma), and 1 μM elicitors flg22 or 200 μg/mL chitin before measurement with a luminometer (Tecan F200) (Zhang et al. 2007).

Co-IP assay

The indicated constructs were expressed in N. benthamiana leaves by Agrobacterium-mediated transient expression system for 2 days. The leaves were collected and grounded in liquid nitrogen, and total protein was then extracted with protein extraction buffer [50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail]. Total protein was incubated with anti-FLAG M2 agrose (Sigma) for 3 h, washed with protein extraction buffer for 6 times, and eluted with 3 × FLAG peptide (Sigma), and the immunoprecipitates were separated on SDS-PAGE gel. Protein interactions were detected with anti-HA and anti-FLAG immunoblots (Wang et al. 2020).

MAPK activity assay

N. benthamiana leaves were infiltrated with 1 μM flg22, 200 μg/mL chitin, or water. After incubation for 0, 8, and 16 min, samples were collected and ground in liquid nitrogen and resuspended in lysis buffer [50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail]. Total proteins were separated on SDS-PAGE gel, and activation of MAPK was examined by anti-pERK immunoblots.

Luciferase complementation image (LCI) assay

The LCI assay was performed following the previously published protocol (Zhao and Zhou 2020). The indicated Cluc and Nluc constructs were expressed in N. benthamiana leaves by Agrobacterium-mediated transient expression. Leaf disks were taken at 2 days post-infiltration, incubated with 1 mM luciferin in a 96-well plate for 10–20 min, and the relative luminescence unit was measured by luminometer (Tecan).

RNA isolation and qPCR analysis

Four to five-week-old N. benthamiana plants were treated with 200 μg/mL chitin, 1 μM flg22, or water for 3 h. Total RNA was extracted using Eastep Supre RNA extraction Kit (Promega) following manufacturer’s instructions. The first strand cDNA was synthesized with the SuperScriptIII First-Strand Kit (Invitrogen) and subjected to qPCR analysis. The indicated primers were listed in Additional file 3: Table S2.

Osmotic stress assays

The N. benthamiana seedlings were vertically grown on 1/2 MS medium at 23 °C under a 16-h day/8-h night cycle for 5 days. Then, the seedlings were transferred to 1/2 MS medium supplemented with different concentrations of NaCl, mannitol, or PEG and incubated for another 7 days. The plates were photographed and root length was measured (Farid et al. 2013).