Tissue-cultured sugarcane cultivar ROC22 plantlets were cultured in a greenhouse with a 14/10-h light/dark cycle at 28°C and were individually inoculated with SCMV at the 5–6 leaf stage, as previously described (Zhang et al., 2019; Yang et al., 2021). The mock-inoculated ROC22 plantlets with 0.01 M phosphate buffer (pH 7.0) were used as the negative control. The inoculated or mock-inoculated leaves were sampled at 0 h, 6 h, 12 h, 24 h, 48 h, 5 d, 8 d and 14 d post-inoculation. For abiotic treatments, ROC22 plantlets of were grown in water for one week and then transferred to conical tubes for the following eight different treatments with a 16 h light/8 h dark cycle at 28°C. The plantlets were treated with 250 mM NaCl, 25% PEG (polyethylene glycol), 10 mM H₂O₂ (Su et al., 2014), 100 µM ABA, 400 mg/L ethylene solution (Chen et al., 2019) (Coolaber, Beijing, China), 25 mg/L BR (Wang et al., 2020), 5 mM SA, or 25 µM MeJA (methyl jasmonate) in 0.1% (v/v) ethanol, 0.05% (v/v) Tween-20, and were incubated for 0 h, 6 h, 12 h, 24 h and 48 h. After the treatments, three sugarcane plantlets per time point were sampled, frozen in liquid nitrogen immediately and kept at −80°C before use. Tissue samples from the leaves, roots and stem piths of 10-month-old healthy plants of ROC22 were also collected.

The genome sequence of Saccharum spontaneum was obtained from published genomic information (http://www.life.illinois.edu/ming/downloads/Spontaneum_genome) (Zhang et al., 2018). A Hidden Markov Model (HMM) of REM (Remorin_N: PF03766; Remorin_C: PF03763) was obtained from the Pfam database (http://pfam.xfam.org/) (Mistry et al., 2021) and was used to identify the REM genes in S. spontaneum. The gff3 files for S. spontaneum were downloaded (Zhang et al., 2018) to acquire the length and location information for the REM genes, then TBtools software was used to visualize the chromosomal locations of the REM genes. The chromosome color was set to black with the remaining parameters set to the default values. MCScanX software was used to analyze the replication pattern of the REM gene family with the parameters set to the default values (Wang et al., 2012).

The phylogenetic tree of REM was constructed by using MEGA6 version with the neighbor−joining method and 1000 bootstrap replicates. The physicochemical properties of the REM protein, including the number of encoded amino acids, molecular weight, isoelectric point, instability index, aliphatic index and grand average of hydropathicity, were predicted by the ProtParam tool of ExPASy (https://web.expasy.org/protparam/) (Gasteiger et al., 2003). The Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) website was used to predict the subcellular localization of REM proteins (Chou and Shen, 2008).

The S. spontaneum REM gene family motif information was acquired from the MEME website (https://meme-suite.org/meme/doc/meme.html) with the parameter was set to show 17 conserved motifs and the rest set as the default values (Bailey et al., 2009). The exon−intron structure information of the REM gene family was acquired from the gff3 files of S. spontaneum. The visualization of conserved motifs or gene structure of the REM gene family was conducted using TBtools software (Chen et al., 2020).

MCScanX software was used to analyze the synteny of the REM gene family with the parameters set to the default values (Wang et al., 2012). TBtools software was used to generate a synteny map within the genomes of S. spontaneum, sorghum (Sorghum bicolor), maize (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), and rice (Oryza sativa). The genome information of sorghum, maize, wheat, barley, and rice was acquired from Ensembl Plants (https://plants.ensembl.org/index.html). TBtools software was used to calculate the values of nonsynonymous (Ka)/synonymous (Ks) between homologous gene pairs based on the synteny relationship (Chen et al., 2020).

The upstream 2,000 bp promoter sequences of the REM gene family members were acquired from the databases of S. spontaneum genome. The prediction of the cis-elements in the REM gene family was conducted through the PlantCARE online website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002), while the visualization of the cis-elements was conducted with TBtools software (Chen et al., 2020).

Sugarcane tissue-specific expression data included leaf and stem data at the seedling stage (35 d), early maturity stage (9 months) and maturity stage (12 months). Materials were taken from SES2086 sugarcane plants for 11 d. Leaves were divided into 15 segments from the base to the tip (a total of 15 cm); each segment was 1 cm long, and RNA was extracted from each leaf segment. For the leaf segment model, three representative regions of each material were taken. All SsREMs FPKMs (fragments per kilobase of transcript per million segments mapped) were used to construct heatmaps and perform cluster analysis through TBtools.

