Combinatorial interactions between viral proteins expand the potential functional landscape of the tomato yellow leaf curl virus proteome

Viruses manipulate the cells they infect in order to replicate and spread. Due to strict size restrictions, viral genomes have reduced genetic space; how the action of the limited number of viral proteins results in the cell reprogramming observed during the infection is a long-standing question. Here, we explore the hypothesis that combinatorial interactions may expand the functional landscape of the viral proteome. We show that the proteins encoded by a plant-infecting DNA virus, the geminivirus tomato yellow leaf curl virus (TYLCV), physically associate with one another in an intricate network, as detected by a number of protein-protein interaction techniques. Importantly, our results indicate that intra-viral protein-protein interactions can modify the subcellular localization of the proteins involved. Using one particular pairwise interaction, that between the virus-encoded C2 and CP proteins, as proof-of-concept, we demonstrate that the combination of viral proteins leads to novel transcriptional effects on the host cell. Taken together, our results underscore the importance of studying viral protein function in the context of the infection. We propose a model in which viral proteins might have evolved to extensively interact with other elements within the viral proteome, enlarging the potential functional landscape available to the pathogen.


Introduction
Viruses are intracellular parasites that need to subvert the host cell in order to enable their own replication and ensure viral spread. For this purpose, viruses co-opt the cell molecular machinery, modulating or redirecting its functions; as a result, infected cells undergo dramatic molecular changes, including heavy transcriptional reprogramming, concomitant to the proliferation of the virus.
Most viruses have small genomes, likely due to bottlenecks in encapsidation and/or withinhost transport, which imposes limitations in coding capacity: viral proteins frequently exhibit small size, and their numbers per viral genome range from a few (<10) to a few dozen (Fig 1). Viral proteins have evolved to be multifunctional, and have been suggested to target hubs in the proteomes of their host cells [1][2][3][4], hence maximizing the impact of the viral-host proteinprotein interactions; nevertheless, how a limited repertoire of small viral proteins can lead to the drastic cellular changes observed during the viral infection remains puzzling. Upon viral invasion, virus-encoded proteins are produced in large amounts in the infected cells, where they co-exist. Therefore, physical or functional interactions among viral proteins might have evolved as a potential mechanism to expand the virus-host functional interface, increasing the number of potential targets in the host cell and/or synergistically modulating the cellular environment. Interestingly, examples of interactions between viral proteins have been recently documented for both animal and plant viruses (e.g. ; see VirHostNet 2.0, http:// virhostnet.prabi.fr/ [27]); some of these interactions are proposed to contribute to viral genome replication and virion assembly. However, the hypothesis that the combination of individual virus-encoded proteins might result in the acquisition of novel functions still lacks experimental support, and therefore the general biological relevance of these protein-protein interactions remains unclear.
Here, we use the plant DNA virus tomato yellow leaf curl virus (TYLCV; Fam. Geminiviridae) to test the idea that combinatorial interactions among viral proteins exist and may underlie an expansion of the functional landscape of the viral proteome. TYLCV is traditionally believed to encode six proteins (C1/Rep, C2, C3, C4, V2, and CP), although additional open reading frames (ORFs) have been recently described in its genome [28]. Local infection by TYLCV in the experimental host Nicotiana benthamiana results in heavy transcriptional reprogramming, with >11,000 differentially expressed genes (DEGs) detected at 6 days post-

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Interactions between viral proteins expand the potential functional landscape of the viral proteome inoculation (dpi) [29]. Although a limited number of viral protein-protein interactions have been described for this virus to date [30][31][32][33][34][35], the intra-viral interactome has not been systematically explored, and the functional impact of these interactions remains elusive. Our results show that, strikingly, viral proteins form prevalent pairwise interactions in the context of the viral infection, displaying a high degree of intra-viral connectivity, as demonstrated by an array of protein-protein interaction techniques. As proof-of-concept for the idea that intraviral protein-protein interactions can expand the functional repertoire of individual viral proteins, we focus on the pair formed by C2 and CP, since the presence of the latter is required and sufficient to shift the subcellular localization of the former from the nucleoplasm to the nucleolus, where the interaction occurs. Our data indicate that the combination of C2 and CP

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Interactions between viral proteins expand the potential functional landscape of the viral proteome results in drastic transcriptional reprogramming in the host plant, which goes beyond the sum of the effects of each of the individual proteins, hence supporting the idea that combinatorial interactions between viral proteins, physical and/or functional, can expand the functional repertoire of the viral proteome. The results obtained here might have important implications for the study not only of plant-geminivirus interactions, but of viral infections in general.

