Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection in plants

doi:10.21203/rs.3.rs-4308500/v1

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Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection in plants

https://doi.org/10.21203/rs.3.rs-4308500/v1

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Potyvirids (viruses in the Potyviridae family) are the largest group of plant RNA viruses. Our recent study has shown that Pelota, a core component of RNA quality controls (RQC), promotes the degradation of potyvirids’ genomic RNA by recognizing a specific G_1-2A_6-7 motif within the P3 cistron. Here, using turnip mosaic virus (TuMV) as a potyvirid model, we demonstrated that potyvirids have evolved a counteracting mechanism to inhibit Pelota-mediated RQC antiviral activities and promote virus infection. In this mechanism, the viral RNA-dependent RNA polymerase (also known as NIb) acts as a SUMOylation decoy to effectively reduce Pelota SUMOylation by competing with SCE1, the only SUMO E2 conjugating enzyme to inhibit Pelota-mediated RQC. TuMV NIb is comprised of two functional SUMO interacting motif (SIM) sites: SIM2 and SIM3. The former is identified as the key site for NIb’s SUMOylation, whereas the latter is responsible for the interaction with SCE1. These two SIMs are conserved among the majority of potyvirids-encoded NIbs. The other potyvirid NIb orthologs and their SIMs have similar functions in interacting with SCE1 and perturbing the Pelota-mediated RQC. Thus, virus protein-mediated SUMOylation decoy strategy to suppress host defense may be a common feature in plant virus pathosystems. Taken together, these findings highlight a dynamic interplay between plant defense mechanism and viral counter-strategy by orchestrating the post-translational modifications of virus and host defense components.

Biological sciences/Microbiology/Virology/Virus–host interactions

Biological sciences/Plant sciences/Plant stress responses/Biotic

Biological sciences/Microbiology/Virology/Viral pathogenesis

Biological sciences/Microbiology/Pathogens

SUMOylation

Pelota

Virus infection

RNA polymerases

Counter-defense

Plants inhabit environments teeming with abiotic and biotic challenges, including frequent exposure to viral infections. Plants have developed multi-layered defense lines to combat viral infections. Among them, RNA-targeting regulation mechanisms are generally considered an essential line of defense against RNA viruses. These include RNA silencing, RNA decay, and translation-coupled RNA quality control (RQC) mechanisms, targeting and degrading viral RNAs^1,2. During the coevolutionary arms race with their hosts, viruses have evolved sophisticated strategies to counteract, evade, or manipulate host antiviral defenses^{3, 4, 5}. A dynamic balance between host defense and viral counter defense determines viral pathogenesis and symptom development. This ongoing tug-of-war highlights the adaptive capabilities of the plant host and the infecting virus.

RNA silencing is a well-characterized RNA-targeting defense mechanism that confers resistance to viruses in plants. Almost all viruses encode viral suppressors of RNA silencing (VSR) forged to fine-tune virus-host coexistence. This suggests a plant-virus coevolution in the molecular arms race between host RNA silencing and virus counter-defense⁶. Due to the paramount roles of VSRs in viral infection, most work has primarily focused on elucidating the molecular mechanisms by which VSRs suppress antiviral RNA silencing in plants^{7, 8}. The contribution of other viral elements or proteins to the destruction of plant RNA-mediated defense mechanisms may have been underestimated¹. Until recently, only a few studies have described the interplay between RQC and viruses in plants^{9, 10, 11}. For instance, nonsense-mediated decay (NMD) has been identified as a viral restriction mechanism against pea enation mosaic virus 2 (PEMV2) with an unusual long 3’ untranslated region (UTR), and potato virus X (PVX) and turnip crinkle virus (TCV) with internally located termination codons (iTCs). Potyvirids appear unaffected by NMD due to their long polyprotein translation strategy⁹. However, the Pelota-mediated RQC can target and promote the degradation of potyvirids’ RNA, and this host anti-viral strategy is also involved in restricting the infection of the human immunodeficiency virus (HIV, a mammalian virus) and bamboo mosaic virus (BMV, a plant virus) ^{11, 12, 13}. Since the RNA recognition mechanisms involved in these antiviral responses are gradually revealed, viruses as successful cellular parasites are expected to evolve counteracting strategies to repress, manipulate or avoid these defenses. Studies have shown that plant viruses can evade NMD through several strategies, including the unstructured region of RNA downstream of the termination codon and the protection of viral transcripts by long-distance movement protein p26¹⁴. However, the question of whether the plant viruses could suppress or evade the Pelota-mediated RQC pathway has not been investigated. Answers to this question will not only contribute to a better understanding of the roles of RQC in plant-microbe interactions, but also assess if RQC can be targeted for crop improvement with antiviral genetic resistance.

