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Article

Transcriptomic Profiling of Host Responses Underlying Synergistic Interaction Between the Phloem-Limited Brassica Yellows Virus and Pea Enation Mosaic Virus 2 in Nicotiana benthamiana

by
Cuiji Zhou
1,2,†,
Xiaoyan Zhang
2,3,† and
Chenggui Han
2,*
1
Department of Horticulture, College of Agricultural and Biological Engineering, Foshan University, Foshan 528225, China
2
State Key Laboratory of Agricultural and Forestry Biosecurity, and Ministry of Agriculture and Rural Affairs Key Laboratory of Pest Monitoring and Green Management, China Agricultural University, Beijing 100193, China
3
School of Horticulture, Ludong University, Yantai 264025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2026, 15(4), 645; https://doi.org/10.3390/plants15040645
Submission received: 25 January 2026 / Revised: 11 February 2026 / Accepted: 16 February 2026 / Published: 19 February 2026
(This article belongs to the Section Plant Molecular Biology)
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Abstract

Phloem-restricted poleroviruses cause substantial yield losses in crops. Co-infection of the polerovirus brassica yellows virus (BrYV) with the umbravirus pea enation mosaic virus 2 (PEMV 2) results in synergistic interactions that enable BrYV to overcome phloem limitation in Nicotiana benthamiana, yet the associated host transcriptional responses remain poorly understood. At 7 days post inoculation (dpi), BrYV RNA accumulation was increased in plants co-infected with BrYV and PEMV 2, although no visible symptoms or detectable cell death were observed. By 14 dpi, extensive cell death was induced in upper leaves infected with BrYV and PEMV 2. Transcriptome analysis at 14 dpi identified 45, 188, and 1962 differentially expressed genes (DEGs) in leaves infected with BrYV, PEMV 2, and co-infected with BrYV and PEMV 2, respectively, compared with mock-inoculated plants. A large number of DEGs, Gene Ontology terms, and KEGG pathways were predominantly observed in co-infected plants. Notably, expression changes were observed in genes related to plasmodesmata-associated processes, RNA silencing, photosynthesis, cell death, and ethylene biosynthesis and signaling during co-infection. These results provide a transcriptome-based overview of host responses during the late stage of BrYV and PEMV 2 co-infection and highlight the complexity of viral synergism between phloem-limited and taxonomically distinct plant viruses.

1. Introduction

The phloem plays a vital role in nutrient transport, connecting source and sink tissues to support plant growth and survival. However, it also serves as a conducive environment for various pathogens [1,2]. Phloem-limited pathogens are rapidly expanding in prevalence. Among these, phloem-limited viruses are capable of long-distance movement within the plant, yet they are confined to the vascular tissues and unable to move beyond them. This restriction is thought to result from a combination of host and viral factors [2], but the underlying mechanisms remain poorly understood.
Plants in natural environments are frequently infected by more than one virus, and such mixed infections often result in outcomes distinct from those caused by single viruses. These interactions are generally classified as synergistic, antagonistic, or neutral [3]. Among these, synergism is of particular importance, as it enhances the replication of one or both viral partners and typically leads to more severe disease symptoms than those induced by individual infections [3,4]. Synergistic interactions have been shown to increase viral titers [5,6,7], broaden host range [8,9], alter tissue tropism [10,11,12,13,14,15,16], exacerbate symptom expression [17,18], and enhance vector transmission rate [19,20], ultimately contributing to significant yield losses.
Polerovirus within the family Solemoviridae represents one of the most economically important groups of plant viruses [21]. Members of this genus are globally distributed and responsible for severe yield and quality losses in a wide range of crops [22,23]. Among them, brassica yellows virus (BrYV) has emerged as a serious threat to crucifer crop production. First identified in China in 2011 [24], it is now prevalent across many Chinese provinces [25,26] and has also been reported in South Korea, Japan and Australia [27,28,29,30]. BrYV naturally infects a broad range of cruciferous hosts and is frequently detected in mixed infections with other major viruses [25].
Poleroviruses possess a positive-sense single-stranded RNA genome approximately 5–6 kb in length [21,31]. An intriguing characteristic of poleroviruses is their restriction to phloem tissues. This phloem limitation prevents mechanical transmission, and infection can occur when the virus is delivered directly into the phloem via aphid feeding, agroinoculation, or grafting [32,33,34].
Co-infection of poleroviruses with taxonomically unrelated viruses can overcome their phloem limitation, a phenomenon likely associated with viral movement and RNA silencing suppression. Members of the genus Umbravirus have been shown to assist poleroviruses in breaching this restriction. For instance, mixed infections of potato leafroll virus (PLRV) with pea enation mosaic virus 2 (PEMV 2) or groundnut rosette virus (GRV), both belonging to the genus Umbravirus, induce severe symptoms in Nicotiana benthamiana and N. clevelandii. In these cases, PLRV accumulates not only in the inoculated leaves but also in parenchyma cells of systemically infected leaves and can be mechanically transmitted, indicating that PEMV 2 and GRV enable PLRV to overcome its phloem restriction [35,36]. Our previous studies demonstrated that co-infection of BrYV with PEMV 2 in N. benthamiana resulted in synergistic effects, including increased BrYV titers, severe symptom development, acquisition of mechanical transmission ability, and broader distribution of BrYV beyond vascular tissues [13]. The movement proteins (MPs) of umbraviruses are essential for this process. A chimeric cucumber mosaic virus (CMV) mutant, CMV(ORF4), in which the movement protein of CMV was replaced with that of GRV, was able to facilitate PLRV movement beyond the phloem. This finding indicates that umbraviral MPs are key determinants in enabling PLRV to overcome phloem restriction, potentially compensating for the inability of PLRV to move between mesophyll cells. However, a recombinant potato virus X (PVX) expressing the GRV MP or a CMV(ORF4) mutant defective in translation of the RNA silencing suppressor 2b fail to allow PLRV to move beyond the phloem, suggesting that the umbraviral MP alone is insufficient to mediate phloem escape [12]. RNA silencing suppressors are also implicated in facilitating PLRV movement beyond phloem tissues. HC-Pro has been shown to play an important role in promoting PLRV infection. Transgenic plants harboring a full-length PLRV cDNA clone show only limited accumulation in a few mesophyll cells. However, when these plants are either infected with potato virus Y (PVY) or co-transformed with the P1/HC-Pro sequence from tobacco etch virus, the number of PLRV-infected mesophyll cells increases significantly [37]. In contrast, infection of plants expressing the HC-Pro of potato virus A (PVA) with PLRV only leads to increased PLRV accumulation within phloem tissues, whereas mixed infection with PLRV and PVA allows systemic infection of all cell types. These findings indicate that RNA silencing suppression alone is insufficient for PLRV to overcome its phloem limitation and invade mesophyll tissues. Additional viral components, such as PVY P1 or factors facilitating intercellular movement, are also required [16]. Understanding how these viruses bypass their phloem limitation, particularly during co-infections, is crucial for development of effective management strategies. However, the host factors involved in these processes remain poorly understood.
Transcriptomic analysis has been widely applied to investigate synergistic interactions among various plant viruses, such as PVX and PVY [38], tomato chlorosis virus and tomato yellow leaf curl virus [39], sweet potato feathery mottle virus and sweet potato chlorotic stunt virus [40], tomato spotted wilt orthotospovirus and hippeastrum chlorotic ringspot orthotospovirus [41], wheat streak mosaic virus and triticum mosaic virus [17], and maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) [42]. However, little information is available regarding transcriptomic alterations during the synergistic interaction between phloem-limited and taxonomically distinct viruses.
In this study, we further examined the progression of BrYV and PEMV 2 co-infection in N. benthamiana. At 7 days post inoculation (dpi), Northern blot analysis revealed increased BrYV RNA accumulation in plants co-infected with BrYV and PEMV 2, despite the absence of visible symptoms or detectable cell death. By 14 dpi, pronounced cell death was observed in the upper leaves of co-inoculated plants. To characterize host transcriptomic responses associated with viral synergism, RNA-seq analysis was performed at 14 dpi to examine gene expression changes during co-infection with BrYV and PEMV 2. This analysis focused on host pathways related to plasmodesmata (PD)-associated processes, RNA silencing, photosynthesis, cell death, and ethylene biosynthesis and signaling. Collectively, this study provides a transcriptome-based overview of host responses associated with synergistic interaction between the phloem-limited BrYV and PEMV 2.