Viral proteins form complexes in the host cell
In order to test whether virus (TYLCV)-encoded proteins associate with one another, we employed a number of protein-protein interaction methods, namely yeast two-hybrid (Y2H), and in planta co-immunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFC), and split-luciferase assays. Several viral protein-protein interactions were identified in yeast (Figs 2A and S1) (Rep-Rep, Rep-C3, C2-C3, C2-C4, C3-C3, C3-C4, C3-CP, C3-V2, C4-C3, V2-V2); of note, the C2-C2 self-interaction could not be evaluated, since full-length C2 fused to the GAL4 binding domain displays auto-activation (Fig 2A), as previously described for other geminiviral C2 proteins [36-37]. Next, pairwise interactions between viral proteins were tested by co-IP assays following transient expression of C-terminally tagged versions of the viral proteins in N. benthamiana. The number of associations between viral proteins found in co-IP, which detects both direct and indirect interactions, was higher (Figs 2B, S2 and S3). The viral infection reshapes the cell environment where the viral proteins coexist: the presence/absence of host proteins, the activation of post-translational modification pathways, or the presence of additional viral proteins might influence the outcome of the tested interactions. Therefore, co-IP assays were performed both in the presence and absence of the virus. Most reproducible interactions detected in the absence of the virus were maintained in the context of the infection (Rep-Rep, C2-C2, C4-C2, C4-C3, C4-C4, C4-V2, CP-C2, V2-C2, V2-V2), and additional interactions were detected in an infection-dependent manner (Rep-C2, Rep-C3, Rep-C4, Rep-CP, Rep-V2, CP-Rep, C4-CP, CP-CP, CP-V2, V2-C3). Viral protein-protein interactions were further tested in planta by BiFC and split-luciferase assays (Fig 2C and 2D). In both assays, the viral proteins were fused to one half of the protein to be reconstituted upon a positive interaction (nYFP and cYFP for YFP, or nLuc and cLuc for luciferase, respectively) and transiently expressed in N. benthamiana leaves; for the BiFC experiments, n-YFP and c-YFP were fused to the C-terminus of the viral proteins, while for split-luciferase experiments nLuc was fused to the C-terminus and cLuc to the N-terminus of the viral protein. Interestingly, BiFC indicates that most of the detected interactions occur in the nucleus, with different distribution patterns, including localization in the nucleoplasm (e.g. Rep-C2), nucleolus (e.g. C3-C3), or nuclear speckles (e.g. Rep-C3) (Figs 2C and S4; additional patterns of interactions observed by BiFC can be found in S5 Fig). This nuclear prevalence of viral protein-protein interactions correlates with the nucleus hosting most of the viral cycle, including replication, transcription of viral genes, and encapsidation [38]. One notable exception is the interaction between C4 and V2, which takes place in intracellular punctate structures outside of the nucleus; this localization may be linked to the proposed role of these proteins in viral movement [39]. All viral proteins were shown to interact with one another (including self-interactions) at least in one direction by BiFC (Fig 2C). Similarly, all viral proteins displayed a positive interaction with each of the viral proteins by split-luciferase assays, with three exceptions (Rep-CP, C4-CP, and V2-CP) ( Fig 2D). The identification of positive interactions in these reconstitution assays that were not detected as such by co-IP could be explained by the potential weak or transient nature of these associations, which could be overcome by artificial stabilization provided by the complementation of the split reporter.

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Interactions between viral proteins expand the potential functional landscape of the viral proteome

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Interactions between viral proteins expand the potential functional landscape of the viral proteome A summary of all detected pairwise interactions between viral proteins is shown in Fig 2E  and 2F; all viral proteins were found to interact with one another, including self-interactions, by at least two independent methods. Importantly, some of these interactions could also be detected in unbiased affinity purification followed by mass spectrometry (AP-MS) experiments with C-terminal GFP-tagged versions of the viral proteins expressed in infected N. benthamiana cells [40] when these datasets were re-analyzed to search for the viral proteins, indicating that viral proteins physically associate with one another in the context of the infection in their native state (S1 Table).
As shown in Fig 2E and 2F, only certain pairwise viral protein-protein interactions could be detected by all used approaches (Rep-Rep, Rep-C3, C2-C4, C3-C4, C3-V2, and V2-V2). The differences in the detected outcomes when using different techniques might be due to specific requirements of each of the assays (e.g. strength or stability of the interaction necessary for this to be detected), or to the effect of the tags used (nature and position) on the interaction.