Potyvirids constitute the largest group of plant-infecting RNA viruses and cause worldwide crop yield losses¹⁵. Most potyvirids are characterized by single-stranded, positive-sense RNA genomes. These genomes encode a large polyprotein, which undergoes processing into nine or ten mature proteins¹⁶. A second small ORF, PIPO, is generated by an RNA polymerase slippage mechanism and is expressed as the trans-frame protein P3N-PIPO^{17, 18}. These viral proteins have different biological functions and promote virus infection partly by hijacking and manipulating host cellular components. Among the known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase (RdRP) required for viral genome replication¹⁹. Beyond its conventional role in RNA replication, NIb has been implicated in regulating virus-host interactions²⁰. Previous studies highlighted the dual functionality of Nib, as a critical component of the viral replication complexes (VRC) and as a recruiter of host proteins to enhance viral replication efficiency^{21, 22, 23}. In a few cases, it has been demonstrated or suggested that NIb can subvert host defense responses. The NIb of turnip mosaic virus (TuMV) undergoes SUMOylation by SUMO-CONJUGATING ENZYME 1 (SCE1) and competes with NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), effectively dampening salicylic acid-mediated immune responses in Arabidopsis^{24, 25}.

We have recently demonstrated that the SUMOylated Pelota contributes to the host restriction of potyvirids by the RQC pathway¹¹. In this study, we showed that Pelota’s SUMOylation is suppressed during viral infection. We presented evidence that the NIb of potyvirids competitively interacts with the only SUMO-E2 enzyme, SCE1, leading to the inhibition of Pelota's SUMOylation. Evolutionary comparisons and molecular experiments revealed the presence of two conserved functional SUMO interacting motif (SIM) sites on NIb: one for SUMO modification and the other for interaction with SCE1 that is responsible for the Pelota modification suppression. These findings shed new light on the interplay of posttranslational modifications (PTMs), RQC, and plant-microbe co-evolution.

TuMV infection decreases the Pelota SUMOylation.

Our previous studies showed that the SUMOylated Pelota-mediated RQC mechanism negatively regulates the infection of potyvirids by targeting a conserved G_1-2A_6-7 motif within the P3 cistron¹¹. Based on the theory of a co-evolutionary arms race between plants and viruses, we speculate that potyvirids might evolve strategies for counter-defending the Pelota-mediated antiviral RQC activity. To test this hypothesis, we first analyzed the Pelota expression pattern under various biotic and abiotic stresses. Wild-type Arabidopsis plants were subjected to wounding (0.5 h and 2 h), cold stress (4 ℃ for 2 h), salicylic acid (SA) treatment (1 mM for 2 h), or TuMV infection at 14 days post-infiltration (dpi). Quantitative RT-PCR (qRT-PCR) revealed a slight upregulation of Pelota at 0.5 h during wounding and cold treatments. However, neither TuMV infection nor SA treatment significantly affected the level of Pelota mRNA (Fig. 1c, Supplementary Fig. 1a). Additionally, no correlation was observed between the expression of Pelota and several plant virus-related systems, including RNA silencing and phytohormone response pathways (Supplementary Fig. 1b, c). These results suggest that the Pelota regulation might be primarily at the level of protein or PTMs.

Considering that the activity of Pelota in RQC relies on its SUMO modification that determines its interaction with Hbs1, we compared the SUMOylation of Pelota under the conditions described above. Subsequent SUMOylation analysis with polyclonal antibodies against SUMO1 showed that TuMV infection significantly reduced the SUMO modification of Pelota in Pelota-YFP: pelota transgenic Arabidopsis plants (Fig. 1b, Supplementary Fig. 1d). Further investigations with transient expression in Nicotiana benthamiana, indicated a correlation between the increased TuMV accumulation and the decreased SUMOylation of Pelota (Fig. 1c). These data demonstrated a significant decrease in the SUMO modification level of Pelota during TuMV infection.