2. Results

2.1. PEMV 2 Enhances BrYV Accumulation at the Pre-Symptomatic Stage and Induces Cell Death at Later Stages in N. benthamiana

N. benthamiana plants were mock inoculated, singly inoculated with BrYV or PEMV 2, or inoculated with both BrYV and PEMV 2, as described previously [13]. As reported earlier, plants co-infected with BrYV and PEMV 2 developed mild leaf curling and chlorotic spots on upper leaves at 14 dpi, whereas plants infected with either virus alone remained symptomless (Figure 1A). No visible symptoms were observed at 7 dpi in any treatment, indicating a pre-symptomatic stage of infection (Figure 1A). To evaluate whether co-infection of BrYV and PEMV 2 induced cell death, trypan blue staining was performed at 7 and 14 dpi. At 7 dpi, no staining was detected in plants infected with BrYV, PEMV 2, or both viruses (Figure 1B). In contrast, at 14 dpi, intense trypan blue staining was observed in upper leaves of co-infected plants, indicative of extensive cell death, whereas only weak staining was detected in singly infected plants (Figure 1C).
We next examined whether BrYV accumulation was enhanced prior to symptom development in co-infected plants. Northern blot analysis of upper non-inoculated leaves revealed that BrYV RNA accumulation were markedly elevated in plants infected with BrYV and PEMV 2 at 7 dpi compared with those infected with BrYV alone (Figure 1D). At 14 dpi, BrYV RNA accumulation in co-infected plants was further increased (Figure 1E), consistent with our earlier findings [13]. In contrast, PEMV 2 RNA levels did not differ significantly between singly infected and co-infected plants at either time point (Figure 1D,E). These results indicated that BrYV accumulation was increased during co-infection at a pre-symptomatic stage of infection.

2.2. Evaluation of RNA-Seq

To characterize host transcriptomic responses associated with BrYV and PEMV 2 single infection and co-infection, upper leaves from plants inoculated with mock, BrYV, PEMV 2 or BrYV + PEMV 2 (co-infection of BrYV and PEMV 2) were collected at 14 dpi and subjected to RNA-Seq analysis.
Raw reads ranging from 46,314,330 to 62,090,154 per library were generated by RNA-Seq (Table 1). After quality filtering, 44,636,542, 54,906,026, 46,618,538, and 59,290,936 clean reads were obtained for mock-, BrYV-, PEMV 2-, and BrYV + PEMV 2-inoculated samples, respectively (Table 1). The Q20 percentage exceeded 97% for all samples. Clean reads were subsequently mapped to the N. benthamiana reference genome, with mapping rates greater than 94% for each library (Table 1), indicating high sequencing quality suitable for transcriptome analysis.

2.3. Identification of Differentially Expressed Genes (DEGs) and Venn Analysis

Compared with mock-inoculated plants, a total of 45 DEGs were identified in BrYV-infected plants, including 31 up-regulated and 14 down-regulated DEGs (Figure 2A). In plants inoculated with PEMV 2, 188 DEGs were identified, with 121 up-regulated and 67 down-regulated DEGs (Figure 2A). Notably, in plants co-infected with both BrYV and PEMV 2, 1962 DEGs were detected, comprising 1250 up-regulated and 712 down-regulated DEGs (Figure 2A).
To identify DEGs associated with synergistic interaction between BrYV and PEMV 2, a Venn analysis was conducted. The results showed that 53% of DEGs (24 out of 45) in BrYV vs. Mock were common among all virus treatments, 27% (12 out of 45) were shared between BrYV vs. Mock and BrYV + PEMV 2 vs. Mock, and 20% (9 out of 45) were unique to BrYV vs. Mock (Figure 2B). In PEMV 2 vs. Mock, 79% of DEGs (148/188) overlapped with BrYV + PEMV 2 vs. Mock, 13% (24/188) were common across all virus treatments, and 8% (16/188) were unique to PEMV 2 vs. Mock (Figure 2B). Notably, in BrYV + PEMV 2 vs. Mock, 91% of DEGs (1778/1962) were unique to this treatment, 8% (148/1962) overlapped with PEMV 2 vs. Mock, and 1% (24/1962) were common across all treatments (Figure 2B). These findings suggested that co-infection of BrYV and PEMV 2 in N. benthamiana triggered more extensive transcriptional changes than infection with either virus alone.

