Over-Expression of Soybean Gm SAUL1 Enhances Disease Resistance in Nicotiana tabacum

: E3 ubiquitin ligases play essential roles in plant defense responses. However, their roles in other plant species have not been investigated extensively. Here, we used a gain-of-function approach to interrogate the function of Gm SAUL1 (Senescence-Associated E3 Ubiquitin Ligase 1) homologs in soybeans. Ectopic over-expression of GmSAUL1a in Nicotiana tabacum resulted in autoimmune responses that could be suppressed by high temperature, which is a hallmark of NLR (nucleotide-binding leucine-rich repeat protein)-mediated resistance. Interestingly, an elevated activation of Nt MPK6, but reduced activation of Nt MPK4, responding to ﬂg22 treatment, was observed in these GmSAUL1a- over-expressing tobacco lines, suggesting that over-expressing GmSAUL1a activates immune signaling through activating Nt MPK6, but suppressing Nt MPK4 kinase activity. Collectively, these results demonstrated that Gm SAUL1 plays a positive and pivotal role in soybean immunity that is likely governed by NLR proteins.


Introduction
Plants fight against pathogens via two layers of defense: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) [1]. Upon sensing and detection of PAMPs by plasma-membrane-localized receptor-like kinases (RLKs) or receptor-like protein (RLPs), PTI is activated [2,3]. Specific recognition of effectors secreted from pathogens into plant cells by host NLRs (nucleotide-binding leucine-rich repeat proteins) activates a stronger and long-lasting ETI [1]). Recently, it has been shown that PTI and ETI share common components and activate a set of overlapping responses [4,5]. In addition, PTI and ETI mutually regulate each other to achieve stronger immunity [6,7].
Ubiquitination is a conserved protein modification in regulating protein stability in eukaryotes [8]. Ubiquitination plays a wide range diverse functions in plants. Protein ubiquitination reactions are sequentially catalyzed by three family of enzymes: an ubiquitinactivating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and an ubiquitin ligase (E3) [8,9]. The substrate specificity is determined by the E3 ligases [10]. Over 1400 E3 ligase-encoding genes have been identified in the Arabidopsis genome [11], which can be classified into five families: HECT, SCF, RING-finger, U-box, and APC [12,13]. It has been shown that U-box E3 ligases play a critical role in plant immunity [12][13][14].
BPMV-mediated VIGS is the most widely used approach in the interrogation of gene functions in soybean [35,36]. Using this approach, we have successfully silenced multiple genes associated with defense responses [33,34,[37][38][39][40][41]. Recently, we showed that silencing two homologous SAUl1 genes simultaneously in soybean (GmSAUL1a and GmSAUL1b) activated defense responses [42]. To further understand the role of GmSAUL1 in immunity, we generated transgenic plants ectopically expressing GmSAUL1a in Nicotiana tabacum. We found that ectopic over-expression of the GmSAUL1a in N. tabacum activated immune responses that could be partially suppressed by high temperature, indicating that the Gm-SAUL1a plays a positive role in immunity. Interestingly, over-expression of the GmSAUL1a in N. tabacum led to activation of NtMPK6, but it led to repression of NtMPK4. Taken together, our results indicated that GmSAUL1a/1b plays a positive role in regulating cell death and immunity.

Transient Over-Expression of GmSAUL1a Induces HR Cell Death on the Leaves of Nicotiana benthamiana Plants
We previously showed that simultaneously silencing two homologous GmSAUL1 genes, GmSAUL1a and GmSAUL1b, resulted in an autoimmune response in soybean [42]. Over-expression of AtSAUL1 in Arabidopsis also activates autoimmunity [19]. To test whether over-expressing GmSAUL1a results in a similar phenotype, we transiently overexpressed the GmSAUL1a driven by a 35S promoter on the leaves of N. benthamiana via agro-infiltration. As anticipated, transient over-expression of GmSAUL1a induced cell death, similar to what is induced by over-expression of GmMEKK1 ( Figure 1A; [38]). As a negative control, infiltration of an agro-bacterial strain, expressing free GFP, did not induce HR-like cell death. This result demonstrated that the function of the SAUL1 homologs from different plant species is conserved.

