Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches
Botanical Studies volume 61, Article number: 22 (2020)
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.
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.
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.
Posttranscriptional gene silencing (PTGS) includes the regulation of microRNA (miRNA) and short-interfering RNA (siRNA) in plant development. The DICER-LIKE 2 (DCL2)/DICER-LIKE 4 (DCL4)-mediated siRNA pathway is a major defense system that inhibits viral infection. 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 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/HCZy plant) or P1/HC-Pro of TuMV (P1/HCTu 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.
Materials and methods
Plant material and transgenic plants
Arabidopsis thaliana ecotype Col-0 and transgenic plants, P1/HCTu plant, and P1/HCZy 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.
Transgenic plant construction
For P1/HCTe plant construction, the P1/HC-Pro gene of TEV was amplified from the pTEV-At17 plasmid (Agudelo-Romero et al. 2008) by polymerase chain reaction (PCR) with the primer set: PteP1 (5′-CACCATGGCACTCATCTT-3′) and MTEHC (5′-TCCAACATTGTAAGTTTT-3′). The PCR fragment was cloned into the pENTR/D-TOPO vector (Invitrogen) to generate pENTR-P1/HCTe. The pENTR vector was transferred into the pBCo-DC vector (Kung et al. 2014) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher) to generate pBCo-P1/HCTe.
For P1Tu plant construction, the TuMV infectious clone was used as a template to amplify the P1Tu gene with the primer set: PtuP1/MTuP1 (5′-TCAAAAGTGCACAATCTT-3′), and the gene was then cloned into the pENTR and pBCo-DC vectors following the above procedures to generate pBCo-P1. For the HCTu plant resistant to Basta, the TuMV infectious clone was used as template to amplify the HCTu gene with the primer set: PTuHC (5′-CACCATGAGTGCAGCAGGAGCC-3′)/MTuHC, and it was then cloned into the pENTR and pBCo-DC vectors following the above procedures to generate the pBCo-HCTu fragment. An NheI site was introduced into the fusion form of the P1HC-Pro gene (P1HCTu−FA) to generate a F362A 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 P1Tu and HCTu plants, respectively. The pBCo-P1/HCTe, pBCo-P1Tu, pBCo-HCTu, and pBCo-P1HCTu-FA binary vectors were transferred into Col-0 by the floral-dipping method with the Agrobacterium tumefaciens ABI strain to generate the P1/HCTe, P1Tu, and HCTu 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.
For the TuMV P1 antibody, the N-terminus of the P1 (1-190 aa) of DNA fragment was amplified with the following primer sets PTuP1-NheI (5′-TATGGCTAGCATGGCAGTAGTTACATTCGC-3′)/MTuP1570-XhoI (5′-GGTGCTCGAGGCTCGCAGAGAGTCCTCCTC-3′). For the ZYMV P1 antibody, the N-terminus of the P1 (1-142 aa) DNA fragment was amplified with primer sets PZyP1- NheI (5′-TATGGCTAGCATGGCCTCAGTTATGATTGG-3′)/MZy426-XhoI (5′-GGTGCTCGAGCACTTCAGGTGGAAGAACAC-3′). For the TEV P1 antibody, the N-terminus of the P1 (1-133 aa) DNA fragment was amplified with primer sets PTeP1-NheI (5′-TATGGCTAGCATGGCACTCATCTTTGGCAC-3′)/MTe399-XhoI (5′-GGTGCTCGAGATCAACCTTTCTCTCGGTGT-3′).
For the TuMV HC-Pro antibody, the internal region of the HC-Pro (1-103 aa) of DNA fragment was amplified with primer sets PTuHC-NheI (5′-TATGGCTAGCACAGGGGAGGAATTCTCACA-3′)/MTuHC309-XhoI (5′-GGTGCTCGAGGATTGCAAGTTTCCGTGACC-3′). For the ZYMV HC-Pro antibody, the internal region of the HC-Pro (1-87 aa) DNA fragment was amplified with primer sets PZyHC-NheI (5′-TATGGCTAGCACACAGGCAACTCAGAATCT-3′)/MZyHC261-XhoI (5′-GGTGCTCGAGAGGATTTATCATAGCCTTGC-3′). For the TEV HC-Pro antibody, the internal region of the HC-Pro (1-99 aa) DNA fragment was amplified with primer sets PTeHC-NheI (5′-TATGGCTAGCACAGGGGCTGATCTCGAAGA-3′)/MTeHC297-XhoI (5′-GGTGCTCGAGTCTGCTACCCCTGATATGTT-3′).