To further validate this observation, we used the YFP-P3 carrying G1-2A6-7 motif (a substrate of Pelota) to examine the functional impact of viral infection on eliminating aberrant RNA mediated by Pelota. GUS (used as control) or Pelota together with P19 (a potent RNA silencing suppressor to exclude the effect of RNA silencing) were co-expressed with YFP-P3 during TuMV infection. Subsequent qRT-PCR tests showed that the Pelota-mediated degradation of YFP-P3 transcripts was substantially blocked (Fig. 1d). Additionally, the decrease in Pelota SUMO modification showed a correlation with a reduced Hbs1 immunoprecipitation (Fig. 1e). Together, the TuMV infection decreases the SUMOylation level of Pelota and disrupts the Pelota mediated RQC.

NIb is the viral factor that inhibits Pelota SUMOylation.

The results above encouraged us to explore this molecular basis driven by TuMV. SCE1 is the only SUMO conjugating enzyme in plants and is responsible for transferring SUMO directly onto Pelota^{11, 26}. We then performed a yeast two-hybrid (Y2H) to identify interactions between TuMV proteins and SCE1. The Y2H assay revealed that viral proteins CI, VPg, NIa, NIb, and CP could interact with SCE1 (Supplementary Fig. 2). Our investigation then focused on which proteins can suppress Pelota SUMOylation. SUMOylation analysis indicated that, among these SCE1-interacting proteins, only NIb significantly reduced the modification level of Pelota (Fig. 2a). Since NIb is known to interact with SCE1 and SUMO3 in the SUMO-modification pathway^{24, 25}, we thus speculated that NIb could affect Pelota-mediated RNA degradation. Increased NIb expression was correlated with decreased Pelota SUMOylation, which parallels the findings in TuMV accumulation (Fig. 1, 2b). The Pelota-mediated degradation of YFP-P3 transcripts, as observed in TuMV-infected cells, was completely inhibited upon expression of NIb. This functional impact of NIb on the Pelota-mediated RQC activity was further confirmed by an agrobacterium-free transient expression assay performed with the protoplasts of TuMV P3 transgenic Arabidopsis (P3-OE #4), the target of Pelota (Fig. 2c-e, Supplementary Fig. 3a-c).

Next, we assessed the impact of NIb expression on Pelota functionality during TuMV infection. GUS, Pelota and Pelota-NIb were co-expressed with TuMV-GFP infectious clones in N. benthamiana plants. Subsequent qRT-PCR analysis revealed that Pelota-mediated antiviral activity was sufficiently blocked in NIb transient-expression plants (Fig. 2f, g). This observation aligns with the effects induced by viral infection, suggesting a robust inhibition of the functional role of Pelota due to NIb expression. Therefore, these findings support the idea that TuMV utilizes NIb to inhibit the Pelota-mediated RQC.

NIb and Pelota form an interaction complex in vivo.

Next, we tried to dissect the molecular link between NIb and Pelota. A bimolecular fluorescence complementation (BiFC) assay was performed to investigate the interaction between NIb and Pelota. Co-expression of constructs encoding YN-Pelota and YC-NIb resulted in a YFP fluorescence signal in the cytoplasm and the nucleus, but not with the P3N-PIPO, as a negative control (Fig. 3a). Luciferase complementation imaging (LCI) assay was further conducted in N. benthamiana leaves, which revealed vigorous luciferase activity when CLuc-NIb and NLuc-Pelota were co-expressed (Fig. 3b). This interaction was further substantiated through a Co-immunoprecipitation (Co-IP) assay, confirming the binding of Pelota with NIb in vivo (Fig. 3c).

However, our Y2H and in vitro pull-down assays showed no direct interaction between NIb and Pelota, indicating NIb and Pelota do not physically interact. Since both NIb and Pelota interact directly with SCE1(Fig. 3d, e), we hypothesized that SCE1 might serve as a mediator in this interaction, bridging the interaction between NIb and Pelota. These findings collectively imply the formation of an NIb-Pelota complex in plant cells.

NIb disrupts Pelota's function by competing with SCE1.