TRIzol was used to isolate total RNA (Connolly et al., 2006). The Primer-Script^® RT−PCR Kit (TaKaRa Biotechnology, Dalian, China) was used to synthesize first-strand cDNA. For the real-time quantitative PCR (RT-qPCR) analysis, three technical replicates were completed for each sample with 25S rRNA and eEF-1a were used as internal references (Iskandar et al., 2004; Guo et al., 2014; Ling et al., 2014). The specificity of each primer pair were confirmed by the melting curve analysis. The relative gene expression was calculated by using the 2^−ΔΔCt method in triplicate and presented as the means ± standard error.

The ChamQ Universal SYBR qPCR Master Mix (Vazyme, Shanghai, China) was used to determine the expression patterns of ScREM1.5e-1/-2 by RT-qPCR with the sugarcane cDNA using as the templates, and 25S rRNA and eEF-1a were used as internal references (Iskandar et al., 2004; Guo et al., 2014; Ling et al., 2014) ( Supplementary Table 1 ). The primers ScREM1.5e-1/-2-qPCR ( Supplementary Table 1 ) were designed by the Primer Premier 5.0 software. RT−qPCR assays were conducted on the An ABI 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). The relative gene expression was calculated by using the 2^−ΔΔCt method in triplicate and presented as the means ± standard error.

The primers used for plasmid generation are listed in Supplementary Table 1 . For the Yeast two hybrid (Y2H) assays, the Y2H vectors and all DNA fragments were individually ligated via Sfi I sites. SCMV-/SCSMV-/SrMV-VPg or ScREM1.5e-1/-2 were individually cloned into the bait vector pGBKT7, and ScREM1.5e-1/-2 were individually cloned into the prey vector pGADT7. For the transient protein expression and bimolecular fluorescence complementation (BiFC) assays, all plasmids were constructed using Gateway technology as previously described (Cheng et al., 2020). All the plasmids generated in the present study were verified by sequencing.

For the Y2H assay, the Matchmaker Gold Yeast Two-Hybrid System (Clontech, Mountain View, CA, USA) was used according to the manufacturer’s instructions. The prey vector pGADT7 and bait vector pGBKT7 harboring the genes to be tested were cotransformed into the yeast (Saccharomyces cerevisiae) strain AH109. The yeast transformants were spread on SD/-Trp/-Leu agar plates and incubated at 30°C for 2–3 d. Colonies grown on SD/-Trp/-Leu agar plates were suspended in SD/-Trp/-Leu liquid medium to an optical density of 0.6 at 600 nm (OD₆₀₀). A 10× dilution series of 5 µL aliquots of yeast transformants were spotted onto SD/-Trp/-Leu or SD/-Trp/-Leu/-His/-Ade agar plates supplemented with 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal). Then the plates were incubated at 30 °C for 2–3 d. The yeast AH109 cotransformed with pGADT7-T and pGBKT7-53, which interact in Y2H assays, were used as positive controls, and the yeast cotransformed with pGADT7-T and pGBKT7-Lam, which do not interact, were used as negative controls.

For the BiFC assays, two YFP fusion constructs were cotransformed into the Agrobacterium tumefaciens GV3101, then cultured to 0.6 at OD₆₀₀. Equal volumes of each culture were mixed and infiltrated into the epidermal cells of N. benthamiana leaves using a needleless syringe. The agroinfiltrated plants were cultured under normal growth conditions for 48 to 72 h.

For Co-IP assays, total proteins were extracted as previously described (Vijayapalani et al., 2012) and then centrifuged at 16,000 × g for 10 min at 4°C for three times. The supernatants were incubated with anti-mCherry Nanobody Magarose Beads (Shenzhen KangTi Life Technology, Shenzhen, China) overnight at 4°C with gentle shaking. Then the Magarose beads were centrifuged at 500 × g for 3 min at 4°C and washed five times with IP buffer. Western blotting was conducted by using mCherry antibodies as described above.