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-P1Tu, pET-P1Zy, pET-P1Te, pET-HCTu, pET-HCZy, and pET-HCTe). 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-P1Tu, α-P1Zy, and α-P1Te was used for the in vivo IP. IP was performed by mixing 30 μl of washed Protein A Mag SepharoseTM 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 in-solution 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.
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.
Total RNAs that were isolated from 10-day-old seedlings of Col-0, P1Tu, HCTu, and P1/HCTu 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.contigviews.bioagri.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/HCTu plants (n = 3) with an 80% passing rate were selected for the assay. Reads with twofold log10 FPKM values of genes under 1.14 were trimmed. At least 10 samples from Col-0, P1Tu, HCTu, and P1/HCTu 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.
Three-week-old Col-0 and P1/HCTu 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 P1Tu and P1/HCTu in PTGS suppression, we generated Arabidopsis transgenic lines expressing P1Tu and P1/HCTu in combinations or individually (Fig. 1a, b). The P1/HCTu plants showed a severe serrated and curled leaf phenotype (Fig. 1b, panel ii). The translated P1/HC-Pro protein contains an F362/S363 cleavage site (Fig. 1a), which can generate separated P1 and HC-Pro proteins through P1 cleavage (Fig. 1c). The P1Tu plant showed normal development similar to that of the Col-0 plants, whereas the HCTu 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 HCTu plant was larger than that of the P1/HCTu plant (Fig. 1b, panels ii and iv).
In addition, an F362A substitution at the F362/S363-P1 cleavage site produced a P1HC-Pro fusion protein (P1HCTu-FA) (Fig. 1a, c). This transgenic P1HCTu-FA plant showed a normal phenotype (Fig. 1b, panel v). Furthermore, a kanamycin-resistant HCTu plant [HCTu(kan) plant] was generated for crossing with the P1Tu plant (Basta resistant) (Fig. 1a). Similar to the HCTu plant, the HCTu(kan) plant showed mildly serrated leaves (Fig. 1b, panel vi). Interestingly, the P1Tu× HCTu (Kan) offspring showed severely serrated and curled leaves, but the P1Tu× HCTu (Kan) plant was larger than that of the P1/HCTu plant (Fig. 1b, panel vii). In addition, only the P1/HCTu plant showed high levels of the P1 and HC-Pro proteins, while the other lines, even the P1Tu× HCTu (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/HCZy plants showed a severe serrated and curled leaf phenotype, whereas P1/HCTe 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, P1Zy/HCTu, P1Te/HCTu, P1Tu/HCZy, P1Te/HCZy, P1Tu/HCTe, and P1Zy/HCTe (Fig. 2d). Except for P1Tu/HCTe plants that show serrated leaves, the other 5 recombinant transgenic plants showed a severe serrated and curled leaf phenotype (Fig. 2e). The represented plants, P1Zy/HCTu and P1Te/HCTu 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/HCTu, P1/HCZy, P1/HCTe, 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 P1HCTu-FA plant, all transgenic lines that contained HCTu showed abnormal miRNA and miRNA* accumulation (Fig. 3a), confirming that HCTu is the dominant player in PTGS suppression. Surprisingly, the P1Tu 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 the transgenic plants because of miRNA misregulation (Kasschau et al. 2003; Kung et al. 2014; Wu et al. 2010). Transcriptome profiles also indicated that miRNA targets were upregulated in HCTu, HCTu (kan), P1Tu× HCTu, and P1/HCTu 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/HCTu plant suppressed most of the miRNA-target regulation (Fig. 3b). We conclude that the P1/HCTu plant has a stronger suppressive effect than the HCTu plants. In addition, the heterologous P1s have conserved function(s) in enhancing HC-Pro-mediated PTGS suppression.
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/HCTu, P1/HCZy, and P1/HCTe plants were used for IP with α-P1Tu, α-P1Zy, and α-P1Te antibodies, respectively. These IP eluates were analyzed by LC–MS/MS. We identified 101 cytoplasmic P1 of TuMV (P1Tu)-interacting proteins (Additional file 1: Data). Furthermore, we identified 56 cytoplasmic P1 of ZYMV (P1Zy)-interacting proteins and 20 cytoplasmic P1 of TEV (P1Te)-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 P1Tu and P1Zy IP profiles, whereas 10 consensus proteins were identified in the P1Tu and P1Te IP profiles (Table 1). Moreover, 5 consensus cytoplasmic proteins were found in the P1Zy and P1Te IP profiles (Table 1).