2.4. Functional Enrichment Analysis of DEGs

Gene Ontology (GO) enrichment analysis was performed to further characterize the functional roles of DEGs identified in BrYV-, PEMV 2- and BrYV + PEMV 2-infected plants at 14 dpi. GO terms were assigned to three major categories: biological process, cellular component, and molecular function. In total, 3, 11 and 38 GO terms were enriched in plants infected with BrYV, PEMV 2 and co-infected with BrYV and PEMV 2, respectively (Figure 3 and Figure S1). Further comparison of enriched GO terms among the three treatments showed that the majority of enriched GO terms (30/38) were specific to the co-infection treatment (Figure S1). These unique terms were primarily associated with the biological process category, particularly those related to metabolic processes, cellular metabolic process, and oxidation-reduction process. In the cellular component category, DEGs were predominantly enriched in terms related to ribosome, photosystem II, and the oxygen evolving complex (Figure 3). Within the molecular function category, DEGs showed enrichment in terms such as cation binding, metal ion binding, and oxidoreductase activity (Figure 3).
KEGG pathway analysis was conducted to explore the function of DEGs in samples singly infected with BrYV or PEMV 2, as well as those co-infected with both viruses. No enriched pathways were detected in BrYV vs. Mock (Figure 4A). In PEMV 2 vs. Mock, several pathways, including metabolic pathways, biosynthesis of secondary metabolites, and photosynthesis, showed enrichment (Figure 4B). In BrYV + PEMV 2 vs. Mock, a larger number of pathways were identified compared with single-virus infections, many of which were specific to the co-infection treatment (Figure 4C). These pathways included carbon fixation in photosynthetic organisms, ribosome, protein processing in endoplasmic reticulum, porphyrin and chlorophyll metabolism, plant-pathogen interaction, glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism, peroxisome, pentose phosphate pathway, biosynthesis of unsaturated fatty acids, protein export, plant hormone signal transduction (Figure 4C).

2.5. Identification of DEGs Related to Plasmodesmata

Plant viruses are known to utilize PD for cell-to-cell movement, thereby facilitating their systemic spread within host plants [43,44]. At 14 dpi, compared with mock-inoculated plants, two PD-associated DEGs were identified in BrYV-infected plants, two in PEMV 2-infected plants, and nine in plants co-infected with BrYV and PEMV 2 (Figure 5, Table S1). β-1,3-glucanase (GLU) has been reported to be involved in regulating callose deposition at PD, a process associated with viral cell-to-cell movement [45,46]. In this study, two GLU-encoding genes were up-regulated under all three virus treatments, with higher expression levels observed in virus co-infection compared with single infections (Figure 5, Table S1). In addition, seven PD-related DEGs were specifically detected in BrYV + PEMV 2 infection. These included genes encoding remorin (REM), expansin and calreticulin (Figure 5, Table S1). These proteins have previously been reported to be associated with viral trafficking or PD-related processes [44,47,48,49,50]. In the present study, two DEGs encoding expansin-like proteins and three DEGs encoding calreticulin-3 (CRT3) were up-regulated in plants co-infected with BrYV and PEMV 2, and two DEGs encoding REM were down-regulated in response to co-infection with BrYV and PEMV 2 (Figure 5, Table S1).

2.6. Identification of DEGs Related to RNA Silencing

DEGs encoding AGO2 and RDR1 were specifically induced in response to infection with BrYV + PEMV 2, compared with mock inoculation (Table 2). AGO2 is known to play a crucial role in antiviral silencing [51], while RDR1 in N. benthamiana contains a 72-bp insertion that disrupts its translation and implies a natural loss-of function of NbRDR1 [52,53].

2.7. Identification of DEGs Related to Photosynthesis

0, 10 and 68 DEGs related to photosynthesis were identified in response to BrYV infection, PEMV 2 infection, and co-infection with BrYV and PEMV 2, respectively, compared with mock-inoculated plants (Table S2). Among these, ten DEGs including eight associated with photosystem I (PSI) and two involved in photosynthetic electron transport, were down-regulated by PEMV 2 infection and co-infection with BrYV and PEMV 2 (Table S2), with a more pronounced suppression observed in the co-infection compared with PEMV 2 infection (Table S2). In addition, 56 photosynthesis-related DEGs were uniquely down-regulated during co-infection with BrYV and PEMV 2, including 25 genes related to PSII, 19 genes involved in PSI, one associated with cytochrome b6/f complex, four implicated in photosynthetic electron transport, and seven related to F-type ATPase (Figure 6, Table S2). One DEG encoding ferredoxin-3 and another encoding ferredoxin-NADP reductase were up-regulated by infection with BrYV + PEMV 2 (Figure 6, Table S2). These transcriptomic changes reflected widespread expression alterations in photosynthesis-related genes during BrYV and PEMV 2 co-infection.

2.8. Identification of DEGs Related to Cell Death

Several DEGs previously reported to be associated with cell death-related processes were specifically up-regulated during co-infection with BrYV and PEMV 2, compared with mock inoculation, including genes encoding probable linoleate 9S-lipoxygenase 5, SGT1 homolog, metacaspase-1, and serine/threonine/tyrosine-protein kinase SOBIR1 (Table 3). Probable linoleate 9S-lipoxygenase 5 has been reported as a molecular marker associated with systemic necrosis [54], whereas SGT1, metacaspase-1, and SOBIR1 have been implicated in programmed cell death-related pathways in previous studies [55,56,57,58]. Notably, one DEG encoding SOBIR1 was induced under both PEMV 2 infection and BrYV + PEMV 2 co-infection (Table 3). In addition, Bax inhibitor-1, a conserved endoplasmic reticulum-resident suppressor of cell death [59], was up-regulated by co-infection of BrYV and PEMV 2 (Table 3). Collectively, these results revealed altered expression patterns of cell death-related genes during BrYV and PEMV 2 co-infection at 14 dpi, when necrotic symptoms were apparent.

2.9. Identification of DEGs Related to Ethylene Biosynthesis and Signaling Pathway

Six DEGs associated with ethylene biosynthesis were specifically up-regulated in response to infection with BrYV + PEMV 2 compared with mock-inoculated plants, including genes encoding 1-aminocyclopropane-1-carboxylate synthase (ACS1), 1-aminocyclopropane-1-carboxylate oxidase (ACO), ACO3, and ACO homolog (ACOH) (Figure 7, Table S3). Two DEGs encoding ACO and ACO3 were up-regulated under both PEMV 2 infection and BrYV + PEMV 2 co-infection, with higher expression levels observed in co-infected plants (Figure 7, Table S3). Additionally, 11 DEGs encoding ethylene-responsive transcription factors (ERF), including ERF1B, ERF3, ERF4, and ERF071, showed increased expression specifically in plants co-infected with BrYV and PEMV 2 compared with mock-inoculated plants (Figure 7, Table S3). One DEG encoding ERF1B was up-regulated in response to PEMV 2 infection and co-infection with BrYV and PEMV 2 compared with mock inoculation (Figure 7).