The constructs harboring the genes to be tested were individually transformed into Agrobacterium, which were then agroinfiltrated into N. benthamiana leaves using needleless syringes. The GV3101 transformants were cultured overnight in Luria–Bertani medium supplemented with the appropriate antibiotics and collected by centrifugation, and were resuspended in 10 mM MgCl₂ supplemented with 100 mM acetosyringone (Sigma, St. Louis, MO, USA). After incubation at room temperature for 2-3 h, the culture was diluted to an OD₆₀₀ of 0.2-0.5 and agroinfiltrated into the leaves of N. benthamiana plants. The agroinfiltrated plants were maintained under normal growth conditions for 48 to 72 h.

Leica SP8 X inverted confocal microscope with an argon laser (Leica, Wetzlar, Germany) was used to image the agroinfiltrated leaf sections at room temperature. YFP was excited at 514 nm, and the emission was captured at 530–590 nm. The RFP was excited at 561 nm, and the emission was captured at 588–648 nm. Images were captured digitally and processed using Leica Application Suite Advanced Fluorescence Lite software (Leica Microsystems). ImageJ (http://rsbweb.nih.gov/ij/) was used to quantify the fluorescence intensity.

A total of 65 SsREM genes were identified in the S. spontaneum genome (Zhang et al., 2018) based on the N-terminal (Pfam ID PF03766) and C-terminal (Pfam ID PF03763) regions of the REM proteins. To investigate the evolutionary relationships of the REM gene family, a phylogenetic tree was constructed to associate the SsREMs with those of five other monocotyledonous plants (Zea mays, Setaria italica, Triticum aestivum, Oryza sativa, and Allium cepa), ten dicotyledonous plants (Arabidopsis thaliana, Solanum tuberosum, Solanum lycopersicum, Populus trichocarpa, Persea Americana, Nicotiana tabacum, Nuphar advena, Medicago truncatula, Mesembryanthemum crystallinum and Amborella trichopoda) and three gymnosperms (Pinus taeda, Pinus pinaster and Picea sitchensis) (Raffaele et al., 2007; Badawi et al., 2019; Wang et al., 2022) ( Supplementary Table 2 ). The 65 SsREM proteins were classified into 6 groups ( Figure 1 ), namely, Group 0.1, Group 0.2, Group 1, Group 4, Group 5, and Group 6, which contained 2, 7, 18, 8, 10, and 20 members, respectively ( Figure 1 ; Supplementary Figure 1A ; Supplementary Table 2 ). These results are similar to those of previous reports (Raffaele et al., 2007; Badawi et al., 2019; Wang et al., 2022).

The online software MEME (http://meme-suite.org/tools/meme) was used to investigate the motifs contained in the SsREM proteins, and a total of 20 conserved motifs were identified ( Supplementary Figure 1B ). With the exceptions of SsREM1.1c, SsREM1.3a, SsREM1.3b, SsREM4.1a, SsREM5.3a/b/c/d, SsREM6.4a/b/c and SsREM6.5c/d, the SsREM proteins contain Motif 1, Motif 3 and Motif 7; Group 0.1 and Group 5 do not contain Motif 2. Moreover, SsREM0.1a, SsREM1.1b/c, SsREM1.3a/b, and SsREM5.3a contain only three motifs, and Group 0.2, Group 5 and Group 6 only contain Motif 8, Motif 9, Motif 15 and Motif 19. SsREM0.1a and SsREM1.1b contain Motif 1, Motif 3 and Motif 7. SsREM1.1c and SsREM1.3a/b contain Motif 1, Motif 2 and Motif 7. SsREM5.3a contains Motif 1, Motif 4 and Motif 6.

The exon−intron distribution patterns of the SsREM genes were also investigated via TBtools ( Supplementary Figure 1C ). The results showed that the number of exons in the SsREM genes varied from 1 to 9, and the exons also varied within the same group. For example, in Group 1, SsREM1.2a had only 1 exon, while SsREM1.1a had 6 exons. Most of the genes contained 4 to 6 exons, which is similar to the findings of previous reports (Badawi et al., 2019).

To investigate the evolutionary mechanisms of the SsREM gene family, the synteny of the SsREM gene family within S. spontaneum itself was analyzed. As shown in Figure 2 , there were 60 pairs of homologous SsREM genes in the genome of S. spontaneum. There were 2, 27, 3, 11 and 17 pairs of homologous SsREM genes in Group 0.2, Group 1, Group 4, Group 5 and Group 6, respectively. However, Group 0.1 contained no gene pairs. To obtain additional information on the evolution of the REM gene family, synteny analysis was conducted on S. spontaneum compared with sorghum, maize, rice, barley, and wheat. The results revealed 37 pairs of homologous genes between S. spontaneum and sorghum, 40 pairs between S. spontaneum and maize, 33 pairs between S. spontaneum and rice, 15 pairs between S. spontaneum and barley, and 69 pairs between S. spontaneum and wheat ( Figure 3 ; Supplementary Table 3 ).