Next, we focused on P1Tu-interacting proteins because the P1/HCTu plant was the model used in this study. In the P1Tu 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/HCTu 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 P1Tu 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 P1Tu 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).
P1 and HC-Pro cause differentially expressed host proteins in transgenic plants
We performed label-free proteomics to identify the differentially expressed host proteins between Col-0 and other transgenic plants. We identified 2757 Arabidopsis proteins in Col-0, P1Tu, HCTu, and P1/HCTu plants (Additional file 1: Data). We found that ADP-GLUCOSE PYROPHOSPHORYLASE (APL3; AT4G39210), 6-PHOSPHOGLUCONOLACTONASE (PGL5; AT5G24420), and TONSOKU (TSK)-ASSOCIATING PROTEIN 1 (TSA1; AT1G52410) were decreased in P1Tu and P1/HCTu plants compared to Col-0 plants but not decreased in HCTu plants (Fig. 4a–c, panel i). However, the transcript of APL3 showed no significant difference among the various transgenic plants, whereas PGL5 and TSA1 were upregulated in HCTu and P1/HCTu plants compared with Col-0 plants (Fig. 4a–c, panel ii). APL3 is a starch biosynthesis enzyme, whereas PGL5 is a catalyzed enzyme in the oxidative pentose-phosphate pathway (OPPP) (Lansing et al. 2020; Liu et al. 2019). TSA1 is induced by methyl jasmonate (MeJA) and triggers endoplasmic reticulum (ER) body formation (Geem et al. 2019; Suzuki et al. 2005). These data indicated that the reduction in the protein levels of APL3, PGL5, and TSA1 occurred in a P1-dependent manner.
Next, we identified differentially expressed proteins between the HCTu and P1/HCTu plants. Nine proteins, including 2 superoxide dismutases [SOD1 (AT1G08830), and SOD2 (AT2G28190)], COPPER CHAPERONE FOR SOD1 (CCS1; AT1G12520), and ENHANCED MIRNA ACTIVITY 1/SUPER SENSITIVE TO ABA AND DROUGHT 2 (EMA1/SAD2; AT2G31660), were increased in P1/HCTu plants compared with HCTu plants (Fig. 4d–g, panel i). EMA1/SAD2 contains an importin-beta domain and negatively regulates in miRNA activity and is involved in abscisic acid (ABA) signaling (Cui et al. 2016; Panda et al. 2020; Wang et al. 2011).
In contrast, levels of 8 photosystem proteins (ATCG00340, AT1G55670, AT1G31330, AT4G12800, AT1G52230, AT1G44575, ATCG00350, and AT2G20260) were decreased in the P1Tu, HCTu, and P1/HCTu plants compared with Col-0 (Fig. 4h–o, panel i). However, their transcript levels were not significantly different (Fig. 4h–o, panel ii). We also found that PATHOGENESIS-RELATED GENE 5 (PR5; AT1G75040) was decreased in P1/HCTu plants compared with Col-0 plants (Fig. 4P, panel i), whereas JASMONATE RESISTANT 1 (JAR1; AT2G46370) was decreased in P1Tu and P1/HCTu plants (Fig. 4q, panel i). Similarly, the transcript levels of PR5 and JAR1 were not significantly different between plants (Fig. 4p, q, panel ii). In summary, many instances of posttranslational regulation occurred in the P1Tu, HCTu, and P1/HCTu plants.
The posttranscriptional and posttranslational regulation of miRNA targets in P1/HCTu 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 HCTu and P1/HCTu 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 HCTu and P1/HCTu plants compared with Col-0 (Fig. 4r, panel ii). Surprisingly, the level of AGO1 protein was decreased via an unknown mechanism in HCTu and P1/HCTu plants (Fig. 4r, panel i). The western blot data also indicated that the level of AGO1 was lower in P1/HCTu plants than in Col-0 plants but was similar to that in Col-0 plants, P1/HCZy and P1/HCTe 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 gene-to-gene correlation network to study PTGS suppression from a different perspective. First, we constructed a network for Col-0 vs. P1/HCTu plants in the ContigViews system. A list of twofold DGEs between Col-0 and P1/HCTu 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 HCTu and P1/HCTu 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 HCTu plants or between Col-0 and P1Tu plants (Fig. 7a, b). The gene positions in the comparative networks were followed with the Col-0 vs. P1/HCTu network for comparison (Figs. 6 and 7). There were 97 genes in the Col-0 vs. P1/HCTu network (Fig. 6); however, there were only 36 genes showed up when we applied the same parameters in the Col-0 vs. HCTu 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. HCTu network (Fig. 7a). This suggested the presence of a basic network backbone in the HCTu-mediated PTGS suppression that occurs without the effects of P1Tu. In contrast, the Col-0 vs. P1Tu network only had 7 genes in 2 small groups that were also present in parts of the Col-0 vs. HCTu or Col-0 vs. P1/HCTu networks (Figs. 6 and 7). Moreover, XTH7 had fewer than 49 connected genes in the Col-0 vs. HCTu network, whereas XTH7 had 61 connections in the Col-0 vs. P1/HCTu 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 P1Tu on HCTu-mediated PTGS suppression. This also explains why the P1/HCTu plant has a severe phenotype because of how many pathways were interfered with.