Furthermore, we found that the NIb expression did not notably alter the subcellular localization of the SCE1-Pelota interaction complex; it did significantly reduce the BiFC fluorescence intensity of this complex (Fig. 4a, b). Considering that both Pelota and NIb can directly interact with SCE1, we assessed the possibility that NIb interferes with the SCE1/Pelota interaction in a competitive manner. Competitive maltose-binding protein (MBP) pull-down and Co-IP assays supported this hypothesis, showing a marked decrease in the amount of Pelota protein immunoprecipitated by SCE1 both in vitro and in vivo as the amounts of NIb increased (Fig. 4c, d). In addition, the dissociation of the Pelota-SCE1 complex was also observed with the accumulation of TuMV (Supplementary Fig. 4a). Moreover, Pelota immunoprecipitation by Hbs1 decreased notably as the amounts of NIb increased (Supplementary Fig. 4b). These results indicate that TuMV encoded NIb inhibits the association of SCE1 and Pelota in a competitive manner, leading to a reduction of Pelota’s SUMOylation.

To further verify whether NIb alone is sufficient to inhibit the function of Pelota in plants, we performed RNA-sequencing (RNA-seq) and comparative transcriptome analysis using Col-0, pelota mutant, and NIb transgenic (35S: Myc-NIb, NIb) Arabidopsis seedlings. We found that NIb or pelota mutants significantly affected gene expression in Arabidopsis (Fig. 4e and Supplementary Fig. 5a). Principal component analysis (PCA) showed the Col-0 samples were substantially different from NIb and pelota. At the same time, significant overlap was observed between the NIb and pelota samples. We identified 1001 genes upregulated in pelota mutants with at least a four-fold change and statistical significance (compared to Col-0), categorizing them as potential targets regulated by Pelota. Remarkably, over 86% of these upregulated genes (865 out of 1001) were also affected by the NIb expression (Fig. 4f-g and Supplementary Fig. 5b). Gene ontology (GO), gene set enrichment analysis (GSEA), and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis also indicated a significant overlap between the differentially expressed genes in pelota mutant and NIb transgenic plant (Fig. 4h-i and Supplementary Fig. 5c-d). Collectively, these data compellingly demonstrate that the expression of NIb impedes the function of Pelota in plants by competing for the interactions with SCE1.

The SIM3 motif in NIb is required for interacting with SCE1.

The GPS-SUMO prediction service identified several SUMO-interacting motifs (SIMs) within NIb (Fig. 5a). Among these SIMs, the SIM2 motif, previously established as essential for NIb’s SUMOylation²⁵, was also predicted. We next generated NIb SIM mutants (NIb^sim1, NIb^sim2 and NIb^sim3), replacing the conserved residues within the SIMs with alanines (A) and assessing their interactions with SCE1. Y2H assays revealed that while NIb^sim1 and NIb^sim2 mutants retained the ability to interact with SCE1, the NIb^sim3 mutant lacked this capacity (Fig. 5b). Pull-down assay using recombinant proteins expressed in E. coli demonstrated that MBP-SCE1 could interact with NIb, but not MBP-YFP. Crucially, NIb^sim1 and NIb^sim2 mutants maintained SCE1 binding ability, whereas the NIb^sim3 mutant exhibited no interaction (Fig. 5c). Previous studies have demonstrated that SIM2 is essential for NIb SUMOylation, thereby facilitating its nucleus-cytoplasm transportation²⁵. The subsequent experiment was conducted to examine the effects of SIM mutants on the SUMOylation. NIb^sims-YFP were transiently expressed in the presence of SCE1, the NIb^sim2 and NIb^sim3 mutations rather than the NIb^sim1 mutations, exerting a significant effect on the SUMO modification (Supplementary Fig. 6a, b). We further confirmed that the NIb^sim3 mutation results in the loss of the ability of NIb to interact with SCE1 in the BiFC and Co-IP assays (Fig. 5d, e and Supplementary Fig. 6c, d). Thus, we conclude that SIM3 serves as the SCE1 binding site. As NIb^sim3 lost its ability to interact with SCE1, it could not inhibit Pelota-SCE1 interaction in the competitive experiments and thereby did not affect Pelota SUMOylation (Fig. 5e, f).

To investigate whether the SIM3 motif in NIb is essential for TuMV infection, we created the TuMV mutant clone TuMV-NIb^sim3, with a variant of NIb defective in interacting with SCE1. The wild-type clone TuMV, TuMV-NIb^sim1, TuMV-NIb^sim2 (the SUMOylation-defective form clone), and TuMV-NIb^sim3 were inoculated into N. benthamiana and Brassica napus (B. napus). Plants infected with TuMV and TuMV-NIb^sim1 exhibit severe virus symptoms, while plants infected with the TuMV-NIb^sim2 and TuMV-NIb^sim3 mutants did not show any obvious virus symptoms (Fig. 5g, h). Furthermore, qRT-PCR analysis showed few viral genomic RNA in the newly developed leaves of plants infected with TuMV-NIb^sim2 and TuMV-NIb^sim3 (Fig. 5i), indicating that the mutated virus lost its capability to inhibit the Pelota-mediated RQC, resulting in ineffective infection to plants. This result is consistent with the previous finding that TuMV systemic infection requires SUMOylated NIb²⁵, indicating that SUMOylation via SCE1 interaction is a pivotal regulatory step during virus infection.

