Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches

Background Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein that was translated from potyviral RNA. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5′-fragment of the PTGS-cleaved RNA degradation product. Second, the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

However, different species of viruses have developed various suppressors to counteract the DCL2/4-mediated siRNA defense system, known as PTGS suppression, making these species capable of surviving and multiplying in the infected plants. Viral suppressors of PTGS not only suppress the siRNA defense system but also inhibit miRNA regulation, resulting in symptom development. Symptoms represent the misregulation of the miRNA phenomena, whereas a mutant virus has a defective suppressor that causes mild symptoms and has a limited Open Access *Correspondence: linss01@ntu.edu.tw 1 Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan Full list of author information is available at the end of the article inhibitory effect on miRNA-regulation (Kung et al. 2014;Wu et al. 2010).
Viral suppressors of PTGS have various approaches to interfere with miRNA biogenesis or miRNA regulation. For instance, 2b of the cucumber mosaic virus (CMV) Q strain and p19 of tomato busy stunt virus (TBSV) binds miRNA and siRNA to prevent those of small RNAs loading into AGO1 (Silhavy et al. 2002;Zhang et al. 2006). P0 of polerovirus has an F-box-like domain to trigger AGO1 degradation (Michaeli et al. 2019). P1/HC-Pro is the first identified viral suppressor of PTGS (Anandalakshmi et al. 1998;Kasschau and Carrington, 1998). HC-Pro is a highly conserved protein in potyvirus that plays a major role in PTGS suppression (Kasschau and Carrington, 1998;Kasschau et al. 2003;Kung et al. 2014;Valli et al. 2006). In contrast, P1 is a highly divergent protein that has variable sequences in each potyvirus. P1 of tobacco etch virus (TEV) can enhance the HC-Pro-mediated PTGS suppression; however, the mechanism is still unclear (Kasschau and Carrington, 1998;Martínez and Daròs, 2014;Valli et al. 2006). Martinez and Daròs (2014) demonstrated that P1 of TEV interacts with the 60S ribosomal subunit and enhances in vitro translation.
Previous studies demonstrated that P1/HC-Pro genes of zucchini yellow mosaic virus (ZYMV) and turnip mosaic virus (TuMV) suppressed miRNA regulation (Kung et al. 2014;Wu et al. 2010). Transgenic Arabidopsis expressing P1/HC-Pro of ZYMV (P1/HC Zy plant) or P1/HC-Pro of TuMV (P1/HC Tu plant) showed severe serrated and curling leaf phenotypes that are related to miRNA misregulation and viral symptom development (Kung et al. 2014;Wu et al. 2010). Moreover, the FRNK motif (highly conserved amino acid sequence) of HC-Pro in TuMV and ZYMV is necessary and sufficient for PTGS suppression (Kung et al. 2014;Wu et al. 2010). The miRNA misregulation in transgenic plant expressing viral suppressor gene, such as 2b, P1/HC-Pro, and P19, is occurred by abnormal miRNA/miRNA* accumulation via an unknown mechanism, resulting in target RNA accumulation (Kasschau et al. 2003;Kung et al. 2014). Therefore, abnormal miRNA/miRNA* and target RNA accumulations are the molecular phenotypes of PTGS suppression.
In this study, we demonstrated that various potyviruses of P1 are necessary and sufficient to enhance HC-Pro PTGS suppression. Through high-throughput omics approaches, several critical genes that interact with P1 or are involved in PTGS were identified from immunoprecipitation (IP) and transcriptomic profiles. We also found that P1/HC-Pro of TuMV triggers Argonaute protein 1 (AGO1) posttranslational degradation. These critical genes offer new directions for further investigation of the PTGS and P1/HC-Pro-mediated suppression.