The gene duplication types or events were identified by MCScanX software. As shown in Supplementary Table 4 , dispersed, tandem and whole-genome duplications (WGD)/segmental duplications were present in the SsREM genes. The main duplication types for the SsREM genes were WGD/segmental (25/65, 38%) and dispersed (22/65, 34%). The Ka/Ks values of the SsREM gene pairs were investigated to evaluate the evolutionary selection pressure on the SsREM gene family. As shown in Supplementary Table 3 , the Ka/Ks ratios of all the gene pairs were < 1, indicating that the homologous genes among S. spontaneum, sorghum, maize, rice, barley and wheat had undergone strong purifying selection.

Chromosomal mapping revealed that the SsREM genes were unevenly distributed on the 23 chromosomes ( Supplementary Figure 2 ; Supplementary Table 5 ), with one on chromosomes 2C, 5A, 6B, 6D, 8A, 8B and 8C ( Supplementary Figure 2 ); two on chromosomes 5C, 5D, 6A and 6C ( Supplementary Figure 2 ); three on chromosomes 1C, 1D, 2B and 5B ( Supplementary Figure 2 ); four on chromosomes 2A, 2D, 4A, 4B and 4C ( Supplementary Figure 2 ); and five, six and seven on chromosomes 4D, 1B and 1A, respectively ( Supplementary Figure 2 ; Supplementary Table 5 ).

The physicochemical properties and subcellular localization of the 65 SsREM proteins were calculated using the ProtParam tool in ExPASy ( Supplementary Table 6 ). The 65 SsREM proteins encoded proteins ranging from 99 to 939 amino acids, with molecular weights ranging from 11.28 kDa to 101.46 kDa. Among them, 53 SsREM proteins were basic proteins, and 12 SsREM proteins were acidic proteins. The instability coefficient indicated that the 65 SsREM proteins were unstable, with the single exception of SsREM4.1b, which was stable. The average hydrophobicity index indicated that all 65 SsREM proteins were hydrophilic. Subcellular localization prediction via Cell-PLoc 2.0 software (online site) revealed that 62 SsREM proteins localize to the plasma membrane, whereas only three SsREM proteins (SsREM6.2a, SsREM6.5b, and SsREM6.6c) localize to the nucleus ( Supplementary Table 6 ).

According to the genome of S. spontaneum, the cis-regulatory elements within the first 2,000 bp upstream of 65 SsREMs were predicted. A total of 17 cis-regulatory elements were screened; these elements are involved in the responses to stress, growth and development, and hormones ( Figure 4 and Supplementary Table 7 ). More than 80% of the SsREM genes contained elements related to anaerobic induction (ARE, 81.5%), methyl jasmonate (TGACG and CGTCA motifs, 86.2%), ABA (ABRE, 83.1%) and light responsiveness (G-box, 84.6%). Among the elements involved in growth and development, the ACE element is present in SsREM1.5e, SsREM5.1e and SsREM6.5a/b; the motif I element is present in SsREM5.1a/b, SsREM6.3b/c and SsREM6.6c, SsREM0.2c; and the cell cycle regulation element (MSA-like) is present in SsREM4.1c, SsREM5.1a/b/c and SsREM6.1a/d. Moreover, only SsREM4.1c and SsREM6.3b/c contain the TATC box. However, SsREM0.1b, SsREM1.3d, SsREM1.4a and SsREM5.2b contain 4 cis-elements (SsREM0.1b: TGACG-motif, LTR, TCA-element and GCN4-motif; SsREM1.3d: MBS, ARE, TCA-element and TC-rich repeats; SsREM1.4a: ARE, ABRE, G-box and GCN4-motif; SsREM5.2b: TGACG-motif, ABRE, G-box and TC-rich repeats) ( Figure 4 ; Supplementary Table 7 ).