Counteraction of Pelota defense machinery by NIb SIM3 is conserved amongst potyvirids.

To further investigate the functional conservation of NIb proteins, we explored the evolutionary dynamics within this family began with a re-analysis of NCBI RefSeqs of potyvirids, encompassing representative viruses from Arepavirus, Bevemovirus, Brambyvirus, Bymovirus, Celavirus, lpomovirus, Macluravirus, Poacevirus, Potyvirus, Roymovirus, Rymovirus, and Tritimovirus genera. Phylogenetic analysis and multiple sequence alignment, focusing on the NIb proteins, disclosed distinct inter-genus clustering patterns, illustrating the sequence diversity of NIb proteins across various genera (Supplementary Fig. 7a). This diversity contrasts with the sequence homogeneity observed within each genus, highlighting the close phylogenetic relationships among different virus species (Fig. 6a).

The multiple sequence alignment, with a specific focus on the presence of isoleucine (I), leucine (L), and valine (V), reaffirmed the presence of three previously verified SIMs (SIM1, SIM2, and SIM3). Among these, SIM2 and SIM3 are highly conserved within the viral genome, indicating a potentially conserved role of SUMOylation in the life cycle of potyvirids (Fig. 6a and Supplementary Fig. 7b). Our data above indicated that the TuMV NIb protein disrupts the Pelota-mediated RQC by reducing the Pelota’s SUMOylation through SIM3-SCE1 competitive interaction. Therefore, the sequence conservation of NIb SIM2 and SIM3 implies that SCE1-mediated NIb SUMOylation could be a strategic approach for viral infection within this family.

To investigate whether this phenomenon reflects a general virulence strategy deployed by viruses in this family, the interactions between NIbs from potato Y virus (PVY, Potyvirus), pepper veinal mottle virus (PVMV, Potyvirus) or areca palm necrotic ringspot virus (ANRSV, Arepavirus) and SCE1 were analyzed by Y2H and BiFC. Figure 6b-c illustrates the conserved binding interactions between the NIbs of PVY, PVMV, and ANRSV with SCE1. The NIb SUMO-modification site, SIM2, did not affect the interaction with SCE1, while the mutation in SIM3 lost this ability. Further experiments showed that these NIbs and SIM2 mutants (but not SIM3 mutants) expression exhibited decreased levels of Pelota’s SUMOylation (Fig. 6d). Since the RNA accumulation of YFP-P3 can serve as a marker for assessing the Pelota activity, we then tested the impact of NIbs (sims) on Pelota function. Consistent with the effect on Pelota SUMO modification, NIbs and SIM2 mutants continue to inhibit the Pelota activity in plants. In contrast, these NIbs carrying SIM3 mutation have lost this capability (Fig. 6e). These results together provide compelling evidence for the evolution of a counter-defense mechanism among potyvirids and plants, where viral NIb proteins employ the conserved SIM3-decoy tactic to interfere with SUMO modification and inhibit the Pelota-meditated RQC mechanism (Fig. 7).