Stable Transgenic N. tabacum Plants Over-Expressing GmSAUL1a Displayed an Auto-Immune Phenotype
Soybeans are recalcitrant for transformation. To circumvent the obstacle and to confirm the result from transient over-expression of GmSAUL1a ( Figure 1A), over 20 independent stable transgenic N. tabacum (Samsun, NN) plants that overexpress the GmSAUL1a were generated. Genomic PCR and RT-PCR analysis confirmed that the GmSAUL1a was successfully transformed and over-expressed in the majority of these transgenic lines ( Figure S1). Consistent with the result from transient over-expression of GmSAUL1a, spontaneous cell death was displayed on the leaves of all the GmSAUL1a-overexpressing plants. Representative images of the whole plants and the leaves from line #9 and #12 are shown in Figure 1B-D. The cell death was observed on the older leaves first and, subsequently, progressed to the younger leaves ( Figure 1B,C). HR-like cell death was accompanied by significantly induced expression of multiple PR genes ( Figure 1E) and over-accumulation of H 2 O 2 ( Figure 1F), suggesting that cell death is reminiscent of what occurred in the GmSAUL1a/b-silenced leaves [42]. Autoimmunity, resulting from the activation of R genes, is usually suppressed by high temperature [43,44]. To further test if the autoimmunity of the GmSAUL1a/1b-silenced plants resulted from NLR activation, the GmSAUL1a-overexpressing N. tabacum plants were treated at 30 • C. As anticipated, HR-like cell death on the leaves of the GmSAUL1aoverexpressing N. tabacum plants could be suppressed by high temperature ( Figure S2), indicating that cell death could be a result of a NLR activation.

GmSAUL1a-Overexpressing N. tabacum Plants Exhibit Enhanced Resistance against Different Biotrophic Pathogens
To examine whether cell death is associated with enhanced resistance, we firstly infected two independent GmSAUL1a-overexpressing tobacco plants with Tobacco mosaic virus (TMV). Because the tobacco cultivar we used for generating transgenic plants carries the N gene, TMV infection induces HR cell death in the leaves of these plants [45]. The size of the HR reflects the extent of TMV replication or movement [46]. As seen in Figure 2A, the number of Trypan blue-stained HR spots formed on the leaves of the GmSAUL1a-overexpressing plants upon TMV infection was significantly reduced compared to that on the WT plants ( Figure 2A). In addition, the sizes of the HRs on the GmSAUL1a-overexpressing plants were significantly reduced compared with those formed on the WT leaves ( Figure 2B). Furthermore, RT-qPCR analysis showed that the transcript level of the TMV capsid protein (CP)-encoding gene was significantly lower in the leaves of the GmSAUL1a-overexpressing plants than in the leaves of the WT plants ( Figure 2C), demonstrating that overexpressing GmSAUL1a enhances resistance to TMV in tobacco. . Overexpression of GmSAUL1a in N. tabacum leads to increased resistance to an agrobacterium strain (GV3101), carrying a construct of the pBI121-GUS; (F). The relative intensity of GUS staining was quantitated by Image J. ** and *** represent significant differences at 0.01 and 0.001 levels, respectively, by Student's t-test.
Next, WT and two independent GmSAUL1a-overexpressing plants were inoculated with a bacterial pathogen Pseudomonas syrangae pv. tomato DC 3000 (Pst DC3000). As anticipated, the multiplication of the Pst DC3000 on the leaves of the GmSAUL1a-overexpressing plants was significantly lower than that on the leaves of the WT plants ( Figure 2D), indicating that overexpression of GmSAUL1a in tobacco enhances resistance to Pst DC3000.
Lastly, we examined the effect of over-expression of GmSAUL1a on the resistance to an Agrobacterial strain carrying a 35::GUS construct. The agro-bacterial strain was infiltrated in the leaves of the WT and the GmSAUL1a-overexpressing tobacco plants. At 2 dpi, the leaf discs from the infiltrated area were stained for GUS activity, which reflects the infectivity of the Agrobacterial strain. As shown in Figure 2E,F, the intensity of the GUS staining on the leaf discs collected from the GmSAUL1a-overexpressing plants was significantly reduced relative to that of the WT plants, indicating that overexpressing GmSAUL1a in tobacco enhances its resistance to Agrobacterium. Collectively, these results indicated that overexpressing GmSAUL1a in tobacco enhances resistance to multiple biotrophic pathogens with different lifestyles and infection strategies, and GmSAUL1a is a positive regulator of immunity.

