Characterization of the Roles of SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 in Sw-5b-Mediated Resistance to Tomato Spotted Wilt Virus

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways.


Introduction
Plants have evolved different layers of defense against pathogen infection [1]. In the first layer, plants use an extracellular pattern recognition receptor (PRR) to recognize a pathogen-associated molecular pattern (PAMP) to trigger PAMP-triggered immunity (PTI). In response, plant pathogens use multiple virulence effectors to suppress PTI. As a second layer of defense, plants have evolved various intracellular nucleotide-binding leucine-rich repeat (NLR) receptors to recognize these effectors and invoke effector-triggered immunity (ETI). NLR proteins are the largest group of resistance genes in plants [2]. Plant NLRs typically have a variable N-terminal domain: a Toll/interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain, a central NB-ARC (nucleotide binding adaptor and APAF-1, R proteins, and CED-4) domain, and a C-terminal leucine-rich repeat (LRR) domain [3,4]. Hereafter, NLRs with CC domains are referred to as CNLs, and NLRs with TIR domains are referred to as TNLs. Effector-mediated NLR activation results in a robust defense response that is typically associated with a hypersensitive response (HR) and constrains pathogens at the site of infection [1,[4][5][6]. However, our understanding of how NLRs activate downstream resistance remains limited. To date, only a few common key downstream immune-signaling components of NLR-mediated resistance have been identified. Tomato spotted wilt virus (TSWV, genus Orthotospovirus; family Tospoviridae) infects more than 1000 different plants in at least 85 families [7] and is among the most destructive plant viruses [8][9][10], causing more than 1 billion USD in crop losses worldwide each year [11]. The Sw-5b gene is the most effective resistance gene for controlling TSWV in tomato [12][13][14][15]. Since it was introgressed from Peruvian tomato, Solanum peruvianum L., it has been used widely in tomato-breeding projects. Sw-5b encodes a protein belonging to a CNL-type resistance gene. It specifically recognizes the movement protein NSm of TSWV [16][17][18]. Sw-5b has also been found to confer broad-spectrum resistance against various American-type tospoviruses by recognizing a highly conserved 21 amino-acid region in NSm (NSm21) [19]. Sw-5b carries an extended N-terminal Solanaceae-specific domain (SD) and develops a multilayered regulatory mechanism to control its autoinhibition and activation [20]. For activation, Sw-5b uses a two-step recognition mechanism involving both SD and the LRR domain in the detection of viral NSm [21]. This two-step recognition increases the specificity of NSm recognition and also significantly enhances the sensitivity of NSm detection. Although our understanding of how Sw-5b recognizes viral elicitor NSm has increased significantly, the process by which Sw-5b activates downstream defense signaling has yet to be elucidated. SGT1 (suppressor of the G2 allele of skp1) has been found to be required for the induction of the immunity mediated by many NLRs [22,23]. As a protein conserved in eukaryotes, SGT1 functions in a variety of biological processes by interacting with different protein complexes [24][25][26]. It regulates the protein accumulation levels of NLR immune receptors [27] and participates in the nucleocytoplasmic distribution of tobacco NLR protein N [28]. SGT1 is associated with RAR1, which is required for Mla12 resistance, and heat-shock protein 90 (HSP90), forming a molecular chaperone complex that is required for appropriate accumulation of NLR proteins [23]. Although SGT1 is required for the establishment of the Sw-5b-mediated HR [29], the requirement for SGT1/RAR1/HSP90 in Sw-5b-mediated induction of systemic resistance to TSWV has not been tested. EDS1 (enhanced disease susceptibility 1) is a lipase-like protein that is needed for all tested TNLs to activate downstream signaling. EDS1 is also necessary for some CNLs, such as the CNL HRT and the CC R-protein RPW8 [30,31]. However, some CNLs, such as RPS2, RPM1, and RPS5, require an alternate protein, NDR1 (non-race-specific disease resistance 1), for immunity [32]. The requirement for EDS1/NDR1 in the Sw-5b/TSWV resistance pathways has not yet been tested.
Salicylic acid (SA) plays an important role in many NLR-mediated resistance mechanisms. In Arabidopsis, NPR1 (non-expresser of pathogenesis-related genes 1) is a central component in SA signaling [33,34]. NPR1 was found to be a receptor of SA [35]. It controls the transcription of approximately 90% of the SA-dependent genes in Arabidopsis [33,[36][37][38]. NPR1 shuttles into the nucleus, where it interacts with TGA transcription factors, thereby activating defense genes to establish plant immunity [39]. Tobacco NPR1 is required for N-mediated resistance to tobacco mosaic virus (TMV) [37]. However, HRT-mediated resistance to turnip crinkle virus (TCV) in Arabidopsis is independent of NPR1 [40]. The role of NPR1 in the Sw-5b resistance pathway is not known.
Recently, many "sensor" NLR proteins have been found to require "helper" NLR proteins to activate downstream immune signals [41,42]. Three types of "helper" NLR proteins have been described in plants: the NRC (NB-LRR protein required for HR-associated cell death) family, ADR1 (activated disease resistance 1) family, and NRG1 (N requirement gene 1) family. All of these are CNL-type proteins [42,43]. The NRC family proteins in tobacco and tomato are required for the induction of HR mediated by a variety of NLRs [29,41,44]. Although NRC2/3/4 have been shown to be required for the induction of HR mediated by Sw-5b [29], whether they are required for induction of systemic resistance to TSWV remains to be elucidated. ADR1 family proteins that function downstream of RPS4/RRS1, RPP2, SNC1, CHS1/SOC3, RPP4, and RPS2 are involved in both TNL-and CNL-mediated immunity [45][46][47][48]. NRG1 was first identified in Nicotiana benthamiana L. using virus-induced gene silencing (VIGS) and was found to be required for N-mediated Viruses 2021, 13, 1447 3 of 16 resistance to TMV [49]. NRG1 is essential for Roq1, N, and RPP1-mediated HR and disease resistance [50]. Ry sto -dependent extreme resistance to potato virus Y also requires NRG1 [51]. NRG1 is likely a conserved key component of TNL-mediated immune signaling pathways. The ADR1 and NRG1 gene families are function redundant, and their N-terminal domains are related to RPW8-CC [46]. Disease susceptibility was enhanced in an adr1 adr1-L1 adr1-L2 nrg1.1 nrg1.2 nrg1.3 sextuple helperless mutant in comparison to adr1 triple and nrg1 triple mutants [47]. ADR1 and NRG1 were also found to be involved in Rx2-mediated resistance to potato virus X (PVX) [46]. However, the requirement for ADR1 and NRG1 in Sw-5b/TSWV has not yet been tested.
In this study, we characterized the requirement for SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated immune signaling pathway using a TRV-based virus induced gene silencing (VIGS) approach. We found that SGT1, but not RAR1, was required for Sw-5b-mediated resistance. Sw-5b function was independent of both EDS1 and NDR1. Suppression of NPR1 did not cause TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. The helper NLRs NRC2/3/4, but not ADR1 and NRG1, were required for Sw-5b-mediated systemic resistance to TSWV infection. These findings provide new insights into the requirement for conserved components in the Sw-5b-mediated signaling pathway.