It is well established that RNA silencing plays a pivotal role in antiviral immunity in eukaryotes^1,2,4,7,8. Emerging evidence suggests that RQC is another RNA-targeting mechanism that functions as a critical regulator in plant virus infection^{1, 2, 27}. Eukaryotes are equipped with RQC systems to monitor the quality of mRNAs during translation. To maximize coding potential, RNA viruses often contain unique structures and coding features, making these viruses ideal targets for RQC. Studies have shown that RQCs play crucial roles in combating viral infections and are recognized as critical virus restriction mechanisms in plants^{1, 2, 28}. Potyvirids use a genome expression strategy consisting of one or two polyprotein open reading frames (ORFs). Without eliciting signals in the viral genome, potyvirids could evade NMD. However, the conserved G_1-2A_6-7 motif within their genome has been identified as a target of the Pelota-mediated RQC^{9, 11}. Nevertheless, the specific mechanisms by which potyvirids inhibit, manipulate, or evade the reorganization of Pelota have not been methodically explored. In plants, the spectrum of gene expression alterations observed in pelota mutants is nearly completely covered by the changes induced by the NIb expression (Fig. 4f-i). Consequently, NIb alone can sufficiently compromise Pelota's RQC function, even without viral infection, indicating that NIb could perturb Pelota's biological functions. Unlike pathogenic bacteria, fungi, and oomycetes, viruses encode a limited number of viral proteins. No specific effector or paralogous decoy like PsXEG1 or PsXLP1 from Phytophthora sojae is generated from viruses only for counterattacking or evading plant defense responses²⁹. Therefore, viral proteins are multifunctional, a feature thought to be an adaptation to the limited viral genome to maximize their utilization. Our study discovered that the NIb of potyvirids acts as a SUMOylation decoy, targeting SCE1, abolishing the SUMOylation of Pelota and thereby facilitating viral infection (Fig. 7). These findings further expand the multiple functionalities of NIb beyond its primary role as an RNA polymerase.

Viruses utilize host cellular components as opportunistic intracellular parasites to facilitate their survival and pathogenicity. SUMO, unlike ubiquitin, can regulate the function (rather than abundance) of target proteins^{30, 31, 32}. Consequently, many viral proteins undergo PTMs mediated by the host machinery. The SUMOylation of NIb promoted TuMV infection by shifting NIb from the nucleus to the cytoplasm and suppressing host antiviral responses through interactions with the nuclear export protein XPO1^{25, 33}. The carrot mottle virus (CMoV) movement protein has also been SUMOylated in plant cells, and this modification helps its localization to plasmodesmata, thereby facilitating virus spread in host plants³⁴. The conservation of SUMO sites in viral proteins hints at a broader role for SUMOylation in enabling viruses to establish systemic infection successfully. Nevertheless, the precise mechanism of SUMOylation in plant-microbe interactions remains largely elusive. Our recent data showed SUMO modification acts as a molecular switch of Pelota-dependent RQC in regulating viral infection¹¹. At the same time, this study demonstrated that the viral virulence factors manipulate the SUMO modifications of host defense components to enhance virus infection. These findings suggest orchestrating the PTMs of host defense components and viral proteins, which likely represents a novel mechanism during the arms race between host and pathogens.

The functional NIb SIM sites (SIM2 and SIM3) were found to be highly conserved across potyvirids, as revealed through homologous sequence comparison (Fig. 6a). NIbs and SIM2 mutants, but not SIM3 mutants, could still interact with SCE1 and reduce the SUMOylation level of Pelota (Fig. 2b, 6b-c). NIb SIM2 is identified as the crucial site for NIb SUMOylation²⁵, while SIM3 is responsible for interacting with SCE1, the only SUMO conjugating enzyme in plants (Figs. 5 and 6). Highly conserved SIM2 and SIM3 sites observed at the genomes of potyvirids may underscore the selective pressures operative during evolution. Given the evolutionary limitation on viral genomes, we hypothesized that the conservation of these sites ensures the maintenance of crucial biological functions within the host, including the antagonism of the plant defense lines. These two SIM sites, each serving a unique function in the SUMO modification process, collectively enhance viral infectivity. The functional importance of these sites may have pushed them to undergo positive selection dynamics during evolution. This finding provides novel insights into the functional complexity of the NIb protein and its essential role in the viral life cycle of potyvirids. Our results also demonstrated overexpression of NIb could affect the expression of more plant endogenous genes than knocking out of Pelota (Fig. 4f-i), suggesting that NIb may have additional functions in virus-infected plants beyond interfering with the Pelota-mediated RQC. Therefore, clarifying the underlying molecular mechanism in the inhibition of plant defense by NIb is essential for a comprehensive understanding of this aspect of host-microbe interactions. Taken together, this study proposes a novel molecular basis in which, through the co-evolutionary arms race, viruses have evolved virulence strategies that subvert host defense responses by interfering with the SUMO modifications of crucial host defense components (Fig. 7).

Plant materials and growth conditions

Col-0 was used as a wild type and pelota (SALK_124403) was described previously¹¹. TuMV-P3 and TuMV-NIb transgenic materials were obtained using the floral-dip method³⁶. Transgenic RFP-H2B N. benthamiana was a gift from Michael M. Goodin (University of Kentucky, USA). The growth conditions consisted of 60% relative humidity and a day/night regime of 16 h in the light at 22ºC followed by 8 h of darkness at 18ºC.