Enhanced Disease Resistance of GmSAUL1a-Overexpressing Tobacco Plants Is Correlated with Elevated Callose Deposition
To understand the molecular mechanism of the enhanced resistance of the GmSAUL1aoverexpressing tobacco plants, we performed the callose deposition assay on the leaves of WT and the GmSAUL1a-overexpressing plants. It was found that callose deposition of the GmSAUL1a-overexpressing plants was significantly increased relative to the WT plants ( Figure 3A,B), suggesting that the enhanced callose deposition is one of the mechanisms explaining the elevated resistance in the GmSAUL1a-overexpressing plants.

Over-Expressing GmSAUL1a in Tobacco Has Opposite Effects on the Activation of NtMPK4 and NtMPK6 Responding to flg22 Elicitation
To test the effect of the GmSAUL1a overexpression on the activation of the MAPK signaling pathways, MAPK kinase assay was performed using a Phospho-p44/42 MAP Erk1/2 antibody. As shown in Figure 4, the activation of NtMPK6 in response to flg22 elicitation was dramatically elevated in the GmSAUL1a-overexpressing plants compared with the WT plants. Interestingly, contrary to NtMPK6, the activation of NtMPK4 was drastically reduced in the GmSAUL1a-overexpressing plants relative to the WT plants. The activation of NtMPK3 was barely detectable on the GmSAUL1a-overexpressing leaves, but it was only visible on the WT leaves at 60 min of flg22 elicitation. Together, these results indicated that over-expression of the GmSAUL1a exerts opposite effects on the activation of NtMAPK6 and NtMPK3/4. . Over-expressing GmSAUL1a in N. tabacum cv. Samsun (NN) enhances the activation of NtMPK6, but it reduces the activation of NtMPK4. Protein samples were prepared, as described previously [38]. Western blotting was performed using a Phosph-p44/p42 MAP Erk1/2 antibody. An Arabidopsis sample treated with 10 µM flg22 for 10 min was used as a positive control. Coomassie blue-stained gel (CBS) was used as a loading control.

Subcellular Localization of GmSAUL1a
To visualize the subcellular localization of GmSAUL1a, the 35S::GFP-GmSAUL1a construct was transiently expressed in N. benthamiana leaves via agroinfiltration. Green fluorescence was observed, not only in the cytosol, but also in the nucleus ( Figure 5). Interestingly, we frequently observed the fluorescent patches at the plasma membrane (PM) ( Figure 5, see white arrows), These GFP patches may represent the sites for tethering the tonoplast and/or MVBs to the PM, as described in Arabidopsis [47][48][49]. In addition, mobile vesicles were also observed in the cytoplasm ( Figure 5, see red arrows), suggesting that the function of SAUL1 homologues might be conserved in different plant species.

The Function of SAUL1 Homologs Is Conserved across Plant Species
In Arabidopsis, either loss function of SAUL1 or ectopic over-expression of SAUL1 results in auto-immune phenotypes [16,17,19,20]. We showed that either ectopic overexpression of GmSAUL1a in N. tabacum (Figures 1-3) or silencing GmSAUL1a/1b in soybean [42] led to similar autoimmunity. The autoimmunity of the Arabidopsis saul1-1 mutant is dependent on PAD4 and EDS1 [16,17], suggesting that the autoimmunity of saul1-1 mutant depends on the SA pathway. The elevated levels of both free and conjugated forms of SA in the GmSAUL1a/1b-silenced soybean plants suggest that the autoimmunity displayed in these plants is also dependent on SA [42]. Consistent with the auto-immune phenotypes, the tobacco GmSAUL1a-overexpressed plants exhibited an increased resistance to different biotrophic pathogens (Figure 2). These results indicated that the function of SAUL1 homologs is highly conserved in different plant species [47]. The previously published results, together with our results, strongly indicated that the homeostasis of SAUL1 must be properly maintained within a certain threshold range to prevent activation of immunity. A level of SAUL1 exceeding the higher end or below the lower end of the threshold range will result in the activation of immune responses.
In Arabidopsis, the Toll interleukin 1 receptor (TIR)-type NLR (TNL), as well as SOC3 (Suppressors of chs1-2, 3), which associates with SAUL1, serve as guards to monitor the homeostasis of SAUL1. Either loss or overexpression of SAUL1 triggers SOC3-mediated autoimmunity [19]. Later, the same group showed that SOC3 pairs with two atypical truncated TIR-NB (TN) proteins to guard the homeostasis of SAUL1 [20]. Over-accumulation of SAUL1 is guarded by the SOC3-TN2 pair, and knocking out or knocking down of SAUL1 is guarded by the SOC3-CHS1 (Chilling Sensitive 1) pair [20]. Autoimmunity, resulting from NLR gene activation, is usually suppressed at high temperature [43,44]. The fact that the auto-immune phenotypes of the GmSAUL1a-overexpressing tobacco plants were significantly alleviated by high temperature treatment ( Figure S2) suggest that GmSAUL1a/1b is similarly guarded by a NLR(s), and the auto-immune phenotype might be a result of a NLR activation. It remains to be examined whether the SOC3-TN2 and SOC3-CHS1 homologues function the same way as in Arabidopsis to guard SAUL1 in soybean or tobacco.