Plant material and transgenic plants
Arabidopsis thaliana ecotype Col-0 and transgenic plants, P1/HC Tu plant, and P1/HC Zy plant (Wu et al. 2010) were used in this study. Arabidopsis seeds were surface sterilized and chilled at 4 °C for 2 days and then sown on Murashige and Skoog (MS) medium with/without suitable antibiotics. The seedlings were transferred into soil after 1 week of germination. All plants were grown at 24 °C in a growth room with 16 h of light/8 h of dark.
For P1 Tu plant construction, the TuMV infectious clone was used as a template to amplify the P1 Tu gene with the primer set: PtuP1/MTuP1 (5′-TCA AAA GTG CAC AAT CTT -3′), and the gene was then cloned into the pENTR and pBCo-DC vectors following the above procedures to generate pBCo-P1. For the HC Tu plant resistant to Basta, the TuMV infectious clone was used as template to amplify the HC Tu gene with the primer set: PTuHC (5′-CAC CAT GAG TGC AGC AGG AGCC-3′)/MTuHC, and it was then cloned into the pENTR and pBCo-DC vectors following the above procedures to generate the pBCo-HC Tu fragment. An NheI site was introduced into the fusion form of the P1HC-Pro gene (P1HC Tu−FA ) to generate a F 362 A substitution. Furthermore, the P1 and HC-Pro genes were amplified from the TuMV infectious clone (Niu et al. 2006) and constructed under the 35S promoter to create the P1 Tu and HC Tu plants, respectively. The pBCo-P1/HC Te , pBCo-P1 Tu , pBCo-HC Tu , and pBCo-P1HC Tu-FA binary vectors were transferred into Col-0 by the floral-dipping method with the Agrobacterium tumefaciens ABI strain to generate the P1/HC Te , P1 Tu , and HC Tu plants, respectively.
For recombined P1/HC-Pro transgenic plant construction, the infectious clones of TuMV, ZYMV, and TEV were used as templates to generate the recombinant P1/HC-Pro constructs. The P1 cleavage site in the recombined gene had to be preserved in the recombined constructs, and the constructs were cloned into the pBCo binary vector (Kung et al. 2014) for Agrobacterium-mediated flower-dipping transformation.
All of the PCR fragments were digested with NheI and XhoI and then ligated with the same restriction enzyme-digested pET-28a vector to generate pET-P1 Tu , pET-P1 Zy , pET-P1 Te , pET-HC Tu , pET-HC Zy , and pET-HC Te ). All of the pET28 plasmids were transformed into the E. coli BL21 strain for recombinant protein expression. All recombinant proteins were purified by fast protein liquid chromatography (FPLC) (AKTApurifier, GE Healthcare). One milligram of recombinant protein with a 1 × volume of complete Freund's adjuvant was injected into New Zealand white rabbits for the first injection. The following three injections consisted of 1 mg of protein mixed with a 1 × volume of incomplete Freund's adjuvant. IgG purification was performed according to the protocol of Chiu et al. (2013). The IgG was collected after 4 injections for western blot detection.

Immunoprecipitation and in-solution protein digestion
To identify the P1-interacting proteins, 250 mg of 10-day-old seedlings (n = 6) were homogenized with 1 mL IP buffer [25 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 5% glycerol, and a protease inhibitor (Roche)], followed by centrifugation for 10 min at 4 °C. IgG of α-P1 Tu , α-P1 Zy , and α-P1 Te was used for the in vivo IP. IP was performed by mixing 30 μl of washed Protein A Mag Sepharose TM Xtra ferrite beads (GE), IgG (30 μl per IP reaction) and lysate. The IP reaction was carried out at 4 °C with gentle mixing for 3 h. The tube was then centrifuged at 300 g to pull-down the beads, which were washed three times with 0.3 mL wash buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.1% Triton-X-100, and a protease inhibitor) to remove nonspecific binding. Finally, the beads were resuspended in 50 μL elution buffer (0.1 M glycine, pH 2.0), and the reaction was mixed on a rotary at 4 °C for 10 min. A total of 10 μL of neutralization buffer (Tris-HCl, pH 8.0) was added to neutralize the reaction.
The proteins were dissolved in 6 M urea. A total of 15 μg of protein from each time point was used for insolution digestion. Proteins were reduced by incubation with 10 mM dithiothreitol (DTT) for 1 h at 29 °C and alkylated by 55 mM iodoacetamide (IAA) at room temperature for 1 h. This step was quenched by 55 mM DTT at 29 °C for 45 min. The concentration of urea was diluted to 1 M before the sample was subjected to proteolysis. Protein digestion was performed overnight at 29 °C using mass spectrometry-grade modified trypsin (Promega) at a 1:50 trypsin/protein ratio. After overnight incubation, 0.1% TFA was added to stop the digestion. Finally, all remaining reagents from the in-solution digestion procedure were removed using a C18 stage tip.