The viral coat protein is required and sufficient to modify the subcellular localization of the virus-encoded C2 protein
Although the proteins encoded by TYLCV display specific localizations in the plant cell, all of them, with the exception of C4 (at the plasma membrane and weakly in chloroplasts), can be clear and consistently found in the nucleus (nucleoplasm and/or subnuclear compartments) in basal conditions when expressed alone fused to GFP (Rep: nucleoplasm; C2: nucleoplasm; C3: nucleoplasm, nucleolus, and nuclear speckles; CP: nucleolus and weakly in the nucleoplasm; V2: Cajal body and weakly in the nucleoplasm-in addition to endoplasmic reticulum) ( Fig  3A). Interestingly, in the presence of the virus, several viral proteins fused to GFP, namely C2, C3, C4, and CP, experienced noticeable changes in their subcellular distribution ( Fig 3A): C2-GFP, which is excluded from the nucleolus in the absence of the virus, accumulates in this compartment in infected cells; C3-GFP, on the contrary, is excluded from the nucleolus in the presence of the virus; C4-GFP is depleted from the plasma membrane and accumulates in chloroplasts; and CP-GFP re-localizes from the nucleolus to the nucleoplasm, where it accumulates in unidentified structures. These changes had been previously reported for C4-GFP and CP-GFP; while in the case of C4-GFP, Rep alone can trigger its re-localization from the plasma membrane to chloroplasts [41], no individual protein was sufficient to modify the subnuclear pattern of CP [31].
Using transient co-expression of C2-GFP and each viral protein fused to RFP at their C-terminus in N. benthamiana leaves, we could determine that CP is sufficient to enable a strong accumulation of C2 in the nucleolus, an effect that can also be triggered by an untagged version of CP (Figs 3B and 3C; S6). Of note, C2 and CP have been shown to interact in the nucleolus ( Fig 2C). Curiously, only C2-GFP, but not GFP-C2, re-localizes to the nucleolus when in  Table. https://doi.org/10.1371/journal.ppat.1010909.g002

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Interactions between viral proteins expand the potential functional landscape of the viral proteome

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Interactions between viral proteins expand the potential functional landscape of the viral proteome the presence of CP, likely due to a positional effect of the GFP tag ( Fig 3C). Although full functionality of C2-GFP or GFP-C2 during the viral infection has not been demonstrated, C-terminal GFP fusions have been used in functional studies with other geminiviral C2 proteins (e.g. [42][43][44][45]). Local infection assays with two TYLCV mutant viruses unable to express CP, TYLCV-CPmut1 and TYLCV-CPmut2, in which early stop codons are introduced and alternative transcriptional initiation sites have been removed (for details, see Materials and Methods), demonstrate that the presence of CP is not only sufficient, but also required for the relocalization of C2-GFP into the nucleolus in infected cells (Fig 3D). These mutants accumulate to wild type-like levels in the transiently transformed leaves (S6B Fig). Since, in the absence of the virus, C2-GFP appears evenly distributed in the nucleoplasm and is excluded from the nucleolus, but it gains strong nucleolar accumulation in the presence of the virus, we reasoned that C2 might perform additional functions in the context of the infection, and decided to use the C2-CP interaction as a proof-of-concept for the idea that viral proteins might have combinatorial functions.

