Identification and Characterization of Plant-Interacting Targets of Tomato Spotted Wilt Virus Silencing Suppressor

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is an economically important plant virus that infects multiple horticultural crops on a global scale. TSWV encodes a non-structural protein NSs that acts as a suppressor of host RNA silencing machinery during infection. Despite extensive structural and functional analyses having been carried out on TSWV NSs, its protein-interacting targets in host plants are still largely unknown. Here, we systemically investigated NSs-interacting proteins in Nicotiana benthamiana via affinity purification and mass spectrometry (AP-MS) analysis. Forty-three TSWV NSs-interacting candidates were identified in N. benthamiana. Gene Ontology (GO) and protein–protein interaction (PPI) network analyses were carried out on their closest homologs in tobacco (Nicotiana tabacum), tomatoes (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). The results showed that NSs preferentially interacts with plant defense-related proteins such as calmodulin (CaM), importin, carbonic anhydrase and two heat shock proteins (HSPs): HSP70 and HSP90. As two major nodes in the PPI network, CaM and importin subunit α were selected for the further verification of their interactions with NSs via yeast two-hybrid (Y2H) screening. Our work suggests that the downstream signaling, transportation and/or metabolic pathways of host-NSs-interacting proteins may play critical roles in NSs-facilitated TSWV infection.


Introduction
Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is the best known member in Orthotospovirus, which is the only genus with plant-infecting viruses in the family Tospoviridae [1]. Belonging to the order Bunyavirales, tospoviruses contain segmented RNA genomes with three single-stranded (ss) RNAs packaged in enveloped virus particles [2]. The large (L) RNA is negative sense, while the medium (M) and the small (S) RNAs possess an ambisense genome organization [3]. As a well-studied and economically important plant virus [4], TSWV causes significant yield losses in a wide range of agronomic and horticultural crops such as beans, lettuce, peanuts (groundnuts), peppers, potatoes, tobacco and tomatoes [5,6].
The TSWV L RNA encodes an RNA-dependent RNA polymerase (RdRp). The M RNA encodes a non-structural movement protein NSm, and the precursor of two structural glycoproteins G N and G C . A nucleocapsid protein (N) and another non-structural protein (NSs) are encoded by the S RNA [7]. Both the M and S RNAs are organized in an ambisense manner [8]. The three genomic RNAs of TSWV and the N protein form ribonucleoproteins encapsulated by the glycoprotein (G N and G C ) envelope. TSWV infects plants via the thrips vector in the field [9].
NSs proteins are widely found in plant-and vertebrate-infecting Bunyaviruses [10]. NSs proteins from different tospoviruses share a common feature of binding both small and long double-stranded (ds) RNAs [11]. As a non-structural protein, TSWV NSs acts as an RNA silencing suppressor for overcoming the host immunity barrier [12]. NSs is an avirulence determinant of the TSWV resistance gene Tsw in peppers [13,14]. Tsw-mediated resistance in peppers can be overcome by a single amino acid change in NSs at position 104 (T-A) [15]. The N-terminal domain in NSs is important for its avirulence and RNA silencing suppression functions [16]. Two conserved motifs, GKV/T at positions 181-183 and YL at positions 412-413, are critical for the silencing suppressor function of NSs [7].
Despite the advancement of structural and functional research on TSWV NSs, its protein-interacting targets in host plants are still largely unknown. In this research, we investigated the NSs-interacting proteins in Nicotiana benthamiana via affinity purification and mass spectrometry (AP-MS) analysis. Gene Ontology (GO) and protein-protein interaction (PPI) network analyses were carried out on their closest homologs in Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum) and tomatoes (Solanum lycopersicum). Network analysis was carried out, followed by experimental validation by using the yeast two-hybrid (Y2H) assay. This approach of using AP-MS and network analysis combined with experimental validation offers an efficient approach for understanding the PPIs underlying virus-host interactions.

Affinity Purification-Mass Spectrometry Analysis Reveals NSs-Interacting Proteins in N. benthamiana
TSWV NSs was fused with an mGFP5 tag at its C-terminal (NSs-GFP) and was transiently expressed in N. benthamiana leaves at the four-leaf stage. Its binding proteins were extracted and analyzed by AP-MS. To identify the host proteins that specifically interact with TSWV NSs, overlapping candidates were selected from two independent AP-MS replicates. The list was then compared to the list of candidates that bind the V2 protein of Croton yellow vein mosaic virus (unpublished), to remove overlapping non-specific interactors. Eventually, 43 N. benthamiana proteins were found to specifically interact with TSWV NSs (Table 1). The list is arranged according to the numbers of peptide spectrum matches (#PSMs), posterior error probability (PEP) values of the PSMs (Sum PEP Score) and sums of the scores of the individual peptides from the Sequest HT search (Score SEQUEST HT) in Replicate 1 (R1).