LC-MS/MS analysis
High-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed on an Orbitrap Fusion Lumos Tribrid quadrupole-ion trap mass spectrometer (Thermo Fisher Scientific) in the Instrumentation Center of National Taiwan University. Peptides were separated on an Ultimate System 3000 NanoLC System (Thermo Fisher Scientific). Peptide mixtures were loaded onto a 75 μm inner diameter (ID), 25 cm length C18 Acclaim PepMap NanoLC column (Thermo Scientific) packed with 2 μm particles with a pore size of 100 Å. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 100% acetonitrile with 0.1% formic acid. A segmented gradient was set over 90 min from 2% to 35% solvent B at a flow rate of 300 nl/ min. Mass spectrometry analysis was performed in a data-dependent mode with full-MS (externally calibrated to a mass accuracy of < 5 ppm, and a resolution of 120,000 at m/z = 200), followed by high-energy collision activated dissociation (HCD)-MS/MS of the most intense ions in 3 s. HCD-MS/MS (resolution of 15,000) was used to fragment multiply charged ions within a 1.4 Da isolation window at a normalized collision energy of 32. An automatic gain control (AGC) target at 5e5 and 5e4 was set for MS and MS/MS analysis, respectively, with previously selected ions dynamically excluded for 180 s. The max injection time was 50 ms.

Identification and quantitation of the proteome by label-free labeling methods
Quantitative proteomics was performed by label-free quantitative proteomic analysis. The raw MS/MS data were searched against the UniProt Knowledgebase/ Swiss-Prot Arabidopsis thaliana protein database (Mar 2019 version) by using the Mascot 2.3 search algorithm via the Proteome Discoverer (PD) package (version 2.2, Thermo Scientific). The search parameters were set as follows: peptide mass tolerance, 10 ppm; MS/MS ion mass tolerance, 0.02 Da; enzyme set as trypsin and allowance of up to two missed cleavages; and variable modifications including oxidation on methionine, deamidation on asparagine and glutamine residues, and carbamidomethylation of cysteine residues. Peptides were filtered based on a 1% FDR. Protein quantification was computed by the abundance of ions extracted from the MS spectra of the corresponding peptides. The normalization method was set to the total peptide amount.

Whole-transcriptome analysis
Total RNAs that were isolated from 10-day-old seedlings of Col-0, P1 Tu , HC Tu , and P1/HC Tu plants (n = 3) were used for whole-transcriptome deep sequencing by the High Throughput Sequencing Core of Academia Sinica. The sequencing was accomplished by paired-end (2 × 125) strand-specific HiSeq sequencing (Illumina). The transcriptome was analyzed by the ContigViews system (www.conti gview s.bioag ri.ntu.edu.tw) of the NGS core of National Taiwan University. For the ContigViews network analysis in this study, the twofold differentially expressed genes (DEGs) between Col-0 and P1/HC Tu plants (n = 3) with an 80% passing rate were selected for the assay. Reads with twofold log 10 FPKM values of genes under 1.14 were trimmed. At least 10 samples from Col-0, P1 Tu , HC Tu , and P1/HC Tu profiles (n = 3) were selected to calculate the Pearson correlation with a 0.95 threshold for positive relation and a 0.9 threshold for negative relation. Notable, parameter determination is according to the highlighted genes and network complexity. These parameters can generate the best network for data mining in ContigViews.

Ethylene detection
Three-week-old Col-0 and P1/HC Tu plants (n = 3) were individually sealed in the 1.5 L chambers at 24 °C with 16 h light/8 h dark. Ethylene gas samples in (1 mL) were withdrawn and collected at 4, 24, 48, and 72 h and were analyzed by GC-8A gas chromatography (Shimadzu) equipped with a flame ionization detector (FID).