Host Plant and Virus
Four to 6 week old N. benthamiana plants were used for the VIGS experiments. The source of the TSWV Yunnan isolate has been described previously [53]. TSWV Yunnan isolates were propagated in N. benthamiana. For long-term preservation, fresh systemically infected leaves of N. benthamiana were stored in a refrigerator at −80 • C. Plant leaves containing TSWV were ground in 0.01 M phosphate buffer (pH 7.4). The Sw-5b-transgenic N. benthamiana line has been described previously [20]. The VIGS-treated and TSWVinoculated plants were maintained in a growth chamber at a temperature of 24 • C under a 16 h light/8 h dark photoperiod.

VIGS Assay
pTRV1, pTRV2, and their derivatives were individually introduced into Agrobacterium strain GV3101 cells by electroporation. Agrobacterium cultures were resuspended in agroinfiltration buffer (10 mM MgCl 2 , 10 mM MES pH 5.6, and 100 µM acetosyringone) and adjusted to a final concentration of OD 600 = 0.5. The Agrobacteria containing TRV RNA1 and RNA2 were mixed at a 1:1 ratio and incubated for 3 to 5 h in the dark at 28 • C. The mixtures were co-infiltrated into fully expanded leaves of N. benthamiana using a 1 mL needleless syringe. Six plants were used for each treatment and/experiment. Each experiment was repeated three times.

Total RNA Isolation and Quantitative RT-PCR Analysis
Total RNAs were extracted from N. benthamiana plant leaves using an RNA Easy Fast Isolation kit (catalog no. DP452, Tiangen, Beijing, China). First-strand cDNAs were  Supplementary Table S1. NbActin and NbEF1a were used as internal controls in the assayed samples.