Gene Ontology Overrepresentation/Enrichment Tests of NSs-Interacting Proteins
To facilitate GO analysis, the closest tobacco, tomato and Arabidopsis homologs inferred from the N. benthamiana NSs-interacting proteins ( Table 2) were used for overrepresentation/enrichment tests. Only the Arabidopsis homologs generated meaningful results in the GO biological process test that classified proteins according to the cellular activities in which they were involved (Table 3). Defense-responsive proteins were found to be enriched by about 10 fold (Table 3), which is consistent with the virulent nature of NSs. The defense-related proteins in the list include a LecRK (AT5G55830), a carbonic anhydrase (AT3G01500), chloroplast photosystem II subunit P1 (PSBP1; AT1G06680), a CaM (AT3G43810), a lipoxygenase (AT1G55020), STPK PBS1 (AT5G13160), a MAPK (AT4G01370), a cysteine proteinase (AT4G39090), a defensin-like protein (AT1G61070), a calcium-transporting ATPase (AT3G57330) and a NB-LRR protein required for hypersensitive response (HR)-associated cell death (NRC) (AT1G53350). The host immunity responses triggered by these defense proteins may be suppressed by the binding of NSs during TSWV infection.

The Protein-Protein Interaction Network of NSs-Interacting Proteins
To explore the indirect and expanded consequences of physical interactions between NSs and plant proteins, a PPI network was constructed for 42 Arabidopsis homologs inferred from N. benthamiana NSs-interacting proteins ( Figure 1A and Figure S1; Table S1). A total of 1346 interactions were predicted. Five major node proteins can be found in the PPI network, including HSP70 (At3G12580), CaM (AT3G43810), MAPK (AT4G01370), STPK (AT3G01090) and importin subunit α (AT4G16143) ( Figure 1A). Interactions between NSs and these five plant signaling, chaperone and transporter proteins may play significant roles in TSWV infection. We further investigated interactions within the 42 Arabidopsis homologs ( Figure 1B). The most reliable interaction was predicted to occur between HSP70 and HSP90 ( Figure 1B). Ten proteins including HSP70 and HSP90 were predicted to have self-interactions ( Figure 1B). As two major nodes in the PPI network, CaM and importin subunit α were selected for the further verification of their interactions with TSWV NSs. Table 5. PANTHER overrepresentation test of GO cellular components using tomato and Arabidopsis homologs inferred from N. benthamiana NSs-interacting proteins. A total of 34,637 proteins (GO Ontology database, doi:10.5281/zenodo.4033054) were included in the tomato reference list. All other parameters are the same as those in Table 3.

Importin Subunit α and Calmodulin 3 Interact with NSs in Targeted Yeast Two-Hybrid Assays
N. benthamiana importin subunit α (A1YUL9) and CaM 3 (U3MW48) were selected to verify their interactions with NSs via targeted Y2H. Both proteins interacted with NSs in the assays (Figure 2), which demonstrates that the AP-MS approach is effective and reliable in identifying host-NSs-interacting proteins.