P1 enhances the severity of the HC-Pro-mediated serrated leaf phenotype and PTGS suppression
To dissect the function of P1 Tu and P1/HC Tu in PTGS suppression, we generated Arabidopsis transgenic lines expressing P1 Tu and P1/HC Tu in combinations or individually ( Fig. 1a, b). The P1/HC Tu plants showed a severe serrated and curled leaf phenotype (Fig. 1b, panel ii). The translated P1/HC-Pro protein contains an F 362 /S 363 cleavage site (Fig. 1a), which can generate separated P1 and HC-Pro proteins through P1 cleavage (Fig. 1c). The P1 Tu plant showed normal development similar to that of the Col-0 plants, whereas the HC Tu plant showed mildly serrated leaves (Fig. 1b, panels iii and iv). In addition to the difference in the severity of the leaf phenotype, the size of the HC Tu plant was larger than that of the P1/HC Tu plant ( Fig. 1b, panels ii and iv).
In addition, an F 362 A substitution at the F 362 /S 363 -P1 cleavage site produced a P1HC-Pro fusion protein (P1HC Tu-FA ) (Fig. 1a, c). This transgenic P1HC Tu-FA plant showed a normal phenotype (Fig. 1b, panel v). Furthermore, a kanamycin-resistant HC Tu plant [HC Tu (kan) plant] was generated for crossing with the P1 Tu plant (Basta resistant) (Fig. 1a). Similar to the HC Tu plant, the HC Tu (kan) plant showed mildly serrated leaves (Fig. 1b,panel vi). Interestingly, the P1 Tu × HC Tu (Kan) offspring showed severely serrated and curled leaves, but the P1 Tu × HC Tu (Kan) plant was larger than that of the P1/HC Tu plant (Fig. 1b, panel vii). In addition, only the P1/HC Tu plant showed high levels of the P1 and HC-Pro proteins, while the other lines, even the P1 Tu × HC Tu (Kan) plant, showed low levels of P1 and HC-Pro (Fig. 1c).
We compared 57 potyvirus amino acid sequences of P1/HC-Pro (Fig. 2a). The alignment results showed that the sequence and length of the P1 protein in different potyviruses are highly diverse (Fig. 2a). Only the C-terminal protease activity site (black boxes) is conserved (Fig. 2a). In contrast, several conserved domains of HC-Pro were found in different species (Fig. 2a). To test whether the P1/HC-Pro from other potyviruses also induce serrated leaf phenotype, we generated Arabidopsis transgenic lines expressing P1/HC-Pro form ZYMV and TEV. P1/HC Zy plants showed a severe serrated and curled leaf phenotype, whereas P1/ HC Te plants showed a minor serrated leaf phenotype ( Fig. 2b). However, both plants had high levels of P1 and HC-Pro (Fig. 2c). The results indicated that the P1/ HC-Pro genes of ZYMV and TEV can also trigger a serrated leaf phenotype.
The next question was whether the function of the HC-Pro from each virus requires the P1 from the same species. We generated 6 recombinant P1/HC-Pro plants in which HC-Pro was fused with a heterologous P1, namely, P1 Zy /HC Tu , P1 Te /HC Tu , P1 Tu /HC Zy , P1 Te / HC Zy , P1 Tu /HC Te , and P1 Zy /HC Te (Fig. 2d). Except for P1 Tu /HC Te plants that show serrated leaves, the other 5 recombinant transgenic plants showed a severe serrated and curled leaf phenotype (Fig. 2e). The represented plants, P1 Zy /HC Tu and P1 Te /HC Tu plants, showed detectable P1 and HC-Pro expression (Fig. 2c). These results suggest that multiple P1 genes have conserved functions in enhancing the HC-Pro-mediated serrated leaf phenotype.

HC-Pro-mediated PTGS suppression
Previous studies demonstrated that an abnormal accumulation of miRNA and miRNA* occurs in several transgenic viral suppressor plants because suppressors interfere with miRNA biogenesis (Kasschau et al. 2003;Kung et al. 2014;Wu et al. 2010). Indeed, P1/HC Tu , P1/ HC Zy , P1/HC Te , and 6 recombinant P1/HC-Pro plants showed abnormal miRNA/miRNA* accumulation (Fig. 2f ). These data suggested that 3 species of viral P1/ HC-Pro and recombinant P1/HC-Pro interfered with miRNA biogenesis. In addition, except for the P1HC Tu-FA plant, all transgenic lines that contained HC Tu showed abnormal miRNA and miRNA* accumulation (Fig. 3a), confirming that HC Tu is the dominant player in PTGS suppression. Surprisingly, the P1 Tu plant also showed miRNA and miRNA* accumulation through an unknown mechanism (Fig. 3a). In addition to miRNA/miRNA* accumulation, miRNA targets were also upregulated in  (Kasschau et al. 2003;Kung et al. 2014;Wu et al. 2010). Transcriptome profiles also indicated that miRNA targets were upregulated in HC Tu , HC Tu (kan) , P1 Tu × HC Tu , and P1/HC Tu plants (Fig. 3b), suggesting that miRNA regulation was blocked by HC-Pro. However, DICER-LIKE 1 (DCL1; miR162 target) and two translation inhibition genes, APETALA 2 (AP2; miR172 target) and SHORT VEGETATIVE PHASE (SVP; miR396 target), showed no change in their transcript levels (Fig. 3b). Except for the DCL1, AP2, and SVP genes, the P1/HC Tu plant suppressed most of the miRNA-target regulation (Fig. 3b). We conclude that the P1/HC Tu plant has a stronger suppressive effect than the HC Tu plants. In addition, the