Silencing Sw-5b by TRV-Based VIGS Abolished the Resistance of Sw-5b-Transgenic N. benthamiana to TSWV Infection
Our laboratory previously generated Sw-5b-transgenic N. benthamiana that confer resistance to TSWV infection [20]. To investigate the downstream defense signaling of Sw-5b-mediated resistance, we adopted a TRV-based VIGS to silence downstream candidate genes in Sw-5b-transgenic N. benthamiana plants, followed by TSWV infection. Before silencing the downstream candidate genes, we first tested silencing the Sw-5b gene in Sw-5b-transgenic plants using a TRV-based VIGS approach, and we examined the process of TSWV infection in the Sw-5b silenced plants. A 500 bp fragment of the Sw-5b gene was amplified by PCR and inserted into the TRV2 vector. As a control, a fragment from the GUS gene was inserted into the TRV2 vector. TRV2 carrying a fragment from NbPDS was also used as a control. Agrobacterium carrying TRV1 mixed equally with Agrobacterium carrying TRV2-Sw-5b, TRV2-GUS, or TRV-NbPDS was co-infiltrated into the leaves of 4-6 week old Sw-5b-transgenic N. benthamiana plants. At 3 weeks post TRV treatment, newly emerged leaves of Sw-5b transgenic N. benthamiana infected with TRV-NbPDS were photobleached ( Figure S1A), implying that NbPDS was silenced. At this stage, the corresponding newly emerged leaves of Sw-5b-transgenic N. benthamiana plants pre-treated with TRV-Sw-5b or TRV-GUS control ( Figure 1A and Figure S1B) were inoculated with crude sap from TSWV-infected tissue. At 14 days post TSWV inoculation (dpi), typical viral symptoms including leaf curling, stunting, and systemic wilt were observed in the Sw-5b-transgenic N. benthamiana silenced for Sw-5b ( Figure 1B). No such symptoms were observed in the plants treated with the TRV-GUS control ( Figure 1B).
Total RNA was extracted from systemic leaves of TSWV-inoculated transgenic N. benthamiana treated with TRV-Sw-5b or TRV-GUS. The RT-PCR analysis showed that the TSWV N gene was detected in systemically infected leaves of the Sw-5b-silenced transgenic N. benthamiana, but not in the TRV-GUS-treated control plants ( Figure 1C). Western blotting assays further confirmed that TSWV N protein accumulated in the systemically infected leaves of Sw-5b-transgenic plants treated with TRV-Sw-5b, but not in those treated with the TRV-GUS control ( Figure 1D). These results imply that silencing Sw-5b by TRV-based VIGS abolished Sw-5b-mediated resistance to TSWV infection in transgenic N. benthamiana plants. blotting assays further confirmed that TSWV N protein accumulated in the systemically infected leaves of Sw-5b-transgenic plants treated with TRV-Sw-5b, but not in those treated with the TRV-GUS control ( Figure 1D). These results imply that silencing Sw-5b by TRVbased VIGS abolished Sw-5b-mediated resistance to TSWV infection in transgenic N. benthamiana plants. 3.2. SGT1, But Not Rar1, Was Required for Sw-5b-Mediated Resistance to TSWV SGT1, RAR1, and HSP90 are required for the induction of disease resistance mediated by a variety of NLR proteins. They form a molecular chaperone complex that is important for regulating the protein stability of plant NLRs [54,55].
To examine whether SGT1, RAR1, and HSP90 play essential roles in Sw-5b-mediated resistance to TSWV, we used TRV-based VIGS to silence NbSGT1, NbRAR1, and NbHSP90 in Sw-5b-transgenic N. benthamiana. TRV-GUS was used as a negative control. At 3 weeks post TRV treatment, in comparison with the TRV-GUS-treated controls, the transgenic N. benthamiana plants treated with TRV-NbSGT1 and TRV-NbRAR1 did not exhibit significant growth defects (Figure 2A and Figure S2). However, the transgenic N. benthamiana treated with TRV-NbHSP90 were deformed, with extremely small newly emerged leaves and inhibited growth, making it difficult to inoculate them further with TSWV ( Figure S3A). The qRT-PCR results showed that mRNA expression of NbSGT1, NbRAR1, and NbHSP90 was significantly reduced in newly emerged leaves of these transgenic plants ( Figure 2B and Figure S3B). The newly emerged leaves of transgenic N. benthamiana silenced for NbSGT1 or NbRAR1 were inoculated with TSWV crude extracts from systemically infected tissues. At 14 days post TSWV inoculation, the TSWV spread to the systemic leaves, causing symptoms including systemic wilting in NbSGT1-silenced plants ( Figure 2C). No systemic infection was observed in the TRV-GUS-treated control plants ( Figure 2C). In the Sw-5b transgenic N. benthamiana silenced for NbRAR1, to our surprise, neither TSWV systemic infection nor viral symptoms were observed ( Figure 2C). RT-PCR assays showed that the TSWV N gene was detected in the systemically infected leaves of NbSGT1-silenced transgenic N. benthamiana, but not in the TRV-NbRAR1 and TRV-GUS pretreated plants ( Figure 2D). Western blotting assays further confirmed that the TSWV N protein accumulated in the systemically infected leaves of NbSGT1-silenced transgenic N. benthamiana plants, but not in the leaves of TRV-NbRAR1-or TRV-GUS-treated plants ( Figure 2E). These findings imply that SGT1, but not RAR1, is involved in Sw-5b-mediated resistance to TSWV.