Discussion
Although NSs is well-known for its RNA silencing suppressor function during the TSWV infection process, the direct protein-interacting targets of NSs in plant hosts are still largely unknown. There is a report that TSWV NSs can suppress jasmonate signaling in plants [17] via direct interactions with three basic-helix-loop-helix (bHLH) transcription factors (TFs): MYC2, MYC3 and MYC4 [18]. In this work, we significantly expanded the reservoir of NSs' physical interactors in plants. The interactions may be critical for TSWV virulence.
Multiple NSs-interacting proteins identified in this research have been demonstrated to regulate plant defenses. For example, cysteine proteinases play prominent roles in plant-pathogen interactions [19]. Notably, tomato aleurain-type cysteine proteinase can be inhibited by the pathogenic oomycete Phytophthora [20]. NSs-interacting cysteine proteinases are critical for lysosome-mediated autophagy function, which acts as an antiviral defense mechanism in plants. Viruses counteract host defenses by hijacking the autophagy pathway [21]. Interactions between NSs and lysosome-localized cysteine proteinases may contribute to the TSWV-induced suppression of autophagy.
N. benthamiana CaM 3 and importin subunit α are two NSs interactors verified by both AP-MS and Y2H assays. CaMs are significant components in plant immunity signaling networks [22]. There are multiple lines of evidence showing that CaMs participate in plant defenses against bacterial [23], fungal [24] and viral [25][26][27][28] pathogens. A tobacco CaM can bind the RNA silencing suppressor encoded by Cucumber mosaic virus and thereby trigger its degradation via the autophagy pathway [25]. On the contrary, an N. benthamiana CaM is required for the RNA silencing suppressor function of βC1, which is encoded by the geminivirus Tomato yellow leaf curl China virus [26]. Thus, the interaction between N. benthamiana CaM 3 and TSWV NSs may lead to either NSs degradation or the activation of its RNA silencing suppressor activity. Further investigations would reveal whether CaM 3 plays a positive or negative role in the NSs-mediated suppression of plant RNA silencing.
Importins are critical for the nuclear import of Agrobacterium virulence proteins [29]. There are multiple reports demonstrating that plant importin subunit α facilitates the nuclear transportation of viral proteins. N. plumbaginifolia importin subunit α can bind the coat/capsid proteins (CPs) of Rice tungro bacilliform virus and Mungbean yellow mosaic virus and transport them into the nucleus [30]. Similarly, tobacco importin subunit α mediates the nuclear import of Cauliflower mosaic virus translational transactivator protein P6, which suppresses plant RNA silencing in the nucleus [31]. N. benthamiana importin subunit α has a similar function of transporting viral proteins. For example, it interacts with the CP of Beet black scorch virus and transports it into the nucleus [32]. The nuclear localization of the Potato mop-top virus Triple Gene Block1 (TGB1) protein is mediated by N. benthamiana importin subunit α, which facilitates systemic virus movement [33]. The Pelargonium line pattern virus p37 protein acts as an RNA silencing suppressor whose nuclear localization is also mediated by N. benthamiana importin subunit α [34]. Taken together, we postulate that the physical interaction between TSWV NSs and importin subunit α may facilitate the nuclear transportation of NSs and the following exertion of its RNA silencing suppressor activity.
Since many plant virus infection events occur in the chloroplast [35] and are regulated by host photosynthetic and photomorphogenic activities [36], it is not surprising that NSs interacts with multiple chloroplast-localized proteins. Chloroplast-localized carbonic anhydrases appeared twice in our refined N. benthamiana NSs interactor list (Table 1). Their antioxidant activity is involved in plant HR defenses [37]. For example, carbonic anhydrase expression is indispensable for potato resistance to the late blight pathogen Phytophthora infestans [38]. It is possible that NSs interacts with plant carbonic anhydrases to suppress their antioxidant function, thereby promoting TSWV infection.
Both HSP70 and HSP90 were found to interact with TSWV NSs in our AP-MS analysis ( Table 1). HSP70 is a major node in the PPI network of NSs-interacting proteins ( Figure 1A). Functional and physical interactions between HSP70 and HSP90 exist ubiquitously in bacteria, yeasts [39] and plants [40]. In Arabidopsis, HSP70 expression can be induced by infections by multiple virus species [41]. In tomatoes, the Tomato yellow leaf curl virus CP interacts with HSP70 to facilitate virus infection [42]. In N. benthamiana, HSP90 is indispensable for plant resistance against Potato virus X and Tobacco mosaic virus [43]. Based on these reports, HSP70 and HSP90 may interact with NSs to positively or negatively regulate TSWV infection.
Overall, the NSs-interacting proteins identified via AP-MS provide multiple clues for dissecting the roles of NSs in TSWV-host interaction. CaM-triggered defense signaling, importin-facilitated protein nuclear transportation, carbonic anhydrase-catalyzed antioxidation and HSP70/HSP90-mediated stress tolerance emerged as principal plant cellular activities in response to NSs invasion. In the future, the molecular mechanisms of how TSWV NSs interacts with these defense-related proteins (e.g., time and spatial patterns); the genetic, biochemical and physiological outcomes of the interactions; the expression changes of downstream genes triggered by the interactions; and the regulatory/regulated proteins up-/downstream of the interaction cascades should be investigated to obtain more details. The obtained results would provide a comprehensive portrait of NSs' activities in the plant cell.

Plasmids and Gene Cloning
The TSWV NSs coding sequence (CDS) was previously described [8]. The NSs CDS was amplified using the PCR primers 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCTAT GTCTTCAAGTGTTTATGAG-3 and 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTGT TTTGATCCTGAAGCATA-3 . The amplified NSs CDS was cloned into the Gateway Donor vector pDONR 207 (Invitrogen) via a BP reaction (insertion of the att-B-sequence-containing PCR product into the att P recombination sites) and then inserted into the destination expression vector pEarleyGate 103 [44] from pDONR 207 via an LR reaction (insertion of the att-L-sequence-containing DNA into the att R recombination sites). In pEarleyGate 103, NSs was fused with an mGFP5 tag at its C-terminal (NSs-GFP). All the PCR-amplified sequences used in this research were verified by sequencing.