Host P1-interacting proteins are involved in PTGS
Because the recombinant P1/HC-Pro plants showed identical serrated leaf phenotypes and heterologous P1s could enhance HC-Pro-mediated PTGS suppression, we hypothesize that various P1 proteins have (a) conserved interacting protein(s) in Arabidopsis that enhance HC-Pro-mediated PTGS suppression. To identify the host P1-interacting proteins, the P1/HC Tu , P1/HC Zy , and P1/ HC Te plants were used for IP with α-P1 Tu , α-P1 Zy , and α-P1 Te antibodies, respectively. These IP eluates were analyzed by LC-MS/MS. We identified 101 cytoplasmic P1 of TuMV (P1 Tu )-interacting proteins (Additional file 1: Data). Furthermore, we identified 56 cytoplasmic P1 of ZYMV (P1 Zy )-interacting proteins and 20 cytoplasmic P1 of TEV (P1 Te )-interacting proteins (Additional file 1: Data). Importantly, only one consensus cytoplasmic protein, VERNALIZATION INDEPENDENCE 3/ SUPERKILLER8 (VIP3/SKI8; AT4G29830), was found in the IP profiles of 3 viral P1s (Table 1). VIP3/SKI8 is a subunit of the RNA exosome complex that is required for degradation of the RISC 5′-cleavage fragment (Branscheid et al. 2015;Orban and Izaurralde 2005). In contrast, 12 consensus cytoplasmic proteins were identified in the P1 Tu and P1 Zy IP profiles, whereas 10 consensus proteins were identified in the P1 Tu and P1 Te IP profiles (Table 1). Moreover, 5 consensus cytoplasmic proteins were found in the P1 Zy and P1 Te IP profiles (Table 1).
Next, we focused on P1 Tu -interacting proteins because the P1/HC Tu plant was the model used in this study. In the P1 Tu IP profile, two TUDOR-SN ribonucleases [(TSN1 (AT5G07350) and TSN2 (AT5G61780)] were uniquely identified 5 to 6 times in a total of 6 IP experiments with P1/HC Tu plants (Table 1, and Additional file 2: Table S1). TSN1 and TSN2 have been suggested to be involved in the regulation of uncapping mRNA and localize to processing bodies (P-bodies) and stress granules (Yan et al. 2014). Therefore, whether P1 Tu could alter the function of TSN1 and TSN2 is an interesting project for the further investigation. Moreover, VARICOSE (VSC; AT3G13300) and MODIFIER OF SNC1,4 (MOS4; AT3G18165), which are involved in RNA regulation, were identified in the P1 Tu IP profile (Table 1 and Additional file 2: Table S1). We also identified the NUCLEAR-PORE ANCHOR (NUA; AT1G79280), two IMPORTIN subunits (AT5G53480 and AT4G16143), and BREFELDIN A-INHIBITED GUANINE NUCLEOTIDE-EXCHANGE PROTEIN 5 (BIG5; AT3G43300), which are involved in protein or nucleic acid transport between the nucleus and cytosol (Table 1 and Additional file 2: Table S1) (Xue et al. 2019). Moreover, VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 29 (VSP29; AT3G47810) was identified, which participates in vacuolar protein trafficking and vacuolar sorting receptor recycling (Table 1 and Additional file 2: Table S1) (Kang et al. 2012).

The posttranscriptional and posttranslational regulation of miRNA targets in P1/HC Tu plants
CCS1 is involved in copper delivery, and SOD1 and SOD2 participate in Cu/Zn superoxide dismutase activities. The transcripts of these three genes are regulated by miR398 (Bouché 2010; Sunkar et al. 2006). However, there were high levels of CCS1, SOD1, and SOD2 accumulation in the HC Tu and P1/HC Tu plants, which corresponded to their transcript levels, indicating P1/ HC-Pro-mediated PTGS suppression (Fig. 4d-f, panel ii). Indeed, the transcript level of miR168-regulated AGO1 (AT1G48410) was increased in HC Tu and P1/HC Tu plants compared with Col-0 (Fig. 4r, panel ii). Surprisingly, the level of AGO1 protein was decreased via an unknown mechanism in HC Tu and P1/HC Tu plants (Fig. 4r, panel  i). The western blot data also indicated that the level of AGO1 was lower in P1/HC Tu plants than in Col-0 plants but was similar to that in Col-0 plants, P1/HC Zy and P1/ HC Te plants (Fig. 5). These data suggested that the P1/ HC-Pro of TuMV has a specific ability to trigger the posttranslational degradation of AGO1.