Suppression of EDS1 and NDR1 Did Not Disrupt Sw-5b-Triggered Immunity
EDS1 is a common signal component in the TNL-mediated ETI process [56], in which it plays an important role downstream of TIR NADase activity [57,58]. EDS1 is also involved in the immunity mediated by some CNLs [30,31]. NDR1 is a key signal component of the plant immune response mediated by some CNLs [59][60][61]. To investigate whether EDS1 and NDR1 are involved in Sw-5b-mediated resistance to TSWV infection, Agrobacterium cultures containing TRV1 were mixed with Agrobacterium carrying TRV-NbEDS1 or TRV-NbNDR1 and co-infiltrated into leaves of Sw-5b-transgenic N. benthamiana. The TRV-GUS was used as a control. At 3 weeks post TRV treatment, the transgenic N. benthamiana treated with TRV-NbEDS1 and TRV-NbNDR1 did not show any differences compared to the plant treated with the TRV-GUS control ( Figure 3A and Figure S4). qRT-PCR confirmed that expression of NbEDS1 and NbNDR1 mRNA transcripts in the corresponding silenced plants was significantly reduced ( Figure 3B). Subsequently, gene-silenced newly emerged leaves of transgenic N. benthamiana plants were inoculated with TSWV. At 14 days post TSWV inoculation, no obvious virus systemic infection was observed in the NbEDS1or NbNDR1-silenced transgenic N. benthamiana plants ( Figure 3C). RT-PCR and Western blot assays confirmed that TSWV was absent in the systemic leaves of the NbEDS1and NbNDR1-silenced transgenic N. benthamiana plants ( Figure 3D,E). These results imply that silencing expression of the NbEDS1 or NbNDR1 genes did not disrupt the resistance of Sw-5b-transgenic N. benthamiana plants to TSWV infection.

Silencing Expression of NPR1 Did Not Result in TSWV Systemic Infection in
Sw-5b-Transgenic N. benthamiana Plants NPR1 acts as a key regulator of SA-signaling pathways [33]. To examine whether NPR1 plays a role in Sw-5b-mediated resistance to TSWV, the mixed Agrobacteria carrying TRV1 and TRV-NbNPR1 were co-infiltrated into Sw-5b-transgenic N. benthamiana. The Agrobacterium carrying the TRV-GUS was used as a control. At 3 weeks post TRV treatment, the growth state of transgenic N. benthamiana plants treated with TRV-NbNPR1 was comparable to that of plants treated with TRV-GUS ( Figure 4A and Figure S5). qRT-PCR assays confirmed that expression of NbNPR1 mRNA was silenced in TRV-NbNPR1-treated plants ( Figure 4B). The silenced plant leaves were inoculated with TSWV. At 14 days post TSWV inoculation, no obvious symptoms were observed in the systemic leaves of NbNPR1-silenced N. benthamiana plants ( Figure 4C). RT-PCR and Western blot assays further confirmed the absence of TSWV in systemic leaves of NbNPR1-silenced Sw-5btransgenic plants ( Figure 4D,E). These data imply that silencing expression of NbNPR1 did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana.