2.10. Validation of RNA-Seq Results by RT-qPCR

Nine DEGs were randomly selected to validate their expression levels at 14 dpi using RT-qPCR in N. benthamiana plants subjected to mock inoculation, BrYV infection, PEMV 2 infection, or co-infection with BrYV and PEMV 2. These genes included NbACO, NbACO3, dehydration-responsive element-binding protein 2A (NbDREB2A), epidermis-specific secreted glycoprotein (NbEP1), NbERF1B, NbERF4, sugar transport protein 13 (NbSTP13), fasciclin-like arabinogalactan protein 9 (NbFLA9) and pentatricopeptide repeat-containing protein (NbPCMP-H11). RT-qPCR analyses were performed using three independent biological replicates. For each biological replicate, total RNA was extracted from a pooled sample of three independently infected plants, and each sample was analyzed with three technical replicates. Overall, RT-qPCR analysis of the nine randomly selected genes showed that the expression trends were largely consistent with the RNA-seq data across the four treatments (mock inoculation, BrYV infection, PEMV 2 infection, and co-infection with BrYV and PEMV 2) (Figure 8). NbACO, NbACO3, NbEP1, NbERF1B, NbSTP13 generally exhibited higher expression levels under PEMV 2 infection and co-infection compared with mock treatment (Figure 8). The expression levels of NbDREB2A and NbERF4 showed induction under co-infection with BrYV and PEMV 2 compared to mock inoculation (Figure 8). NbFLA9 displayed reduced expression in plants infected with PEMV 2 and plants co-infected with BrYV and PEMV 2, whereas NbPCMP-H11 showed reduced expression across all virus treatments relative to mock inoculation (Figure 8). Although variability among biological replicates was observed for several genes, the overall expression patterns detected by RT-qPCR were similar to those obtained from RNA-seq, supporting the reliability of the expression trends identified by transcriptome analysis.