Comparative gene-to-gene network and transcriptome analysis
In the transcriptome analysis, we constructed a geneto-gene correlation network to study PTGS suppression from a different perspective. First, we constructed a network for Col-0 vs. P1/HC Tu plants in the ContigViews system. A list of twofold DGEs between Col-0 and P1/ HC Tu plants was used to generate a Pearson correlation network (Fig. 6). A group of positive correlations (red lines) and a group of negative correlations (green lines) were highlighted in the network (Fig. 6). The output of the network showed that AGO1, AGO2 (AT1G31280), and AGO3 (AT1G31290) were present in the group of negative correlations (Fig. 6). AGO2 and AGO3 were positively correlated with each other (red line) but had an indirect correlation with AGO1 through XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 7 (XTH7; AT4G37800) (Fig. 6). Notably, the transcripts of AGO1, AGO2, and AGO3 were upregulated in the HC Tu and P1/ HC Tu plants, but the XTH7 transcripts were downregulated, suggesting that the AGOs and XTH7 might have opposite functions in PTGS (Fig. 4r, panel ii; Fig. 8a-c).
Next, we constructed two comparative networks, which were generated by a list of twofold DEGs between Col-0 and HC Tu plants or between Col-0 and P1 Tu plants (Fig. 7a, b). The gene positions in the comparative networks were followed with the Col-0 vs. P1/HC Tu network for comparison (Figs. 6 and 7). There were 97 genes in the Col-0 vs. P1/HC Tu network (Fig. 6); however, there were only 36 genes showed up when we applied the Two-asterisk (**) indicates cross-reaction of the α-HC Zy or α-HC Te antibodies. Three-asterisk (***) indicates common bands. The @ symbol indicates RUBISCO as an internal control same parameters in the Col-0 vs. HC Tu network (Figs. 6 and 7a). In addition, the main genes involved in PTGS, such as AGO1, AGO2, AGO3, and XTH7, remained in the Col-0 vs. HC Tu network (Fig. 7a). This suggested the presence of a basic network backbone in the HC Tu -mediated PTGS suppression that occurs without the effects of P1 Tu . In contrast, the Col-0 vs. P1 Tu network only had 7 genes in 2 small groups that were also present in parts of the Col-0 vs. HC Tu or Col-0 vs. P1/HC Tu networks (Figs. 6 and 7). Moreover, XTH7 had fewer than 49 connected genes in the Col-0 vs. HC Tu network, whereas XTH7 had 61 connections in the Col-0 vs. P1/HC Tu network (Figs. 6 and 7a). These data indicated that the XTH7 connection is variable in different networks and might play an important role in PTGS suppression. Overall, the comparative network analysis highlights the effects of P1 Tu on HC Tu -mediated PTGS suppression. This also explains why the P1/HC Tu plant has a severe phenotype because of how many pathways were interfered with.

Critical genes in the Col-0 vs. P1/HC Tu network that are involved in PTGS
The importance of XTH7 is not only in the number of gene connections it has or that it is connected with AGO1 and AGO2; XTH7 also had a negative correlation with several miRNA targets in the Col-0 vs. P1/HC Tu network, such as 2 auxin response transcription factor genes [ARF3 (AT2G33860) and ARF8 (AT5G37020)], PHOSPHATE 2 (PHO2; AT2G33770), GROWTH-REGULATING FACTOR 1 (GRF1; AT2G22840), CCS1, Fig. 6 The gene-to-gene network of Col-0 vs. P1/HC Tu plants. The gene profiles of twofold DEGs between Col-0 and P1/HC Tu plants were used to generate the Pearson correlation network. The different circle sizes indicate the numbers of correlated genes. A positive correlation (> 0.95) between the two genes is indicated by a red line, whereas a green line indicates a negative correlation (< −0.9). The red circles indicate the genes involved in calcium signaling and are grouped with a red background. The blue circles indicate the genes involved in the defense response and are grouped with a blue background. The green circles indicate the genes involved in the PTGS pathway and are grouped with a green background. The yellow circles indicate the genes that are the miRNA targets and are grouped with a yellow background. The gray circles indicate the genes involved in the JA, ABA, and ethylene biosynthesis pathways and are grouped with a gray background SOD1, and SOD2 (Fig. 5). However, ARF3, ARF8, PHO2, GRF1, CCS1, SOD1, and SOD2 formed a positive correlation in the network (Fig. 6). These miRNA target transcripts were upregulated in HC Tu and P1/HC Tu plants because of PTGS suppression (Fig. 4e-f; panel ii; Fig. 8d-g). Moreover, SEP3 (AT1G24260) showed negative correlations with XTH7, ARF3, ARF8, and SOD1 (Fig. 7). In addition, SEP3 transcript levels were lower in P1 Tu , HC Tu , and P1/HC Tu plants compared to Col-0 plants (Fig. 8h). Notably, SOD1 was shown to have a physical interaction with P1 Tu and P1 Te (Table 1) and was also highlighted in the network, suggesting the importance of SOD1 in PTGS suppression.