Transcriptomic data were used to explore the expression patterns of SsREM genes in different tissues and developmental stages of S. spontaneum (Li et al., 2021). The expression levels of the SsREM genes in Group 1 and Group 4 were higher than those in Group 0.1, Group 0.2, Group 5 and Group 6. Moreover, the SsREM genes of Group 0.1, Group 0.2, Group 5 and Group 6 were expressed at very low levels or were undetectable in the premature stage and mature stage ( Figure 5A ). Notably, the SsREM genes in Group 1 (SsREM1.5ea/b/c/d/e) and Group 4 (SsREM4.1a/b/c/d/e) were expressed at very high levels in the stems during the premature or mature stage ( Figure 5A ).

To further investigate the functions of the SsREM genes in the photosynthetic tissues of S. spontaneum, we analyzed the transcriptomes of the leaves along a continuous developmental gradient as described in previous studies (Li et al., 2021). The results showed that the expression levels of the SsREM genes were very high in Group 1 and Group 4 but very low in the other groups. Five genes (SsREM1.5c/e and SsREM4.1b/d/e) exhibited very high expression levels, suggesting that these genes play a key role in leaf development in S. spontaneum. SsREM1.5e presented the highest expression levels across the 15 sections of the leaf, whereas SsREM4.2a exhibited very low or undetectable expression levels ( Figure 5B ). The results indicate that the SsREM genes may function differently at various developmental stages, thereby affecting biological processes in different tissues.

Two Group 1 REM alleles were cloned from the sugarcane cultivar ROC22 and designated ScREM1.5e-1 and ScREM1.5e-2, respectively, based on phylogenetic tree analysis with the 65 SsREM genes ( Supplementary Figure 3 ). The open reading frame (ORF) of ScREM1.5e-1 (OR805348) is 555 bp in length and encodes 184 amino acids. While, the ORF of ScREM1.5e-2 (OR805349) is 420 bp in length and encodes 139 amino acids. The genomic structures of ScREM1.5e-1 or ScREM1.5e-2 are similar to that of Group I REM in other plants, with five exons and four introns ( Figure 6 ). The difference between ScREM1.5e-1 or ScREM1.5e-2 at the genomic level is that the fourth intron of ScREM1.5e-1 is 29 bp longer than that of ScREM1.5e-2 ( Figure 6 ). The fourth intron of ScREM1.5e-1 is 239 bp in length with the first 2 nucleic acids at the 5´ terminus are GT and the last 3 nucleic acids at the 3´ terminus are TAG. As GT and AG are conserved splicing sites, the fourth intron of ScREM1.5e-1 could be successfully be spliced, resulting a typical REM protein, ScREM1.5e-1, which contains conserved Remorin_N domain (PF03766, 17-68), Remorin_C domain (PF03763, 71-177) domain (in which contains a coiled-coil domain) ( Figure 6 ). A conserved C-terminal anchor (RemCA) was also found at the C-terminal (155-182) of ScREM1.5e-1 by sequence alignment with the well-studied StREM1.3 or AtREM1.4 (Perraki et al., 2012; Raffaele et al., 2013) ( Figure 6 ). However, for the ScREM1.5e-2, the splicing sites AG at the end of the fourth intron mutated to AA, resulting failure in splicing. Whereas, the splicing occurred at the A₁₄₃G₁₄₄ of the fourth intron, resulting in retainment of the 66 bp of the fourth intron in the ORF of ScREM1.5e-2 and a meaningful termination codon TAA ( Figure 6 ). Thus, ScREM1.5e-2 protein losses whole RemCA and partial Remorin_C or coiled-coil domain ( Figure 6 ).

The expression patterns of ScREM1.5e-1/-2 in different tissues of the sugarcane cultivar ROC22 were determined via RT−qPCR analysis. Low expression levels of ScREM1.5e-1 were observed in the root and 8^th internode. However, a greater expression level (> 6-fold greater than that in the roots) of ScREM1.5e-1 was found in the leaf rolls and 1^st leaves than in the 8^th internode and roots ( Figure 7 ). In addition, the 8^th internode presented the lowest ScREM1.5e-1 expression level ( Figure 7 ). However, the lowest expression level of ScREM1.5e-2 was found in the roots, and the expression levels were significantly greater in the leaves (> 10-fold higher than those in the roots) and internodes ( Figure 7 ). These results suggest that ScREM1.5e-1/-2 may have different functions in the same tissues.