3. Discussion

At early stages of infection (7 dpi), no visible symptoms or cell death were detected in upper leaves of plants co-infected with BrYV and PEMV 2. Pronounced cell death was observed in upper leaves at 14 dpi, as revealed by trypan blue staining. Similar systemic necrosis has been reported in several mixed viral infections, including those involving PVX and various potyviruses [60,61], mixed infections of turnip mosaic virus (TuMV) with PVX and PVY [62], panicum mosaic virus (PMV) and its satellite virus (SPMV) [63], and MCMV with SCMV [64]. These results suggest that systemic necrosis is a common outcome of synergistic interactions between plant viruses. Several P0 proteins encoded by poleroviruses have been shown to possess multifunctional roles. In addition to acting as viral suppressor of RNA silencing, some P0 proteins also capable of inducing cell death in infiltrated tissues of N. benthamiana or N. glutinosa [65,66,67,68]. Consistent with these findings, infiltration of N. benthamiana leaves with either a BrYV P0 construct or an infectious clone of BrYV results in visible necrosis in inoculated areas [69,70]. Because PEMV 2 enables BrYV to overcome phloem restriction [13], the cell death observed during co-infection may be associated with P0 expression in non-phloem tissues. Furthermore, BrYV RNA accumulation was elevated at the pre-symptomatic stage in plants co-infected with BrYV and PEMV 2, indicating that the synergistic effect began before visible symptoms occur. This observation aligns with other studies where viral accumulation does not always correlate with symptom severity. For example, PVX accumulation increases by co-infection of PVX and TEV without proportionally stronger symptoms [60]. In contrast, severe symptoms are observed in zucchini squash co-infected with zucchini yellow mosaic virus and CMV. Despite this severe symptoms, CMV RNA accumulation is only slightly higher in mixed infection compared to single-virus infections, highlighting that severe pathological outcomes can occur even with minimal changes in viral RNA levels [71].
Poleroviruses are typically restricted to the phloem. Co-infection with umbraviruses, such as PEMV 2, enables them to overcome this limitation, allowing invasion of mesophyll tissues and mechanical transmission [12,13,35]. Previous studies have attributed this to umbravirus-mediated enhancement of viral movement and suppression of RNA silencing [12,16,35,36,37], but the host factors contributing to this process remain largely unclear. In the present study, transcriptome analysis identified several PD-related DEGs. Compared with mock inoculation, two DEGs encoding GLU were induced by all three virus treatments, with higher expression levels observed during co-infection with BrYV and PEMV 2 than under single infections. Promotion of virus cell-to-cell movement by GLUs has been documented in tobamoviruses, potexviruses, cucumoviruses, and potyviruses [45,72,73,74,75]. The observed induction of GLU-encoding genes during co-infection was therefore consistent with previously reported roles of GLUs in PD-associated processes. Similarly, two DEGs encoding expansin-like were specifically induced by co-infection of BrYV and PEMV 2. Expansin, known to reside at PD, can relax cell walls and promote viral movement in potyvirus infections. Transient overexpression of NbEXPA1 enhances viral amplification and intercellular trafficking of TuMV and plum pox virus, whereas silencing NbEXPA1 diminishes TuMV accumulation and restrains long-distance spread [76]. Conversely, two DEGs encoding REM were uniquely down-regulated by infection with BrYV + PEMV 2. REM, which accumulate in plasma membrane nanodomains and at PD, restricts viral movement by interacting with viral movement proteins and restricting the PD aperture [77,78]. Elevated REM expression has been shown to inhibit the cell-to-cell movement of several viruses. For instance, overexpression of StREM1.3, NbREM1.2, or NbREM1.3 interferes with PVX movement, and NbREM1.5 inhibits TMV propagation through direct interaction with its movement protein [48,77]. The concurrent induction of GLU and expansin, together with the suppression of REM in plants co-infected with BrYV and PEMV 2, was consistent with previously reported functions of GLU, expansin and REM in PD-associated processes. However, the observed transcriptional changes represent correlations and do not, on their own, demonstrate a causal role for these host factors in BrYV movement beyond the phloem. Further experimental work will be needed to test this hypothesis directly. In particular, virus-induced gene silencing of GLU in co-infected plants could help clarify whether these enzymes contribute to BrYV intercellular movement. In addition, three DEGs encoding CRT3 were up-regulated in BrYV + PEMV2-infected plants. CRT, a Ca2+-binding protein localized in the endoplasmic reticulum and PD, can interfere with the targeting of viral movement proteins such as that of TMV, thereby restricting viral cell-to-cell movement [49,50]. The induction of CRT3 observed here may reflect a host response associated with PD regulation during co-infection.
RNA silencing plays a crucial role in plant antiviral defense. In this study, DEG encoding AGO2 was uniquely up-regulated at 14 dpi in plants co-infected with BrYV and PEMV 2 compared with mock inoculated plants. AGO2 plays an essential role in defense against multiple viruses, including CMV, turnip crinkle virus, PVX, and tomato bushy stunt virus [79,80,81,82]. Virus infections can trigger AGO2 expression, as demonstrated in rice plants infected by rice stripe virus and in N. benthamiana during early infection with tomato ringspot virus [83,84]. Elevated AGO2 transcript levels have also been reported in maize plants infected with MCMV and SCMV, with a more pronounced increase during co-infection [64]. The up-regulation of the DEG encoding AGO2 observed here therefore likely reflects the activation of host RNA-silencing antiviral responses during BrYV and PEMV 2 co-infection. However, this defensive response may not be fully effective, because BrYV encodes the P0 protein, a well-characterized suppressor of RNA silencing that triggers the degradation of AGO1 [85,86]. Given that P0 proteins of poleroviruses have been reported to target multiple ARGONAUTE proteins including AGO2 for degradation in other viral systems [87,88,89], it is highly plausible that BrYV P0 similarly inhibits AGO2 function at the post-translational level despite the increased AGO2 mRNA level. Thus, elevated transcription of AGO2 may not lead to functionally active AGO2 protein or efficient antiviral silencing during co-infection. Further studies examining AGO2 protein accumulation, stability, and activity will be required to clarify its role in the synergistic interaction between BrYV and PEMV 2.
Viral infections often cause mosaic and chlorotic symptoms associated with the downregulation of photosynthesis-related genes [90,91,92,93,94]. Consistent with the chlorosis symptom observed in plants co-infected with BrYV and PEMV 2, a total of 68 photosynthesis-related DEGs showed reduced expression compared to mock-inoculated plants, of which 56 were uniquely down-regulated during the co-infection. This broad transcriptional change is consistent with previous reports showing that CMV infection is accompanied by reduced expression of photosynthesis-related genes [95]. Moreover, previous studies have reported that the extent of downregulation of photosynthesis-associated genes correlates with the severity of chlorosis and thylakoid membrane deformation [94]. Taken together, these observations suggested that transcriptional changes in photosynthesis-related genes were associated with the development of chlorotic symptoms during BrYV and PEMV 2 co-infection in N. benthamiana. It should be noted that RNA-seq analysis in this study was performed using RNA extracted from whole leaves. During co-infection with BrYV and PEMV 2, the expansion of BrYV into mesophyll tissues likely increases the proportion of infected cells within the sampled tissue. Consequently, some of the observed transcriptional changes, including the downregulation of photosynthesis-related genes, may partly reflect a dose effect resulting from an increased proportion of infected cells rather than specific transcriptional regulation. Therefore, the present dataset did not allow us to clearly separate the effects of altered tissue composition from those of transcriptional regulation alone.
Several DEGs associated with ethylene biosynthesis (ACS1, ACO, ACO3, and ACOH) and signaling (ERF1B, ERF3, ERF4, and ERF071) showed increased expression in PEMV 2-infected plants and during co-infection with BrYV and PEMV 2 compared with mock-inoculated plants. Ethylene is a versatile phytohormone that regulates diverse physiological processes and mediates plant responses to both abiotic and biotic stresses [96,97,98,99,100]. Accumulating evidence suggests that ethylene plays dual roles in plant-virus interactions, acting either positively or negatively depending on the specific host-virus system. In some cases, ethylene has been reported to facilitate virus infection. For instance, Arabidopsis thaliana mutants defective in ethylene perception or signaling (etr1-1, etr1-3, ein2-1) exhibit enhanced resistance and restrict systemic movement of cauliflower mosaic virus [101]. Similarly, mutations in ethylene biosynthesis or signaling genes such as acs1, erf106, and ein2 conferred resistance to TMV [102], whereas exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid enhances viral accumulation [103]. In rice dwarf virus infection, a viral protein promotes host SAMS1 activity and ethylene production, thereby increasing viral susceptibility [104]. Conversely, ethylene has also been implicated in antiviral defense. Ethylene signaling is required for systemic resistance to chilli veinal mottle virus in tobacco [105], and in watermelon, activation of the ClACO5-mediated ethylene biosynthesis pathway enhances resistance to cucumber green mottle mosaic virus through the ClWRKY70-ClACO5 regulatory module [106]. Taken together, these findings highlight the complex and context-dependent roles of ethylene in plant-virus interactions. Further functional analyses will be required to determine whether ethylene acts as a positive or negative regulator during BrYV and PEMV 2 co-infection.
In summary, this study provided a transcriptomic overview of N. benthamiana plants co-infected with a phloem-limited polerovirus and an umbravirus. The results indicated that co-infection with BrYV and PEMV 2 elicited a distinct host response that differed markedly from that of single-virus infections, highlighting the complexity of viral synergism in plants. During co-infection, coordinated changes were observed in genes associated with PD-related processes, photosynthetic processes, and cell death. These transcriptional patterns may be relevant to viral spread beyond phloem and symptom development. Further studies of functional assays will help to elucidate the molecular mechanisms underlying virus-host interactions and the role of host factors in viral synergism.

4. Materials and Methods

4.1. Agrobacterium-Mediated Inoculation

N. benthamiana plants were grown and maintained at 18 °C under a 16-h light/8-h dark photoperiod. Agrobacterium-mediated inoculation was conducted as previously described [13]. Briefly, Agrobacterium tumefaciens strain GV3101 harboring the empty vector pCass4-Rz [107], PEMV 2 infectious clone pCaPE2 [13,69], and BrYV infectious clone BrYV-A [69] were cultured separately at 28 °C with shaking at 200 rpm. The agrobacterium cultures were harvested when they reached an OD600 of approximately 0.5 and resuspended in infiltration buffer containing 10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone. For single virus inoculation, cell suspensions were adjusted to an OD600 of 0.5. For co-inoculation, equal volumes of A. tumefaciens cultures harboring the respective viral constructs were mixed such that each strain was present at a final OD600 of 0.5 prior to infiltration.

4.2. Northern Blot Analysis

5 μg of total RNA extracted from N. benthamiana leaves was denatured at 65 °C for 10 min, separated on 1.2% formaldehyde-agarose gels, and subsequently transferred onto Hybond-N+ membranes (Amersham Hybond-NC, GE Healthcare, Chicago, IL, USA). Membranes were hybridized with α-32P-dCTP-labeled DNA probes specific for nucleotides 5161–5620 of BrYV or 2797–3202 of PEMV 2, as previously described [13,108].

4.3. Trypan Blue Staining

Trypan blue staining was performed as described [109]. Briefly, upper infected N. benthamiana leaves were immersed in 100% ethanol for 2–3 min, followed by incubation in staining solution consisting of equal volumes of 100% ethanol and trypan blue solution. Samples were boiled in water for 10–15 min, incubated at room temperature overnight, and subsequently destained with chloral hydrate for three times.