Plasmid construction

Unless otherwise noted, all plasmids used in this study were constructed using Gateway technology (Invitrogen) according to the manufacturer’s protocol. The recombinant viruses TuMV and TuMV-GFP were previously described¹¹. The viral mutants were constructed by overlapping PCR with specific primers. Primers used in this study are listed in Supplementary Table 1.

Agroinfiltration and viral inoculation

Agrobacterium infiltration assays were performed as previously described³⁷. Briefly, the recombinant constructs with target genes were transformed individually into the Agrobacterium strain GV3101. A. tumefaciens cultures harboring the target gene-expressing vectors were resuspended in infiltration buffer [10 mM MgCl₂, 10 mM MES (pH 5.6), 0.1 mM AS] at an OD₆₀₀ of 0.6 before use.

Mesophyll protoplasts were isolated and transfected essentially as described previously. Briefly, mesophyll protoplasts were prepared from 4-week-old Col-0 or P3-OE Arabidopsis leaves. Protoplasts were transfected with the indicated plasmids (10 µg) in PEG solution (40% w/v PEG 4000 containing 0.2 M mannitol and 100 mM CaCl₂) at room temperature for 15 min. The transformed protoplasts were washed and resuspended in W5 buffer [154 mM NaCl, 125 mM CaCl₂, 5 mM KCl and 2 mM MES (pH 5.7)] and harvested for RNA extraction at 36 hpi.

Yeast two-hybrid (Y2H), BiFC and subcellular localization

Y2H were performed according to the manufacturer’s manual (Clontech). Briefly, Y2H Gold yeast cells harboring the co-transformed plasmids were simultaneously plated on a selective medium to analyze the interactions.

BiFC and subcellular localization experiments were performed as described³⁷. Briefly, the indicated constructs were transiently expressed in wild-type or RFP-H2B transgenic N. benthamiana leaves for 36-72h. Fluorescence signals for each combination were visualized with an inverted confocal microscope (Zeiss LSM980).

RNA library construction and sequencing

RNA-seq involved three genotypes of Arabidopsis thaliana: the TuMV-NIb transgenic plants, pelota mutant plants, and Col-0 wild-type plants. Healthy, mature plants were selected as experimental samples for each genotype. All plants were grown under controlled conditions with a constant temperature of 22°C, a photoperiod of 16 hours light/8 hours dark, and 60% relative humidity. Total RNA was isolated from the Arabidopsis samples using Trizol reagent (Invitrogen, CA, USA), following the manufacturer's instructions strictly. The quality and quantity of RNA were determined using a Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit (Agilent, CA, USA), with a requisite RNA Integrity Number (RIN) exceeding 7.0. Approximately 200 µg of total RNA from each sample was subjected to poly-A mRNA isolation using poly-T oligo-attached magnetic beads (Invitrogen). The purified poly-A mRNA fractions were then fragmented into oligonucleotides of approximately 100 nucleotides in length. Divalent cations facilitated this fragmentation under elevated temperatures. The fragmented mRNA was used as a template for cDNA synthesis. An average insert size of approximately 100 ± 50 bp for library construction was targeted. High-throughput sequencing was then performed on an Illumina Novaseq™ 6000 platform, using a paired-end 2×150 bp sequencing protocol as recommended by the manufacturer.

Analysis of the sequencing data

The raw sequencing reads were initially subjected to quality control using FastQC (version 0.11.9). Low-quality reads and adapter sequences were removed using Trimmomatic (version 0.39) with the parameters SLIDINGWINDOW:4:15 and MINLEN:36. The cleaned reads were subsequently mapped to the reference genomes of Arabidopsis thaliana (TAIR10) utilizing HISAT2 (version 2.2.1) with default parameters. Differential expression analysis was conducted using the DESeq2 package (version 1.28.1) in R, with genes exhibiting an adjusted p-value < 0.05, as determined by the Benjamini-Hochberg procedure, considered differentially expressed. Gene Ontology (GO) enrichment analysis was performed on differentially expressed genes using the topGO package (version 2.54.0) in R. Enrichment was determined using a Kolmogorov-Smirnov-like approach, with Fisher's exact test p-values < 0.05 considered significant. Pathway analysis was conducted using the KEGG.db package (version 2.7.1) in R, identifying enriched metabolic and signaling pathways among the DEGs. Pathways with Fisher's exact test-adjusted p-values < 0.05 were marked as significantly enriched. The GO network visualization was implemented using Cytoscape (version 3.10.1) and ClueGO (version 2.5.10) software.