Critical genes in the Col-0 vs. P1/HCTu 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/HCTu 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, 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 HCTu and P1/HCTu 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 P1Tu, HCTu, and P1/HCTu plants compared to Col-0 plants (Fig. 8h). Notably, SOD1 was shown to have a physical interaction with P1Tu and P1Te (Table 1) and was also highlighted in the network, suggesting the importance of SOD1 in PTGS suppression.
Four miRNA targets, including TARGET OF EARLY ACTIVATION TAGGED 2 (TOE2; AT5G60120) and 2 squamosa promoter-binding protein-like genes [SPL13A (AT5G50570), and SPL13B (AT5G50670)], were also found in the group of negative correlation areas, whereas ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 15 (ATHB-15; AT1G52150) and PHABULOSA (PHB; AT2G34710) were found in the boundary between the positive and negative correlation groups (Fig. 6). Notably, miR172b regulated TOE2 modules in regulating plant innate immunity (Zou et al. 2018). In the Col-0 vs. P1/HCTu network, CYCLING DOF FACTOR 2 (CDF2; AT5G39660) and 2 carbon catabolite repressor 4 (CCR4)-associated factor genes [CAF1A (AT3G44260) and CAF1B (AT5G22250)] that are involved in RNA regulation were identified in the Col-0 vs. P1/HCTu network (Fig. 6). CAF1A and CAF1B catalyze mRNA deadenylation, whereas CDF2 interacts with DCL1 for miRNA biogenesis (Liang et al. 2009; Sun et al. 2015; Walley et al. 2010). These genes were also upregulated in HCTu and P1/HCTu plants (Fig. 8q, r, and y).
Eight calcium signaling genes were identified in the group of positive correlations in the network and were significantly upregulated in P1/HCTu plants (Figs. 6 and 8i–p). In the network, CALMODULIN-LIKE 24 (CML24; AT5G37770), and CAM-BINDING PROTWIN 60-LIKE G (CBP60G; AT5G26920) have significant functions in the regulation of autophagy and innate immunity, respectively (Qin et al. 2018; Tsai et al. 2013). In addition, jasmonic acid (JA) signaling and defense genes were highlighted in the positive correlation and their transcripts were upregulated in HCTu and P1/HCTu plants (Figs. 6 and 8q–v). In addition, the network indicated that FUMARASE 2 (FUM2; AT5G50950) and BARELY ANY MERISTEM 2 (BAM2; AT3G49670) were present in the boundary region between groups of positive and negative correlation (Fig. 6). BAM2 is a CLAVATA1-related receptor kinase and promotes the differentiation of stem cells on the meristem flank (DeYoung et al. 2006). FUM2 transcripts were upregulated in HCTu and P1/HCTu plants, whereas BAM2 transcripts were decreased (Fig. 8w and x). We also showed that CDF2, which is involved in miRNA biogenesis (Sun et al. 2015), is in the negative correlation group and is indirectly connected (negative correlation) to AGO1 through HB2 (AT3G10520) and XTH7 (Fig. 6). The CDF2 transcripts were upregulated in HCTu and P1/HCTu plants (Fig. 6y). The functions of critical genes in the Col-0 vs. P1/HCTu network are listed in Additional file 3: Table S2.