Affinity Purification-Mass Spectrometry Analysis of NSs-Interacting Proteins
NSs-GFP was transiently expressed in N. benthamiana leaves by agroinfiltration. Leaf samples were collected two days after infiltration, and the expression of NSs-GFP was verified by Western blotting. AP-MS was carried out using the GFP-Trap beads (Chromotek, Germany) as previously described [45,46]. Briefly, infiltrated leaves were ground into fine powder in liquid nitrogen, mixed with protein extraction buffer (1 mL per 500 mg of tissue) and then thawed on ice. After incubation and centrifugation at 4 • C, the extract supernatant was cleaned by filtration and then mixed with the GFP-Trap beads. After 1 h of incubation at 4 • C, the mixture was subsequently washed with wash buffer 3-5 times. The mass spectrometry (MS) analysis of the immunoprecipitated proteins was performed at the BGI Americas MS Service Center. The MS data were searched against the most updated Uniprot N. benthamiana database (2020_05) [47] using SEQUEST HT 2013 [48].

Refinement of the NSs-Interacting Protein Candidate List
Two NSs-GFP AP-MS biological replicates as well as two non-NSs AP-MS control replicates were performed for NSs-GFP. Since the Gateway-compatible pEarleyGate 103 cannot express mGFP5 without gene insertion, a pEarleyGate 103 construct expressing an mGFP5-fused V2 protein from Croton yellow vein mosaic virus was used as the non-NSs AP-MS control. Overlapping NSs-interacting protein candidates were identified from the two NSs-GFP AP-MS replicates. Non-specific NSs interactors were then removed from the list, including mGFP5, ubiquitin and proteins that were found to also interact with V2 in the control samples. This step helped to exclude non-specific NSs-interacting proteins that are expressed at high levels in N. benthamiana.

Verification of NSs-Interacting Proteins by Targeted Yeast Two-Hybrid Assay
Two N. benthamiana proteins in the interacting list, importin subunit α (A1YUL9) and CaM 3 (U3MW48), were selected to verify their interactions with NSs via targeted Y2H. The Y2H procedure has been described previously [49]. In brief, the NSs CDS was cloned into the Gateway-compatible prey vector pACT2-GW (pACT2-GW-NSs, with leucine selection marker) and then used to transform yeast strain A. After testing the transformed yeast clones for self-activation, the importin subunit α or CaM 3 CDS was cloned into the Gatewaycompatible bait vector pBTM116-D9 with tryptophan selection marker and then used to transform a selected yeast line harboring pACT2-GW-NSs. Empty pBTM116-D9 was used as a negative control. Positive interactions were implied by the observation of the yeast's growth on synthetic defined (SD) selection medium minus four elements of uracil, histidine, leucine and tryptophan (SDIV) and its tolerance to the His3p enzyme inhibitor 3-aminotriazole (3-AT).

Gene Ontology Analysis of Inferred Tobacco, Tomato and Arabidopsis Homologs
Since there is currently no available ontology data and analysis tool for N. benthamiana, the GO analysis of NSs-interacting proteins was performed using their closest homologs in tobacco, tomatoes and Arabidopsis. Tobacco is a close relative of N. benthamiana, and the genome of tomatoes has been well-studied compared to other species in the Solanaceae family. However, neither tobacco nor tomatoes have annotation information sufficient for a comprehensive GO analysis. Thus, Arabidopsis homologs were still used for all the GO enrichment tests for biological processes, molecular functions and cellular components. The test results for tobacco and tomato homologs were included if they contained meaningful information. All the tests were performed using the PANTHER (Version 15.0) GO Term Enrichment tools [50].

Protein-Protein Interaction Network Analysis of Inferred Arabidopsis Homologs
Arabidopsis homologs were used for the protein-protein interaction (PPI) network analysis due to the availability of PPI data and analysis tools. The PPI analysis of the Arabidopsis homologs inferred from the N. benthamiana NSs-interacting proteins was performed using the Bio-Analytic Resource (BAR) Arabidopsis Interactions Viewer [51]. The queries included interaction data and predictions from BioGrid (Version 4.1) [52], IntAct (Version 4.2.16) [53] and BAR (Version 20-04) [51]. The results of protein-DNA interactions from the BAR were also included.