Discussion
Here, we used the TRV-based VIGS system to characterize the roles of the SGT1/RAR1/ HSP90, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in Sw-5b-mediated resistance to TSWV. We found that chaperone SGT1 was essential for Sw-5b function; however, co-chaperone RAR1 was not. Suppression of both EDS1 and NDR1 did not affect Sw-5bmediated resistance to TSWV. Moreover, suppression of NPR1 did not result in TSWV systemic infection in Sw-5b-transgenic plants. However, silencing the helper NLRs NRC2/3/4 compromised systemic resistance to TSWV infection in Sw-5b-transgenic N. benthamiana plants. Silencing another two helper NLRs, ADR1 and NRG1, did not affect Sw-5b-mediated immune signaling.
We explored the role of the SGT1-RAR1-HSP90 chaperone complex in Sw-5b-mediated immunity. Scofield et al. found that all three chaperone molecules, SGT1-RAR1-HSP90, were required for Lr21-mediated resistance. Silencing any of the three compromised resistance [63]. In our study, silencing HSP90 resulted in significant morphological defects in the transgenic N. benthamiana, which led us to discontinue further investigation into the role of HSP90. Silencing SGT1 compromised Sw-5b function; however, silencing RAR1 did not. SGT1-RAR1-HSP90 forms a chaperone complex. However, Sw-5b-mediated immune signaling is independent of RAR1. A previous study on Mi-1.2 also reported that Mi-1.2-mediated pest resistance required SGT1 but not RAR1 [64]. SGT1 has diverse roles in cellular pathways. For example, it plays an important role in nucleoplasm partitioning of tobacco N NLR [28]. We hypothesized that SGT1 may help in stabilizing Sw-5b immune receptor. In addition to functioning as a chaperone, SGT1 may play other roles in Sw-5band Mi-1.2-mediated defense signaling, and these roles of SGT1 may be independent of RAR1.
We tested the role of EDS1/NDR1 in Sw-5b-mediated resistance to TSWV. Early studies revealed that EDS1 was necessary for some CNLs. We found that the Sw-5b-mediated immune response was independent of EDS1. Arabidopsis CNLs such as RPS2, RPM1, and RPS5 are dependent on NDR1 for immunity. Our Sw-5b-mediated immunity was not dependent on NDR1. Arabidopsis CNLs, including HRT, ZAR1, RPP7, and RPP8, have also been found to be NDR1-independent [65]. In addition, the CNLs RPP13 and ZAR1 were independent of both EDS1 and NDR1 [66,67]. These findings imply that Sw-5b may induce EDS1-and NDR1-independent immune signaling pathways that remain to be uncovered.
We also explored the role of NPR1 in Sw-5b-mediated resistance. SA plays important roles in plant innate immunity and system acquired resistance (SAR). NPR1 acts as a receptor of SA and is a core player in SA signaling pathways. In our study, silencing expression of NPR1 did not disrupt Sw-5b resistance to TSWV. However, NPR1 has been shown to be required for the function of the tobacco N gene. Our results imply that Sw-5b-mediated resistance may not rely on the NPR1-dependent SA signaling pathway.
Lastly, we tested the function of "helper" NLR proteins in Sw-5b-mediated systemic resistance to TSWV infection. NRCs are required for HR mediated by various NLRs in Solanaceae and disease resistance mediated by Rpi-blb2 and Rx [29]. Here, we found that silencing the helper NLRs NRC2/3/4 compromised Sw-5b-mediated resistance to TSWV, further supporting the essential role of NRC in NLR-mediated resistance in Solanaceae. ADR1 proteins are involved in both TNL-and CNL-mediated immunity [45][46][47][48]. NRG1 is a key component involved in TNL-mediated immune signaling pathways. ADR1 and NRG1 function redundantly in mediating NLR immune signaling [62]. Silencing ADR1 and NRG1 simultaneously partially compromised resistance to PVX [46]. We found that silencing NRG1, ADR1, or both had no effect on Sw-5b-mediated resistance to TSWV.
Mixed infection by two different viruses may have synergistic or suppressive effects. Our presented data are based on the use of TRV for gene silencing, followed by TSWV infection. We cannot rule out the possibility that infection of Sw-5b transgenic plants by TRV constructs may have enhanced or reduced the resistance to TSWV infection. In the future, gene knockout mediated by CRISPR/Cas9 in Sw-5b transgenic plant is needed to confirm the results obtained in this study.
In summary, we systematically investigated the requirement for the SGT1/RAR1/HSP90, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in Sw-5b mediated immune signaling. We demonstrated that SGT1 and NRCs are conserved essential components of Sw-5bmediated resistance to TSWV. Our results showing the independence of EDS1/NDR1 and NPR1 also indicate that Sw-5b has some unique and undiscovered defense signaling pathway that remains to be elucidated. As Sw-5b-mediated resistance is dependent on NRCs, we proposed that Sw-5b may either directly recruit downstream helper NLRs or indirectly transfer the upstream pathogen perception signals to downstream helper NLRs to amply and induce robust defense signaling.
Author Contributions: Z.C., Q.W. and X.T. conceptualized and designed the experiments; Z.C., Q.W., C.T., H.C., D.M., X.Q., X.Z. and L.J. performed the experiments; Z.C., Q.W. and X.T. analyzed the data and wrote the paper. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: The data set analyzed for the current study is available from the corresponding author upon reasonable request.