The auxin, ethylene, and ABA signaling pathways in PTGS suppression
The auxin response can induce ethylene and concomitantly trigger ABA biosynthesis (Hansen and Grossmann 2000). Importantly, the auxin, ethylene, and ABA signaling genes could be found in the Col-0 vs. P1/HCTu network (Fig. 6). MiRNA-regulated ARF3 and ARF8 targets are also auxin response genes, which were highly expressed in HCTu and P1/HCTu plants (Fig. 8d, e). In contrast, BAM2 expression is antagonistic with auxin transporters (Cecchetti et al. 2015) and its transcripts were downregulated in HCTu and P1/HCTu plants (Fig. 8x). Ethylene signaling genes, 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) SYNTHASE 6 (ACS6; AT4G11280), SCARECROW-LIKE 13 (SCL13; AT4G17230), and 8 ethylene responsive element binding factors [ERF1 (AT4G17500), ERF4 (AT3G15210), ERF5 (AT5G47230), ERF6 (AT4G17490), ERF105 (AT5G51190), ERF104 (AT5G61600), ERF12 (AT1G28360), ERF8 (AT1G53170)] were present in the group of positive correlations and their transcripts were upregulated in HCTu and P1/HCTu plants (Figs. 6 and 8z-ai; and Additional file 3: Table S2). Moreover, the results from endogenous ethylene emission experiments showed higher ethylene levels were detected in P1/HCTu plants than those in Col-0 plants at each time point (Fig. 9). ABA signaling genes, SULFATE TRANSPORTER 3;1 (SULTR3;1; AT3G51895), and 2 DIVARICATA genes [DIV1 (AT5G58900), and DIV2 (AT5G04760)] were also highlighted in the Col-0 vs. P1/HCTu network (Fig. 6) (Chen et al. 2019; Fang et al. 2018). These data suggested that P1/HC-Pro-mediated PTGS suppression also interferes with plant hormone signaling pathways.
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 P1Tu (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 P1Tu, HCTu, and P1/HCTu 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 P1Tu× HCTu (Kan) plants were similar to those of HCTu 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/HCZy and P1/HCTu 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 P1Tu (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. HCTu network, we identified a basic backbone network in HCTu-mediated PTGS suppression. However, the effects of P1-enhanced HC-Pro suppression were highlighted in the Col-0 vs. P1/HCTu network upon comparing the two networks. The Col-0 vs. P1/HCTu 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/HCTu 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 homeodomain-leucine 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/HCTu 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/HCTu plants might be related to endogenous auxin accumulation.
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.
Availability of data and materials
All data generated or analyzed in this study are in this published article.
1-Aminocyclopropane-1-carboxylic acid synthase 6
Automatic gain control
ADP-glucose pyrophosphorylase 3
Auxin response transcription factor gene 3
Auxin response transcription factor gene 8
Autophagy gene 4b
Autophagy gene 8
Arabidopsis thaliana homeobox protein 15
Barely any meristem 2
Brefeldin A-inhibited guanine nucleotide-exchange protein 5
Carbon catabolite repressor 4 (CCR4)-associated factor gene 1A
Carbon catabolite repressor 4 (CCR4)-associated factor gene 1B
Cam-binding protwin 60-like G
Copper chaperone for SOD1
Cycling dof factor 2
Differentially expressed genes
DIVARICATA gene 1
DIVARICATA gene 2
Enhanced MIRNA activity 1/super sensitive to ABA and drought 2
Ethylene responsive element binding factors
Flame ionization detector
Fragments per kilobase of transcript per million
Fast protein liquid chromatography
Growth-regulating factor 1
Jasmonate resistant 1
Modifier of SNC1: 4
Murashige and Skoog
Oxidative pentose-phosphate pathway
Polymerase chain reaction
Pathogenesis-related gene 5
Posttranscriptional gene silencing
RNA-induced silencing complex
Superoxide dismutase 1
Superoxide dismutase 2
Squamosa promoter-binding protein-like gene 13A
Squamosa promoter-binding protein-like gene 13B
Sulfate transporter 3;1
Short vegetative phase
Tobacco etch virus
Target of early activation tagged 2
Tonsoku (TSK)-associating protein 1
TUDOR-SN ribonucleases 1
TUDOR-SN ribonucleases 2
Turnip mosaic virus
Vernalization Independence 3/superkiller 8
Vacuolar protein sorting-associated protein 29
Xyloglucan endotransglucosylase/hydrolase 7
Zucchini yellow mosaic virus
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We acknowledge the mass spectrometry technical research services from the National Taiwan University Consortia of Key Technologies and National Taiwan University Instrumentation Center and deep sequencing from the High Throughput Sequencing Core of Academia Sinica.
This study was supported by the cooperation funding of CH Biotech company (08HT654004) and Council of Agriculture (109A3031). This article was subsidized by the Ministry of Science and Technology and National Taiwan University (NTU), Taiwan.
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Hu, SF., Wei, WL., Hong, SF. et al. Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches. Bot Stud 61, 22 (2020). https://doi.org/10.1186/s40529-020-00299-x
- Viral suppressor
- Posttranscriptional gene silencing
- Comparative network