As the Group 1 remorin are extensively involved in the response to pathogen infection, we investigated the transcription profiles of ScREM1.5e-1/-2 in ROC22 plantlets challenged by SCMV using RT−qPCR. The results showed that the expression levels of ScREM1.5e-1 were significantly downregulated 12 h after SCMV inoculation and subsequently upregulated more than 8-fold at 8 or 14 d ( Figure 7 ). However, the expression of ScREM1.5e-2 was significantly downregulated at 12 h after SCMV inoculation, then significantly (but not more than 1.5-fold) upregulated at 5 d, then downregulated to levels significantly lower than those in the controls ( Figure 7 ).

As many cis-elements involved in plant responses to stress and hormones were found in the promoters of Group 1 remorin, we investigated the expression patterns of ScREM1.5e-1/-2 in the sugarcane cultivar ROC22 under various abiotic stresses and hormonal treatments by RT−qPCR. Generally, the expression patterns of ScREM1.5e-1/-2 were similar under treatment with 25% PEG, 100 µM ABA or 5 mM SA but were different under treatment with 250 mM NaCl, 10 mM H₂O₂, 400 mg/L ETH, 25 mg/L BR or 25 µM MeJA ( Figure 7 ). In addition, ScREM1.5e-1/-2 was downregulated at 6 h and then upregulated to the level of the control or slightly lower than the control level under 25% PEG and 100 µM ABA. However, under 5 mM SA, the expression levels of ScREM1.5e-1/-2 at 6 h and 12 h were 6- to14-fold greater than those in the controls. 250 mM NaCl treatment significantly suppressed the expression of ScREM1.5e-1 but had no impact on the expression of ScREM1.5e-2 except at 48 h. Treatment with 10 mM H₂O₂ also significantly suppressed the expression of ScREM1.5e-1 but significantly promoted the expression of ScREM1.5e-2 at 6 h. In particular, 25 mg/L BR or 25 µM MeJA did not impact the expression levels of ScREM1.5e-1 but significantly upregulated the expression of ScREM1.5e-2. In the 400 mg/L ETH treatment, ScREM1.5e-1 had a greater expression level than the control at 6 h, 12 h, 24 h, and 48 h. Inversely, ScREM1.5e-1 had a higher expression level than the control only at 6 h.

In our previous study, we found that the VPg of TuMV interacts with AtREM1.2/1.3 to facilitate virus infection, and this interaction relocalizes AtREM1.2/1.3 from the plasma membrane to the cytoplasm (Cheng et al., 2020). To test the possible influence of SCMV-/SCSMV-/SrMV-VPg on the subcellular localization of ScREM1.5e-1/-2, we first investigated the subcellular localization of ScREM1.5e-1/-2 or SCMV-/SCSMV-/SrMV-VPg in the epidermal cells of N. benthamiana leaves. The recombinant plasmids YFP-ScREM1.5e-1/-2 and mCherry-AtREM1.2, HDEL-RFP or H2B-RFP were subsequently transformed into N. benthamiana leaves by agroinfiltration as previously described (Cheng et al., 2020). Confocal observation was conducted at 48 hpi. The YFP-ScREM1.5e-1 fluorescence was strongly distributed on the plasma membrane and merged with the red fluorescence of RFP-AtREM1.2, a PM marker, indicating the PM localization of ScREM1.5e-1 ( Supplementary Figure 4A ). However, the fluorescent signal of YFP-ScREM1.5e-2 merged with the fluorescent signal of HDEL-RFP, a cytoplasmic marker, or H2B-RFP, a nuclear marker ( Supplementary Figure 4A ), indicating the cytoplasmic or nuclear localization of ScREM1.5e-2. For SCMV-/SCSMV-/SrMV-VPg-YFP, the fluorescent signals merged with those of HDEL-RFP or H2B-RFP, indicating the cytoplasmic and nuclear localization of SCMV-/SCSMV-/SrMV-VP, respectively ( Supplementary Figures 4B, C ). All the localization assay results were further confirmed by fluorescence intensity measurements ( Supplementary Figure 4 ).