4.4. cDNA Library Construction and Illumina High-Throughput Sequencing

For RNA-Seq analysis, leaf tissues from three individual plants per treatment were collected at 14 dpi and pooled prior to RNA extraction. Total RNA was isolated from each pooled sample, and 3 µg of total RNA was used for library construction. cDNA libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to manufacturer’s guidelines. Briefly, mRNA was purified with poly-T oligo-attached magnetic beads (Life technologies, Carlsbad, CA, USA). Following fragmentation, first-strand cDNA was synthesized with M-MLV reverse transcriptase and random oligonucleotides. Second-strand cDNA was synthesized using DNA Polymerase I and RNase H. The resulting cDNA fragments were purified with AMPure XP beads system (Beckman Coulter, Brea, CA, USA) to obtain cDNA fragments approximately 200 bp. DNA fragments with ligated adaptor sequences on both ends were selectively enriched via a 10-cycle PCR amplification using NEB Universal PCR Primer and Index Primer. The libraries were sequenced on an Illumina Hiseq 2000 platform to generate 100 bp paired-end reads. The sequencing data were deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1391876.

4.5. Reads Mapping to the Reference Genome and Differential Expression Analysis

Raw sequencing data were processed to obtain high-quality clean reads by removing adapter sequences, poly-N reads, and low-quality reads. N. benthamiana reference genome sequence was downloaded from the Sol Genomics Network (http://solgenomics.net/organism/Nicotiana_benthamiana/genome, accessed on 20 March 2013). Clean reads were aligned to the reference genome using TopHat v2.0.9. Read counts for each library were normalized using the edgeR package, and differential expression analysis was performed with the DEGSeq R package (version 1.12.0). p values were adjusted using the Benjamini & Hochberg method. DEGSeq estimates p values based on a Poisson distribution, and in the absence of independent biological replicates, biological variance may not be fully captured. Genes with a corrected p value < 0.005 and |Log2 (fold change)| > 1 were regarded as putative DEGs, and the statistical analysis was used to facilitate the identification of candidate genes.

4.6. GO and KEGG Enrichment Analysis of DEGs

GO enrichment analysis of DEGs was performed using the Goseq R package to identify GO categories among the putative DEGs. GO terms with a corrected p value < 0.05 were used to describe enriched functional categories. KEGG pathway analysis was conducted using the KOBAS software to examine pathways associated with the identified DEGs. Pathways with a Q value < 0.05 were reported as enriched pathway categories.

4.7. RT-qPCR Analysis

To confirm the RNA-Seq results, nine DEGs were randomly selected for expression analysis using RT-qPCR. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 2 μg of total RNA with M-MLV reverse transcriptase (Promega, Madison, WI, USA) and primer 18TR following the manufacturer’s instructions. RT-qPCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each reaction was carried out in a total volume of 16 μL, containing 1 μL of cDNA, 8 μL of SsoFast EvaGreen Supermix (Bio-Rad), 0.4 μL of each primer (10 μM), and 6.2 μL of nuclease-free water. The amplification protocol consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 10 s, and a final melting curve analysis from 65 °C to 95 °C. NbPP2A expression levels were utilized as an internal control. Relative gene expression levels were calculated from Ct values using the 2−ΔΔCT method [110]. Primers used for RT-qPCR are listed in Table S4. RT-qPCR was performed using three independent biological replicates. Each biological replicate was derived from a separate infection experiment. For each treatment, total RNA was extracted from a pooled sample of three independently infected plants. RT-qPCR reactions were carried out with three technical replicates for each biological replicate.

5. Conclusions

In conclusion, this study described a synergistic interaction between BrYV and PEMV 2 that is associated with pronounced changes in host responses in N. benthamiana. This synergism was initiated at a pre-symptomatic stage, as indicated by enhanced BrYV RNA accumulation prior to the appearance of visible symptoms at 7 dpi. Transcriptomic analyses conducted at 14 dpi revealed a distinct and broader host gene expression response in co-infected plants compared with single-virus infections, with many DEGs, GO terms, and KEGG pathways identified predominantly in co-infected plants. Genes associated with PD-related processes, RNA silencing, photosynthesis, cell death, and ethylene biosynthesis and signaling showed marked expression changes during co-infection of BrYV and PEMV 2. Collectively, these findings provide a transcriptome-based framework for understanding host responses during polerovirus-umbravirus co-infection. Further experimental validation will be necessary to clarify the functional relevance of the transcriptomic changes observed during co-infection.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040645/s1, Figure S1. Venn diagram of enriched Gene Ontology (GO) terms. Table S1: List of DEGs related to plasmodesmata; Table S2: List of DEGs related to photosynthesis; Table S3: List of DEGs related to ethylene biosynthesis and signaling pathway; Table S4: Primers used for RT-qPCR validation.

Author Contributions

C.Z. and C.H. conceived and designed the experiments; C.Z. and X.Z. performed the experiments and analyzed the data; C.Z., X.Z. and C.H. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Scientific Research Program of the Guangdong Higher Education Institutions (2019KQNCX165), and funded in part by the National Natural Science Foundation of China (32272494 and 31371909) and the Natural Science Foundation of Shandong Province (ZR2024QC127).

Data Availability Statement

The raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) with accession number PRJNA1391876. All data will be made available upon request.