The C2/CP module specifically reshapes the host transcriptome
With the purpose of assessing if the functional landscape of C2 might be expanded when in the presence of CP, and considering that the C2 protein from geminiviruses has been previously described to impact host gene expression [46-51], we decided to investigate the transcriptional changes triggered by C2 in the presence or absence of CP as a readout for the activity of the former. To this aim, we transiently expressed C2, CP, or C2+CP in N. benthamiana leaves and determined the resulting changes in the plant transcriptome by RNA sequencing (RNA-seq). As shown in Fig Table; validation of the RNA-seq results by RT-qPCR is presented in S7C Fig.). Strikingly, however, the identity and behavior of DEGs was dramatically changed by the presence of CP (Fig 4B and 4C), indicating that C2 and CP have a synergistic effect on the host transcriptome. Functional enrichment analysis unveiled that addition of CP indeed shifted the functional gene ontology (GO) categories transcriptionally reprogrammed by C2, and that certain categories appear as statistically over-represented in the subset of down-regulated genes only when both viral proteins are simultaneously expressed (Fig 4D and 4E; S3 Table).
To investigate the relevance of the re-localization of C2 to the nucleolus (Fig 3B and 3C) for this effect, we selected DEGs specifically affected by the co-expression of C2 and CP, and tested the ability of C2-GFP (which re-localizes to the nucleolus in the presence of CP) or GFP-C2 (which does not re-localize to the nucleolus in the presence of CP) to affect their transcript accumulation when transiently co-expressed with CP in N. benthamiana leaves, as measured by RT-qPCR. As shown in Fig 5, only C2+CP and C2-GFP+CP, but not GFP-C2+CP, affect the expression of the selected genes compared with C2, C2-GFP, or GFP-C2, respectively. This result suggests that the modification in subnuclear localization of C2 mediated by CP is likely required for the impact of the combination of these proteins on host gene expression.
Next, we investigated the contribution of C2 and CP to the virus-induced transcriptional reprogramming in the context of the viral infection. We reasoned that, if C2 and CP together affect the transcriptional landscape of the host in a different manner than C2 or CP alone, then the transcriptional changes triggered by mutated versions of the virus unable to produce either C2 or CP should present overlapping differences compared to the changes triggered by the

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Interactions between viral proteins expand the potential functional landscape of the viral proteome wild-type (WT) virus. Following this rationale, we compared the transcriptome of N. benthamiana leaves infected with the WT virus or mutated versions unable to produce C2 (TYLCV-C2mut) or CP (TYLCV-CPmut1), with respect to the empty vector (EV) control ( Fig  6A) or to the WT virus ( Fig 6B). As expected, both point mutants were unable to establish a full systemic infection, indicating that the corresponding viral proteins are most likely not produced from the mutated genes (S8A and S8B Fig). Of note, although the CP null mutant (TYLCV-CPmut1) accumulated to lower levels in these assays, no significant changes in the accumulation of viral transcripts were detected among these viral variants in local infection

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Interactions between viral proteins expand the potential functional landscape of the viral proteome assays (S6B and S8C- S8F Fig). Importantly, and despite the fact that expression of CP alone did not result in detectable transcriptional changes (Fig 4A), mutation of CP in the viral genome led to the differential expression of 3,256 genes when compared to the WT infection, supporting the notion that CP modulates host gene expression in combination, physical or functional, with other viral proteins; remarkably, 2,591 of these DEGs (79.5%) overlapped with those caused by the loss of C2 (Figs 6C, 6D and S8G; S2 Table; validation of the RNA-seq results is presented in S8H Fig), indicating that C2 and CP cooperatively mediate changes in host gene expression during the infection. Functional categories over-represented among the up-regulated genes in the presence of the WT virus appear as down-regulated in the subset of DEGs commonly triggered by the C2-and CP-deficient viruses compared to the WT version (Figs 6E and S9; S4 and S5 Tables), suggesting that the C2/CP module is responsible for the transcriptional changes of genes associated to these GO terms. A complete overview of the functional enrichment in the different subsets of DEGs can be found in S9 Fig and S5 Table. Taken together, our results demonstrate that TYLCV proteins form an intricate network of interactions that potentially vastly increase the complexity of the virus-host interface, and that viral proteins can have additional effects on the host cell when in combination. Given that intra-viral protein-protein interactions have been reported for viruses belonging to independently evolved families and infecting hosts belonging to different kingdoms of life, we propose that this might be an evolutionary strategy of viruses to expand their functional repertoire

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Interactions between viral proteins expand the potential functional landscape of the viral proteome while maintaining small genomes, which would call for a reconsideration of our approaches to the study of viral protein function and virus-host interactions.