To test the interaction of ScREM1.5e-1/-2 with SCMV, SCSMV, and SrMV-VPg, Y2H assays based on GAL4 and BiFC assays were conducted. For the Y2H assays, pGBKT7-SCMV-/SCSMV-/SrMV-VPg was individually cotransformed with pGADT7-ScREM1.5e-1/-2 into the yeast strain AH109. The combination of pGBKT7-53 and pGADT7-T was used as a positive control, while the combination of pGBKT7-Lam and pGADT7-T was used as a negative control. The results showed that the yeast cells cotransformed with the combination of pGBKT7-SCMV-/SCSMV-/SrMV-VPg with pGADT7-ScREM1.5e-1/-2 produced blue colonies on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade culture medium supplemented with X-α-Gal as the positive control ( Figure 8A ), indicating an interaction between SCMV-/SCSMV-/SrMV-VPg and ScREM1.5e-1/-2, while no colonies developed for the negative control ( Figure 8A ).

For the BiFC assays, the fusion construct SCMV-/SCSMV-/SrMV-VPg-YC was individually cotransformed with ScREM1.5e-1/-2-YN into the Agrobacterium strain GV3101 and then agroinfiltrated into N. benthamiana leaves. Confocal observation was performed at 48 hpi. The results showed that SCMV-VPg, SCSMV-VPg and SrMV-VPg interact with ScREM1.5e-1/-2 individually; SCMV-VPg, SSMV-VPg and SrMV-VPg interact with ScREM1.5e-1 mainly in the cytoplasm, whereas the interaction with ScREM1.5e-2 mainly occurs in the nucleus ( Figure 8B ). As expected, the negative controls ScREM1.5e-1/-2-YN and YC emitted no fluorescence signals ( Figure 8B ).

To further confirm the interaction of SCMV-/SCSMV-/SrMV-VPg with ScREM1.5e-1/-2, Co-IP assays were performed. We found that SCMV-/SCSMV-/SrMV-VPg could be individually pulled down by ScREM1.5e-1/2 but not GUS ( Figures 8C, D ). Collectively, these results demonstrated the interaction of ScREM1.5e-1/-2 with SCMV-/SCSMV-/SrMV-VPg both in vitro and in vivo ( Figure 8 ).

As previous studies have shown that REMs can form homo or hetero-oligomers (Keinath et al., 2010; Martinez et al., 2019; Cai et al., 2020; Ma et al., 2022; Su et al., 2023), we performed Y2H, BiFC and Co-IP assays to evaluate the self-interaction of ScREM1.5e-1/-2 or the interaction between ScREM1.5e-1 and ScREM1.5e-2. For the Y2H assays, the combinations of pGBKT7-ScREM1.5e-1 plus pGADT7-ScREM1.5e-1, pGBKT7-ScREM1.5e-1 plus pGADT7-ScREM1.5e-2, and pGBKT7-ScREM1.5e-2 plus pGADT7-ScREM1.5e-2 were individually cotransformed into the yeast strain AH109. Like yeast cells cotransformed with the positive control plasmids pGBKT7-53 and pGADT7-T, yeast cells cotransformed with the combination of pGBKT7-ScREM1.5e-1 and pGADT7-ScREM1.5e-1, pGBKT7-ScREM1.5e-1 and pGADT7-ScREM1.5e-2, pGBKT7-ScREM1.5e-2 and pGADT7-ScREM1.5e-2 produced blue colonies on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade culture medium supplemented with X-α-Gal, indicating interactions between ScREM1.5e-1 and ScREM1.5e-2 and self-interactions ( Figure 9A ), and the negative control cotransformed with plasmids pGBKT7-Lam and pGADT7-T did not produce blue colonies ( Figure 9A ). For the BiFC assays, the fusion construct ScREM1.5e-1/-2-YN/-YC was individually cotransformed with ScREM1.5e-1/-2-YN/-YC into the Agrobacterium strain GV3101 and then agroinfiltrated into N. benthamiana leaves. At 48 hpi, yellow fluorescence corresponding to YFP was observed via confocal microscopy. The results confirmed the ScREM1.5e-1/-2 interaction and their self-interactions ( Figure 9B ). As expected, the negative controls ScREM1.5e-1/-2-YN/-YC and YC/YN emitted no fluorescence signals ( Figure 9B ).

To further confirm the interaction between ScREM1.5e-1/-2 and the self-interactions, we conducted Co-IP assays. The results showed that ScREM1.5e-1/-2 could be pulled down by ScREM1.5e-1/-2 but not by GUS ( Figure 9C ). Collectively, these results demonstrate the interaction between ScREM1.5e-1 and ScREM1.5e-2 and the self-interactions of each protein both in vitro and in vivo.