Acknowledgments

We are grateful to Michael Taliansky (The James Hutton Institute, Scotland) for kindly providing the pPEMV 2 construct, and to A.L.N. Rao (University of California, Riverside, USA) for the pCass4-Rz vector. We also thank David Baulcombe for generously supplying the A. tumefaciens strain GV3101. We thank Jia-Lin Yu, Da-Wei Li, Ying Wang, Xian-Bing Wang, and Yong-Liang Zhang at China Agricultural University for their valuable suggestions for this work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Symptoms, trypan blue staining, and accumulation of BrYV and PEMV 2 RNAs in Nicotiana benthamiana plants at 7 and 14 days post inoculation (dpi). (A) Symptoms of N. benthamiana plants inoculated with Mock, BrYV, PEMV 2, or BrYV + PEMV 2 (co-infection of BrYV and PEMV 2) at 7 and 14 dpi. Mock: plants inoculated with Agrobacterium tumefaciens carrying the empty vector pCass4-Rz. (B,C) Trypan blue staining of upper leaves from N. benthamiana plants infected with BrYV, PEMV 2, or BrYV + PEMV 2 at 7 dpi (B) and 14 dpi (C). Leaf discs were stained with trypan blue, and cell death was visualized as darkly stained areas. (D,E) Northern blot analysis of BrYV and PEMV 2 RNA accumulation in upper leaves at 7 dpi (D) and 14 dpi (E). The positions of genomic (gRNA) and subgenomic RNA (sgRNA) are indicated on the right. Detection of BrYV and PEMV 2 RNAs was performed using 32P-labeled DNA probes corresponding to nt 5161–5620 of BrYV and nt 2797–3202 of PEMV 2. Methylene blue-stained ribosomal RNA (rRNA) bands served as the loading control.
Figure 1. Symptoms, trypan blue staining, and accumulation of BrYV and PEMV 2 RNAs in Nicotiana benthamiana plants at 7 and 14 days post inoculation (dpi). (A) Symptoms of N. benthamiana plants inoculated with Mock, BrYV, PEMV 2, or BrYV + PEMV 2 (co-infection of BrYV and PEMV 2) at 7 and 14 dpi. Mock: plants inoculated with Agrobacterium tumefaciens carrying the empty vector pCass4-Rz. (B,C) Trypan blue staining of upper leaves from N. benthamiana plants infected with BrYV, PEMV 2, or BrYV + PEMV 2 at 7 dpi (B) and 14 dpi (C). Leaf discs were stained with trypan blue, and cell death was visualized as darkly stained areas. (D,E) Northern blot analysis of BrYV and PEMV 2 RNA accumulation in upper leaves at 7 dpi (D) and 14 dpi (E). The positions of genomic (gRNA) and subgenomic RNA (sgRNA) are indicated on the right. Detection of BrYV and PEMV 2 RNAs was performed using 32P-labeled DNA probes corresponding to nt 5161–5620 of BrYV and nt 2797–3202 of PEMV 2. Methylene blue-stained ribosomal RNA (rRNA) bands served as the loading control.
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Figure 2. Identification of differentially expressed genes (DEGs) and Venn analysis. (A) Number of DEGs identified in comparisons between BrYV-infected plants and mock-inoculated plants (BrYV vs. Mock), PEMV 2-infected plants and mock-inoculated plants (PEMV 2 vs. Mock), and co-infected plants and mock-inoculated plants (B + P vs. Mock). B + P indicates co-infection of BrYV and PEMV 2. Genes with a p value < 0.005 and |log2 (fold change)| > 1 were identified as putative DEGs. (B) Venn diagram showing the overlap of DEGs among the three comparisons. The blue, yellow, and red circles represent DEGs identified in B + P vs. Mock, BrYV vs. Mock, and PEMV 2 vs. Mock, respectively.
Figure 2. Identification of differentially expressed genes (DEGs) and Venn analysis. (A) Number of DEGs identified in comparisons between BrYV-infected plants and mock-inoculated plants (BrYV vs. Mock), PEMV 2-infected plants and mock-inoculated plants (PEMV 2 vs. Mock), and co-infected plants and mock-inoculated plants (B + P vs. Mock). B + P indicates co-infection of BrYV and PEMV 2. Genes with a p value < 0.005 and |log2 (fold change)| > 1 were identified as putative DEGs. (B) Venn diagram showing the overlap of DEGs among the three comparisons. The blue, yellow, and red circles represent DEGs identified in B + P vs. Mock, BrYV vs. Mock, and PEMV 2 vs. Mock, respectively.
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Figure 3. Gene Ontology (GO) enrichment analysis of DEGs in response to infection with BrYV, PEMV 2 and co-infection with BrYV and PEMV 2. (A–C) Enriched GO terms in BrYV vs. Mock (A), PEMV 2 vs. Mock (B), and B + P vs. Mock (C) were shown. B + P indicates co-infection of BrYV and PEMV 2. GO terms with a corrected p value < 0.05 are considered enriched among the identified DEGs. The X-axis displays the names of GO terms, and the Y-axis shows the number of DEGs. The numbers above the bars indicate the number of DEGs annotated to each GO term.
Figure 3. Gene Ontology (GO) enrichment analysis of DEGs in response to infection with BrYV, PEMV 2 and co-infection with BrYV and PEMV 2. (A–C) Enriched GO terms in BrYV vs. Mock (A), PEMV 2 vs. Mock (B), and B + P vs. Mock (C) were shown. B + P indicates co-infection of BrYV and PEMV 2. GO terms with a corrected p value < 0.05 are considered enriched among the identified DEGs. The X-axis displays the names of GO terms, and the Y-axis shows the number of DEGs. The numbers above the bars indicate the number of DEGs annotated to each GO term.
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Figure 4. KEGG pathway analysis of DEGs. (A–C) KEGG pathway profiles of DEGs in BrYV vs. Mock (A), PEMV 2 vs. Mock (B), and B + P vs. Mock (C). B + P indicates co-infection of BrYV and PEMV 2. The rich factor represents the ratio of DEGs associated with a given pathway to the total number of annotated DEGs. The circle size reflects the number of DEGs associated with each pathway, and the Q values reflect the degree of pathway enrichment.
Figure 4. KEGG pathway analysis of DEGs. (A–C) KEGG pathway profiles of DEGs in BrYV vs. Mock (A), PEMV 2 vs. Mock (B), and B + P vs. Mock (C). B + P indicates co-infection of BrYV and PEMV 2. The rich factor represents the ratio of DEGs associated with a given pathway to the total number of annotated DEGs. The circle size reflects the number of DEGs associated with each pathway, and the Q values reflect the degree of pathway enrichment.
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Figure 5. DEGs associated with plasmodesmata in N. benthamiana plants infected with BrYV, PEMV 2 or co-infected with BrYV and PEMV 2. Compared with mock-inoculated plants, the color scale indicates log2 (fold change) values ranging from −5 to +5, with red representing upregulation and blue indicating downregulation. Each row corresponds to an individual DEG, while columns represent the different virus treatments (BrYV, PEMV 2, and B + P). B + P indicates co-infection of BrYV and PEMV 2.
Figure 5. DEGs associated with plasmodesmata in N. benthamiana plants infected with BrYV, PEMV 2 or co-infected with BrYV and PEMV 2. Compared with mock-inoculated plants, the color scale indicates log2 (fold change) values ranging from −5 to +5, with red representing upregulation and blue indicating downregulation. Each row corresponds to an individual DEG, while columns represent the different virus treatments (BrYV, PEMV 2, and B + P). B + P indicates co-infection of BrYV and PEMV 2.