Plant material
Nicotiana benthamiana plants were grown in a controlled growth chamber in long-day conditions (16 h light/8 h dark) at 25˚C.

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Interactions between viral proteins expand the potential functional landscape of the viral proteome

Bacterial strains and growth conditions
Escherichia coli strain DH5α was used for general cloning and subcloning procedures; strain DB3.1 was used to amplify Gateway-compatible empty vectors.
For in planta expression, Agrobacterium tumefaciens strain GV3101 harbouring the corresponding binary vectors were liquid-cultured in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) with the appropriate antibiotics at 28˚C overnight.
The TYLCV infectious clone has been previously described [58]. Using the wild-type (WT) infectious clone as template, the TYLCV C2 null mutant (TYLCV-C2mut), carrying a C-to-G mutation in the 14th nucleotide of the C2 ORF, was generated, converting the fifth codon (encoding a serine) to a stop codon, with the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Cat #210518). Similarly, the TYLCV CP null mutant 1 (TYLCV-CPmut1), carrying a C-to-A mutation in the fourth nucleotide of the CP ORF, was generated, converting the second codon (encoding a serine) to a stop codon. The TYLCV-CP-mut2 infectious clone, containing two premature stop codons in positions 2 and 15 and in which the nine potential alternative starting sites (ATG) have been removed, was synthesized. In both cases, the mutations in the CP ORF do not affect the overlapping V2 ORF.
All primers and plasmids used for cloning are summarized in S6 and S7 Tables, respectively.

Agrobacterium-mediated transient gene expression in N. benthamiana
Transient expression assays were performed as previously described [40] with minor modifications. In brief, all binary plasmids were transformed into A. tumefaciens strain GV3101; A. tumefaciens clones carrying the constructs of interest were liquid-cultured in LB with the appropriate antibiotics at 28˚C overnight. Bacterial cultures were collected by centrifugation at 4,000 x g for 10 min and resuspended in the infiltration buffer (10 mM MgCl 2 , 10 mM MES pH 5.6, 150 μM acetosyringone) to an OD 600 = 0.2-0.5. Next, bacterial suspensions were incubated at room temperature in the dark for 2-4 hours before infiltration into the abaxial side of 4-week-old N. benthamiana leaves with a 1 mL needleless syringe. For experiments that required co-infiltration, the A. tumefaciens suspensions carrying different constructs were mixed at 1:1 ratio before infiltration.

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Interactions between viral proteins expand the potential functional landscape of the viral proteome

Protein extraction and immunoprecipitation assays
Fully expanded young leaves of 4-week-old N. benthamiana plants were co-infiltrated with A. tumefaciens carrying constructs to express Rep-, C2-, C3-, C4-, CP-, and V2-FLAG, with Rep-, C2-, C3-, C4-, CP-, or V2-GFP. To analyze these protein-protein interactions in the context of the viral infection, A. tumefaciens carrying the infectious TYLCV clone were co-infiltrated in the respective experiments. Two days after infiltration, 0.7-1 g of infiltrated N. benthamiana leaves were harvested. Protein extraction, co-immunoprecipitation (co-IP), and western blot were performed as previously described [59]. For western blot, the following primary and secondary antibodies were used at the

Bimolecular Fluorescence Complementation (BiFC)
Fully expanded young leaves of 4-week-old N. benthamiana plants were co-infiltrated with A. tumefaciens clones carrying the appropriate BiFC plasmids using a 1 mL needleless syringe and imaged two days post-infiltration with a Leica TCS SMD confocal microscope (Leica Microsystems) using the preset settings for YFP (Ex: 514 nm, Em: 525-575 nm). For nuclei staining, leaves were infiltrated with 5 μg/mL Hoechst 33258 (Sigma) solution and incubated in the dark for 30-60 minutes before observation by using the corresponding preset settings (Ex: 355 nm, Em: 430-480 nm).

Yeast two-hybrid
pGBKT7-and pGADT7-based constructs were co-transformed into the Y2HGold yeast strain (Clontech) using Yeastmaker Yeast Transformation System 2 (Clontech) according to the manufacturer's instructions. The co-transformants were selected on minimal synthetic defined (SD) media without leucine and tryptophan; interactions were tested on SD media without leucine, tryptophan, histidine, and adenine. pGADT7-T (expressing the SV40 large T-antigen) and pGBKT7-p53 (expressing murine p53) constructs were used as positive control; empty vectors were used as negative control.