Immunoprecipitation and immunoblotting analyses

Immunoprecipitation of protein extracts was conducted as described previously³⁷. Briefly, plant samples were collected at two days post-transformation, grounded in liquid nitrogen, and homogenized in IP buffer. Equal amounts of proteins were incubated with equal amounts of GFP-Trap for 2 h at 4ºC. The beads were washed five times at 4°C for 5 min with IP buffer after incubation, then boiled with SDS loading buffer. Immunoblotting analyses were carried out above with anti-GFP, anti-Myc or anti-Flag.

Pull-down and SUMOylation assays

Pull-down and SUMOylation assays were performed as previously described¹¹. For in vitro expression of the transformed proteins, E. coli was incubated at 37°C until the OD₆₀₀ reached 0.6, followed by adding 0.5 mM IPTG (Sangon Biotech). After an approximately 12 h induction at 18°C, cells were collected and sonicated in PBS with 0.4% NP40 buffer for protein purification. FLAG-tagged proteins were purified using anti-FLAG M2 affinity gel (Sigma). GST-tagged proteins were purified by glutathione-Sepharose 4B beads (GE Healthcare), and MBP-tagged proteins were purified by Amylose Resin (New England Biolabs). For pull-down assays, amylose resin or glutathione-Sepharose 4B beads were used to bind protein complexes in binding buffer [25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, protease inhibitor] at 4°C for 1 h. Then, the beads were collected and washed five times with washing buffer [25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM DTT 150 mM NaCl, 0.1% Triton X-100]. The beads were collected and boiled in 1× SDS sample-loading buffer for immunoblotting analysis. In vitro E. coli SUMOylation assay, equal amounts of eluted proteins were analyzed by immunoblotting with anti-Flag from Abmart (1:5000, M20026), anti-MBP from Transgene (1:2000, HT701), anti-His from Transgene (1:2000, HT501) and anti-SUMO (ABclonal Technology) antibodies.

Evolutionary analysis of NIb proteins

The NIb protein sequences from viruses in the Potyviridae family were retrieved from the National Center for Biotechnology Information (NCBI) database. Detailed virus species and accession numbers are provided in the supplementary material. The collected NIb protein sequences were aligned using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) tool (version 3.8.31). The alignment was executed with default parameters, ensuring optimal alignment quality for phylogenetic analysis. The phylogenetic tree of the aligned NIb protein sequences was constructed using the Maximum Likelihood method implemented in MEGA X (version 10.1.8). The best-fitting model of protein evolution was determined using the MEGA X built-in model selection feature. A bootstrap analysis with 1000 replicates was conducted to assess the robustness of the phylogenetic tree. The final tree was visualized and annotated using ggtree (version 3.10.0), highlighting significant clades and conserved sites.

Acknowledgments

We thank Dr. Andrew O. Jackson for valuable suggestions and English editing of the manuscript and Dr. Rosa Lozano-Dura´ n for stimulating discussions and helpful advice. We also thank Dr. Yule Liu (Tsinghua University, China) for providing the TRV VIGS vector; Dr. Jianxiang Wu (Zhejiang University, China) for providing anti-TuMV CP antibodies; and Dr. Yuhai Cui (Agricultural and Agri-Food Canada) for the modified Gateway vectors. The authors acknowledge support from the National Natural Science Foundation of China (32320103010 and 31972244) to FL, the National Natural Science Foundation of China (32302318) to LG, and the National Natural Science Foundation of China (32001868) to HS.

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Viral RNA polymerase as a SUMOylation decoy inhibits RNA quality control to promote potyvirus infection in plants

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Abstract

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Introduction

Results

Discussion

Methods

Plant materials and growth conditions

Plasmid construction

Agroinfiltration and viral inoculation

Yeast two-hybrid (Y2H), BiFC and subcellular localization

RNA library construction and sequencing

Analysis of the sequencing data

Immunoprecipitation and immunoblotting analyses

Pull-down and SUMOylation assays

Evolutionary analysis of NIb proteins

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Acknowledgments

References

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Supplementary Files

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