The Roles of GmSAUL1s in PTI and ETI
An enhanced resistance to virulent pathogens was observed in the GmSAUL1aoverexpressing tobacco lines, which suggests that PTI is activated in these plants ( Figure 2). If the homeostasis of GmSAUL1 is similarly guarded by SOC3-TN2 and SOC3-CHS1 homologous pairs in soybean [19,20], the activated immunity observed in the 35S::GmSAUL1a tobacco plants (Figure 1) actually results from activated NLRs. High temperature rescued the autoimmune phenotype of the GmSAUL1a-overexpressing plants ( Figure S2), suggesting that certain NLR(s) likely act as guard(s) to monitor the level of GmSAUL1s in soybean or tobacco plants. Because PTI and ETI share overlapping components and are mutually required to maintain a prolonged and stronger immunity [6,7], it is not surprising that the enhanced disease resistance observed in the GmSAUL1a-overexpressing tobacco plants (Figure 2) could be a consequence of the activated ETI.
Interestingly, although both the saul1-1 mutant and the 35S::SAUL1 transgenic lines display autoimmunity [19,20], macroscopic lesions were only seen on the leaves of saul1-1 plants, but not on the leaves of the 35S::SAUL1 plants [20]. Different from the case in Arabidopsis, massive cell death was observed on the leaves of 35S::GmSAUL1a plants ( Figure 1B-D). This disparity is probably caused by differences in the downstream signaling pathways between Arabidopsis and tobacco.

Silencing GmSAUL1a/1b in Soybean and Over-Expressing GmSAUL1a in N. tabacum Activate Immunity through Different Downstream MPKs
Silencing GmSAUL1a/1b caused a reduced activation of GmMPK6 in soybean [42], whereas over-expression of GmSAUL1a results in an elevated activation of NtMPK6 in N. tabacum (Figure 4), indicating that the positive effect of GmSAUL1 on MPK6 activation is consistent between soybean and tobacco. Silencing GmSAUL1a/1b has contrasting effects on the flg22-induced activation of GmMPK3 and GmMPK6 (Figure 4). In Arabidopsis, the enhanced resistance is usually correlated with the enhanced MPK3/MPK6 activity [25]. In Arabidopsis, MPK3 and MPK6 can compensate for each other's function in mpk3 and mpk6 mutants, respectively [50].
Based on these studies, it is reasonable to postulate that the cell death and the activated immune responses observed in the GmSAUL1a-overexpresssing tobacco plants are a result of combined effects of the reduced activation of NtMPK4 and enhanced activation of NtGMPK6. Collectively, our results demonstrated that silencing GmSAUL1a/1b and over-expressing GmSAUL1a activate immune responses through different downstream MAPK modules (Figure 6, [42]). Instead of through a simple linear MAPK pathway, the SAUL1 regulates cell death and immunity through a MAPK network consisting of different downstream MAPKs. It remains to be determined whether the differential activation and repression of downstream MAPK modules resulted from silencing GmSAUL1a/1b in soybean and overexpression of GmSAUL1a in tobacco are associated with the activation of the SOC3-TN2 and SOC3-CHS1, respectively.