P1 enhances HC-Pro-mediated PTGS suppression
In potyvirus, P1 is a hypervariable protein with poorly understood its function. Previous studies suggested that P1 modulates virus replication, determines pathogenicity in a host-dependent manner, and triggers the host defense response (Maliogka et al. 2012;Pasin et al. 2014).
In this study, we demonstrated that 3 viral P1s have a conserved function in enhancing HC-Pro-mediated PTGS suppression. From the perspective of P1-host protein interaction, VIP3/SKI8 turns over the 5′-fragment of RISC-cleaved target RNA, whereas TSN1, TSN2, and VSC are involved in mRNA decapping in stress granules and P-bodies (Branscheid et al. 2015;Deyholos et al. 2003;Gutierrez-Beltran et al. 2015;Sorenson et al. 2018;Xu and Chua 2009). Moreover, a MOS4 modifier, 2 IMPORTINs, and BIG5, which are involved in RNA splicing and RNA transportation, respectively, also interact with P1 Tu (Helizon et al. 2018;Kitakura et al. 2017;Luo et al. 2013;Xu et al. 2012). In addition, EMA1/SAD2 contains an importin-beta domain that negatively regulates miRNA activity (Wang et al. 2011). EMA1/SAD2 protein levels were upregulated in P1 Tu , HC Tu , and P1/HC Tu plants, but their transcript levels did not differ those in Col-0, suggesting P1 stabilized or increases EMA1/SAD2 levels to help inhibit miRNA regulation (Fig. 4g). Moreover, transcriptome data mining also showed that the CAF1A/B deadenylases and the CDF2 zinc finger protein had a strong correlation with PTGS suppression. To summarize these findings, posttranscriptional RNA regulation occurs in stress granules and P-bodies, and many RNA regulatory components were identified among the proteins that interacted with P1 or were highlighted in the PTGS suppression network, which suggests that P1 is extremely vital for HC-Pro to enhance suppression.
Although P1 functions in regulating PTGS, it seems that P1 needs to be generated from the P1/HC-Pro fusion protein to have better enhance HC-Pro suppression. It is a cyclic effect in which lower levels of HC-Pro cause less efficiency in PTGS suppression, resulting in lower levels of HC-Pro. Indeed, the HC-Pro levels of P1 Tu × HC Tu (Kan) plants were similar to those of HC Tu plants (Fig. 1c), suggesting ectopically expressed P1 did not have the same effect on enhancing HC-Pro as did P1 released from the fusion protein. Why must P1 be released from the fusion form to enhance HC-Pro ability? It is still unclear.

P1/HC-Pro of TuMV specifically primes posttranslational AGO1 degradation
AGO1 degradation has been reported to be controlled by selective autophagy (Kobayashi et al. 2019;Li et al. 2019;Michaeli et al. 2019). The P0 viral suppressor of Polerovirus is thought to trigger autophagic AGO1 degradation (Michaeli et al. 2019). In our study, P1/HC-Pro of TuMV specifically triggered AGO1 posttranslational degradation, but the same effect was not observed in P1/HC Zy and P1/HC Tu plants, suggesting that P1/HC-Pro triggering AGO1 degradation does not occur in all potyviruses. In the other words, AGO1 degradation might not be essential for P1/HC-Pro-mediated PTGS suppression.
Autophagy works with vacuoles to allow for the degradation of large protein complexes. VSP29 is involved in the trafficking of vacuolar proteins and in the recycling of vacuolar sorting receptors and specifically interacts with P1 Tu (Table 1) (Kang et al. 2012). Moreover, CML24 interacts with AUTOPHAGY GENE 4b (ATG4b), which primes AUTOPHAGY GENE 8 (ATG8) by removing the C-terminus and exposing a glycine residue during autophagy (Tsai et al. 2013). CML24 was found to be present in the group of positive correlations of the PTGS network. Therefore, we implied that P1/HC-Pro of TuMV might also trigger AGO1 posttranslational degradation through autophagy.