Split-luciferase complementation imaging assay
A. tumefaciens strains carrying the appropriate plasmids were agroinfiltrated into 4-week-old N. benthamiana plants using a 1 mL needleless syringe. Two days post-infiltration, the same leaves were infiltrated with 1 mM D-luciferin solution and kept in the dark for 5 min before imaging. The luminescence images were captured using a CCD camera (NightShade LB 985, Berthold). For a full protocol, see [57].
Confocal imaging for co-localization of C2-GFP and TYLCV proteins fused to RFP was performed on a Leica TCS SP8 point scanning confocal microscope using the pre-set

TYLCV infection
For TYLCV local infection assays, fully expanded young leaves of 4-week-old N. benthamiana plants were infiltrated with A. tumefaciens carrying the TYLCV infectious clone (WT or mutants). Samples were collected at 2.5 days post-inoculation (dpi) to detect viral accumulation.
For TYLCV systemic infection assays, A. tumefaciens clones carrying the TYLCV infectious clones (WT or mutants) were syringe-inoculated in the stem of 2-week-old N. benthamiana plants. Leaf discs from the three youngest apical leaves were harvested at 21 dpi to detect viral accumulation.

Determination of viral accumulation by quantitative PCR (qPCR)
To determine viral accumulation, total DNA was extracted from N. benthamiana leaves using the CTAB method [60]. The DNA from local infection assays was treated with DpnI at 37˚C for 1 hour prior to further analysis. Quantitative PCR (qPCR) was performed with primers to amplify Rep [31]. The qPCR reaction was performed with Hieff qPCR SYBR Green Master Mix (Yeasen), with the following program: 3 min at 95˚C, and 40 cycles consisting of 15 s at 95˚C, 30 s at 60˚C. As internal reference for DNA detection, the 25S ribosomal DNA interspacer (ITS) was used [61]. qPCR was performed in a BioRad CFX96 real-time system as described previously [31]. The primers used are described in S8 Table.

Reverse transcription quantitative PCR (RT-qPCR)
RNA was extracted using the Plant RNA kit (OMEGA Bio-Tek); cDNA was prepared using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions. The qPCR reaction was performed with Hieff qPCR SYBR Green Master Mix (Yeasen), with the following program: 3 min at 95˚C, and 40 cycles consisting of 15 s at 95˚C, 30 s at 60˚C. Elongation factor-1 alpha (NbEF1α) [62] or Actin2 (NbACT2) [63] were used as reference genes, as indicated. The primers used are described in S8 Table.

RNAseq and analysis
Transcriptome sequencing in N. benthamiana was performed as previously described [29]. Four biological replicates were used per sample. The paired-end reads were cleaned by Trimimomatic [64] (version 0.36). Clean read pairs were retained for further analysis after trimming the adapter sequence, removing low quality bases, and filtering short reads. The N. benthamiana draft genome sequence (v1.0.1) [65] was downloaded from the Sol Genomics Network (https://solgenomics.net/ftp/genomes/Nicotiana_benthamiana/assemblies/). Clean reads were mapped to the genome sequence by HISAT [66] (version 2.1.0) with default parameters. The number of reads that were mapped to each N. benthamiana gene was calculated with the htseq-count script in HTSeq [65]. Differentially expressed genes (DEGs) with at least 1.5 fold change in expression and a FDR < 0.05 between control and experiment samples were identified by using EdgeR [67].
The heatmap with hierarchical clustering was drawn by R package pheatmap. Venn diagrams were drawn by Venny (http://bioinformatics.psb.ugent.be/webtools/Venn/) and modified in Adobe Illustrator. The Arabidopsis thaliana homologous genes of the DEGs identified

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Interactions between viral proteins expand the potential functional landscape of the viral proteome in N. benthamiana were used for Gene Ontology (GO) term enrichment analysis in AgriGO v2.0 [68].
Supporting information S1 Fig. Yeast two-hybrid co-transformation control. The minimal synthetic defined (SD) medium without leucine (Leu) and tryptophan (Trp) was used to select co-transformants.  Table. (TIF) S1

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Interactions between viral proteins expand the potential functional landscape of the viral proteome S4