Plant Materials
Nicotiana tabacum cv. Sumsun (NN) and Nicotiana benthamiana were used in this study. Tobacco plants were grown in the growth room or growth chamber at 22 • C with a 16-h light/8-h dark photoperiod. High temperature treatment was performed at 30 • C.

Histochemical Assays
Plant cell death on the infected leaves was visualized by staining with trypan blue in lactophenol and ethanol, as described [53].

Generation of Transgenic N. tabacum Samsun (NN) Plants Over-Expressing GmSAUL1a
The full-length cDNA of GmSAUl1a was amplified using following pair of primers with KpnI and XmaI sites (in bold) attached to the 5 end and 3 end, respectively: GmSAUL1a-pE1776-F: aagGGTACCATGATGGCTGCGAGCTG GmSAUL1a-pE1776-R: ttgCCCGGGTTCATCCCATGTTTGGAAAGATT The amplified fragment was double-digested with KpnI and XmaI and subsequently cloned into the pE1776 vector [55] predigested with the same pair of restriction enzymes. The pE1776-GmSaul1a was transformed into the Agrobacterium strain GV3101 and then transformed into N. tabacum Samsun (NN), as described [56][57][58]. Briefly, tobacco leaves were sequentially sterilized with 70% ethanol and 0.5-2% NaClO 3 , respectively. Leaf discs from the sterilized leaves were pre-cultivated in MS medium for 3 days. After this, the leaf discs were co-incubated with Agrobacterium (GV3101) suspension solution for about a half hour, followed by cultivating in MS medium containing 1 mg/L Indole-3-acetic acid (IAA) and 1 mg/L 6-BA for 2 days. Afterward, these leaf discs were transferred to MS medium containing 1 mg/L IAA, 1 mg/L 6-BA, 50 mg/L Hygromycin, and 25 mg/L rifampicin. The shoots emerging from the callus were subsequently transferred to rooting medium containing IAA (1 mg/L), 50 mg/L Hygromycin, and 25 mg/L Timentin. After rooting, the seedlings were transferred to soil.

The Pst DC3000 Infection
The Pst DC3000 of OD600 = 0.00001 was infiltrated into the N. tabacum leaves, as described previously [59]. A colony-forming unit (cfu) at different days of infiltration (dpi) was counted and calculated, as described [59].

RNA Isolation and RT-qPCR
RNA isolation and RT-qPCR were performed, as described [34]. The RT-qPCRs were performed with three biological replicates and three technical replicates using an ABI550 Real-Time PCR machine (Applied Biosystems, Thermo Fisher Scientific, Auxtin, TX, USA) and the 2× SYBR Green qPCR Mix (Aidlab, Beijing, China). The annealing temperature for the RT-qPCRs was 60 • C. The comparative CT method was used for analysis.

Callose Deposition
Callose deposition in N. tabacum leaves was stained 24 h, as described by [59]. Callose deposits were visualized by ultraviolent epifluorescence using a fluorescence microscope (Olympus, Center Valley, PA, USA).

GUS Staining
GUS staining was performed, as described by [60]. A stereo microscope (Olympus SZH10, Center Valley, PA, USA) was used to take photographs of the GUS-stained leaves.

Sub-Cellular Localization of GFP-GmSAUL1a
The full-length cDNA of GmSAUL1a was amplified by RT-PCR by using the following pair of primers: pGDG-F-Sal1: aaaGTC GAC ATGATGGCTGCGAGCTGG pGDG-R-BamH1: ttgGGA TCC TCATCCCATGTTTGGAAAGATTC The PCR product was double-digested with SalI and BamHI and cloned into pGDG vector [61] predigested with the same set of restriction enzymes to generate the GFP-GmSAUL1a fusion construct. This 35S:: GFP-GmSAUL1a construct was infiltrated onto N. benthamiana leaves, as described [62]. Images were captured with a confocal laser-scanning microscope (Carl Zeiss, LSM 880, Jena, Germany).

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/agronomy13061533/s1, Figure S1: Screening of transgenic tobacco lines that ectopically expressing GmSAUL1. Figure S2: The cell death phenotype of the GmSAUL1aoverexpressing tobacco line is suppressed at 30 • C.