Network of HC-Pro-mediated PTGS suppression
The comparative gene correlation network provides a 4-dimensional perspective, which includes gene expression, gene correlation, position, and time course. This information is helpful to interpret and identify critical genes in pathways of interest. In the Co-0 vs. HC Tu network, we identified a basic backbone network in HC Tu -mediated PTGS suppression. However, the effects of P1-enhanced HC-Pro suppression were highlighted in the Col-0 vs. P1/HC Tu network upon comparing the two networks. The Col-0 vs. P1/HC Tu network specifically highlighted the relationship between AGOs and viral resistance. Previous studies demonstrated that AGO2 and AGO3 upregulated to enhance the viral resistance (Alazem et al. 2017;Harvey et al. 2011;Zheng et al. 2019). AGO2 is a target of miR403, which is negatively regulated by AGO1 (Harvey et al. 2011), suggesting the upregulation of AGO2 in response to AGO1 degradation in P1/HC Tu plants. However, although we have no explanation for AGO3 upregulation, we assume that the AGO2/AGO3 antiviral system was activated and complemented AGO1 degradation. Indeed, AGO2 and AGO3 are directly positively correlated in the network.
Surprisingly, several miRNA targets, such as CCS1, SOD1, SOD2, PHO2, ARF3, and GFR1, showed a positive correlation in the network. These genes were indirectly negatively correlated with AGO1 through XTH7. In addition, other miRNA targets, such as TOE2, SPL13A/B, ATHB-15, and PHB were also present in the network. SPL13A and SPL13B had direct positive correlations. ATHB-15 and PHB, which belong to the homeodomainleucine zipper (HD-ZIP) transcription factor (TF) family, were also positively correlated. To summarize, the gene correlation network had significant accuracy in data mining.
Calcium signaling has been demonstrated to be involved in the suppression of gene silencing (Anandalakshmi et al. 2000;Nakahara et al. 2012). Anandalakshmi et al. (2000) demonstrated that the calmodulin-related protein (rgs-CaM) in tobacco interacts with HC-Pro and that it suppresses gene silencing similar to HC-Pro. Nakahara et al. (2012) demonstrated that tobacco rgs-CaM counterattacked various viral suppressors by binding to RNA-binding domains. In addition, rgs-CaM triggers autophagic viral suppressor degradation (Nakahara et al. 2012). Indeed, CML24 has physical interaction with ATG4b, suggesting that there is crosstalk between calcium signaling and autophagy (Tsai et al. 2013). CML24 was present in the group with a positive correlation, which was opposite to the AGOs that were present in the group with a negative correlation, suggesting that calcium signaling might counteract gene silencing.
We noted that several genes, such as XTH7, FUM2, and BAM2, had a significant number of connected genes (> 50 connected genes) (Fig. 6). XTH7 has been defined as a xyloglucan endotransglucosylase/hydrolase; however, little is known about its function in PTGS suppression. In addition, the cytosolic fumarase FUM2 is essential for Arabidopsis acclimation to low temperatures (Dyson et al. 2016). BAM2 is a CLAVATA1-related receptor kinase, and a little is known about its involvement in anther and meristem development (DeYoung et al. 2006;Hord et al. 2006). Although the functions of these genes were not explicitly linked with PTGS or defense, they were present in critical positions within the network with a large number of connected genes, which provides information for future research directions to investigate PTGS.

Auxin and ethylene signaling in the serrated leaf phenotype
Current studies have indicated that the treatment with a high dose of auxin elicits endogenous ethylene production. In P1/HC Tu plants, 3 auxin signaling genes (ARF3, ARF8, and SUTR3;1) were upregulated; therefore, we assume that ethylene was accumulated along with the increased expression of ethylene signaling genes. In addition, Hay et al. (2006) demonstrated that auxin can initiate marginal serrations in leaves, suggesting that the serrated leaf phenotype of P1/HC Tu plants might be related to endogenous auxin accumulation.

Conclusion
In this study, we used a transgenic plant approach to investigate the functions of P1 and HC-Pro. By mining high-throughput data from proteomic and transcriptomic profiles, P1-interacting proteins and critical genes in PTGS suppression were identified. Instead of traditional DEG identification, the comparative gene correlation network provides a four-dimensional perspective to identify critical genes, and provides new ideas and directions for further investigation. We believe plant molecular viology and plant molecular biology, like two hands, can be used together to efficiently investigate the PTGS mechanism.