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Figure 6. DEGs involved in the photosynthesis pathway in response to co-infection with BrYV and PEMV 2 compared with mock-inoculated plants. The principal components in this pathway are illustrated. Blue and red color fillings indicate down-regulated and up-regulated gene expression, respectively. Red dashed arrows indicate light (hv). Blue dashed arrows represent electron transport, and solid black arrows indicate proton transfer. Enzyme commission (EC) numbers are shown in colored rectangles.
Figure 6. DEGs involved in the photosynthesis pathway in response to co-infection with BrYV and PEMV 2 compared with mock-inoculated plants. The principal components in this pathway are illustrated. Blue and red color fillings indicate down-regulated and up-regulated gene expression, respectively. Red dashed arrows indicate light (hv). Blue dashed arrows represent electron transport, and solid black arrows indicate proton transfer. Enzyme commission (EC) numbers are shown in colored rectangles.
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Figure 7. DEGs associated with ethylene biosynthesis and signaling pathway. Log2 (fold change) values are displayed using a color gradient ranging from −6 to +6, where red denotes up-regulated DEGs and blue indicates down-regulated DEGs. Rows represent individual DEGs, and columns correspond to the different virus treatments (BrYV, PEMV 2, and B + P), with DEGs identified in comparison with mock-inoculated plants. B + P indicates co-infection of BrYV and PEMV 2.
Figure 7. DEGs associated with ethylene biosynthesis and signaling pathway. Log2 (fold change) values are displayed using a color gradient ranging from −6 to +6, where red denotes up-regulated DEGs and blue indicates down-regulated DEGs. Rows represent individual DEGs, and columns correspond to the different virus treatments (BrYV, PEMV 2, and B + P), with DEGs identified in comparison with mock-inoculated plants. B + P indicates co-infection of BrYV and PEMV 2.
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Figure 8. RT-qPCR validation of DEGs identified by transcriptome analysis. (A–I) Nine DEGs (NbACO, NbACO3, NbDREB2A, NbEP1, NbERF1B, NbERF4, NbSTP13, NbFLA9, and NbPCMP-H11) were randomly selected to assess their expression patterns under four treatments: Mock, BrYV, PEMV 2, and B + P. B + P indicates co-infection of BrYV and PEMV 2. The expression of NbPP2A was used as the internal reference gene for normalization. Statistical significance is denoted by asterisks (p ≤ 0.05) based on two-tailed Student’s t-tests. Error bars indicate standard deviation (SD) of three independent biological replicates. Each dot represents one biological replicate, with RNA extracted from a pooled sample of three independently infected plants. RT-qPCR technical triplicates were performed for each biological replicate.
Figure 8. RT-qPCR validation of DEGs identified by transcriptome analysis. (A–I) Nine DEGs (NbACO, NbACO3, NbDREB2A, NbEP1, NbERF1B, NbERF4, NbSTP13, NbFLA9, and NbPCMP-H11) were randomly selected to assess their expression patterns under four treatments: Mock, BrYV, PEMV 2, and B + P. B + P indicates co-infection of BrYV and PEMV 2. The expression of NbPP2A was used as the internal reference gene for normalization. Statistical significance is denoted by asterisks (p ≤ 0.05) based on two-tailed Student’s t-tests. Error bars indicate standard deviation (SD) of three independent biological replicates. Each dot represents one biological replicate, with RNA extracted from a pooled sample of three independently infected plants. RT-qPCR technical triplicates were performed for each biological replicate.
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Table 1. Reads mapping to Nicotiana benthamiana genome.
Table 1. Reads mapping to Nicotiana benthamiana genome.
SampleRaw ReadsClean ReadsTotal MappedQ20 (%)GC (%)
Mock46,314,33044,636,54242,044,625 (94.19%)98.0144.50
BrYV56,877,83854,906,02651,637,007 (94.05%)98.4143.99
PEMV 248,262,91046,618,53843,972,759 (94.32%)98.7744.11
BrYV + PEMV 262,090,15459,290,93655,892,237 (94.27%)98.4843.75
Table 2. List of DEGs related to RNA silencing.
Table 2. List of DEGs related to RNA silencing.
Gene ID Description Log2 Fold Change 1
BrYVPEMV 2B + P 2
NbS00006644g0116 Protein argonaute 2 (AGO2) --2.21
NbS00009618g0001 RNA-dependent RNA polymerase 1 truncated protein (RdRp1m) --1.47
1 corrected p-value < 0.005 was used as a threshold to identify putative differentially expressed genes. 2 B + P indicates co-infection of BrYV and PEMV 2.
Table 3. List of DEGs related to cell death.
Table 3. List of DEGs related to cell death.
Gene ID Description Log2 Fold Change 1
BrYV PEMV 2 B + P 2
NbS00001604g0013 Probable linoleate 9S-lipoxygenase 5 --1.83
NbS00053961g0017 Probable linoleate 9S-lipoxygenase 5 --1.72
NbS00031070g0015 SGT1 homolog A --1.06
NbS00019485g0004 SGT1 homolog --1.44
NbS00016903g0003 Metacaspase-1 --5.88
NbS00037616g0014 Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR1 --3.82
NbS00033954g0001 Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR1 -1.433.23
NbS00033954g0002 Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR1 --3.48
NbS00009323g0008 Bax inhibitor 1 --6.58
NbS00044481g0005 Bax inhibitor 1 --3.51
1 corrected p-value < 0.005 was used as a threshold to identify putative differentially expressed genes. 2 B + P indicates co-infection of BrYV and PEMV 2.
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Zhou, C.; Zhang, X.; Han, C. Transcriptomic Profiling of Host Responses Underlying Synergistic Interaction Between the Phloem-Limited Brassica Yellows Virus and Pea Enation Mosaic Virus 2 in Nicotiana benthamiana. Plants 2026, 15, 645. https://doi.org/10.3390/plants15040645

AMA Style

Zhou C, Zhang X, Han C. Transcriptomic Profiling of Host Responses Underlying Synergistic Interaction Between the Phloem-Limited Brassica Yellows Virus and Pea Enation Mosaic Virus 2 in Nicotiana benthamiana. Plants. 2026; 15(4):645. https://doi.org/10.3390/plants15040645

Chicago/Turabian Style

Zhou, Cuiji, Xiaoyan Zhang, and Chenggui Han. 2026. "Transcriptomic Profiling of Host Responses Underlying Synergistic Interaction Between the Phloem-Limited Brassica Yellows Virus and Pea Enation Mosaic Virus 2 in Nicotiana benthamiana" Plants 15, no. 4: 645. https://doi.org/10.3390/plants15040645

APA Style

Zhou, C., Zhang, X., & Han, C. (2026). Transcriptomic Profiling of Host Responses Underlying Synergistic Interaction Between the Phloem-Limited Brassica Yellows Virus and Pea Enation Mosaic Virus 2 in Nicotiana benthamiana. Plants, 15(4), 645. https://doi.org/10.3390/plants15040645

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