Analysis of bidirectional promoter activity and structure of the large intergenic region (LIR) in mulberry crinkle leaf virus

Analysis of bidirectional promoter activity and structure of the large intergenic region (LIR) in mulberry crinkle leaf virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of bidirectional promoter activity and structure of the large intergenic region (LIR) in mulberry crinkle leaf virus JingJing Yang, Ying Huang, MengSi Zhang, William Kojo Smith, ZiJun Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8036347/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Plant Molecular Biology → Version 1 posted 8 You are reading this latest preprint version Abstract The mulberry crinkle leaf virus (MCLV) is a representative member of the genus Mulcrilevirus of the family Geminiviridae . Here, the bidirectional promoter activity of the long intergenic region (LIR) of MCLV and the roles of a specific element consisting of five GAAAAA repeats [(GAAAAA) 5 ] in LIR were investigated using Agrobacterium-mediated transient expression and transfection of Nicotiana benthamiana protoplasts. Transient expression results demonstrated that, similar to other geminiviruses, MCLV's LIR also exhibits bidirectional promoter activity. The promoter activity of the C-sense is significantly higher in the experimental host, N. benthamiana , while slightly higher than that of the V-sense in the natural host, mulberry ( Morus spp .). However, the promoter activity of LIR is inconsistent between the experimental and natural hosts, especially its C-sense promoter activity, which is slightly lower in the experimental host but higher in the natural host than that of the cauliflower mosaic virus (CaMV) 35S. The (GAAAAA) 5 element does not affect the activity and type of LIR promoter, but its absence significantly reduces the replication of the MCLV genome in protoplasts. Based on the constructed truncated mutants, a fragment of 232 nts (named P232) lacking (GAAAAA) 5 was deduced to be the minimal sequence required for sustaining LIR promoter activity. These findings lay the foundation for further research on MCLV and transgenic breeding of mulberry plants. Geminiviruses Mulberry crinkle leaf virus Large intergenic region Bidirectional promoter (GAAAAA)5 sequence Replication Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Geminiviruses are a class of economically important DNA viruses that infect a wide range of plants. Their genomes consist of single-component or two-component circular single-stranded DNA molecules ranging in size from 2.5 to 5.2 kb (Fiallo-Olivé et al., 2021). More than 700 geminiviral species reported to date were classified into 14 genera established by the International Committee on Taxonomy of Viruses (ICTV): Becurtovirus , Begomovirus , Capulavirus , Citlodavirus , Curtovirus , Eragrovirus , Grablovirus , Maldovirus , Mastrevirus , Mulcrilevirus , Opunvirus , Topocuvirus , Topilevirus , and Turncurtovirus (Fiallo-Olivé et al., 2021; Roumagnac et al., 2022 ). Geminiviruses can infect various plants including economically important crops, such as wheat (Xie et al., 2007 ), maize (Shepherd et al., 2010 ), and tomato (Czosnek and Laterrot, 1997 ), as well as fruit trees and woody plants, such as citrus (Loconsole et al., 2012 ), apple (Liang et al., 2015 ), mulberry (Lu et al., 2015 ) and paper mulberry (Qiu et al., 2020 ). Their infection not only impairs crop growth and reduces yield but also compromises product quality, thereby significantly undermining agricultural productivity and economic development (Moffat, 1999 ; Navas-Castillo et al., 2011 ). In addition to several genes encoding proteins associated with viral replication, movement, virion packaging, and pathogenicity, the genomes of all reported geminiviruses also contain a large intergenic region (LIR) that lies between the complementary-sense (C-sense) and virion-sense (V-sense) strands. LIRs contain the origin (v -ori ) required for the replication of geminiviral genomes, which locates in the putative stem-loop with nonanucleotide motif 5′-TAAT(G)ATT↓A(C)C-3′ (the arrow indicates the location of the DNA origin of replication). The v -ori serves as the recognition and nicking site for the viral replication-associated protein (Rep) to initiate rolling-circle replication (RCR) (Rizvi et al., 2015 ). Additionally, both V-sense and C-sense sequences of LIR contain several of cis -acting elements that maintain promoter activity, such as the core promoter elements TATA box and CAAT box, as well as elements related to light, biotic and abiotic stress response, and tissue-specific elements (Eagle and Hanley-Bowdoin, 1997 ). Structural and sequence properties of geminiviral LIRs determine their importance for viral replication and bidirectional transcription (Ashraf et al., 2014 ; Choi and Stenger, 1996 ; Hanley-Bowdoin et al., 2000 ). Mulberry crinkle leaf virus (MCLV), identified from mulberry plants, is a representative species of the genus Mulcrilevirus in the family Geminiviridae (Lu et al., 2015 ; Ma et al., 2015 ; Roumagnac et al., 2022 ). MCLV is primarily transmitted by leafhoppers and the infected vegetative propagation materials (Lu et al., 2022 ; Yu, 2021 ). There are two MCLV variants (MCLV and MCLV vII) present in naturally infected mulberry plants (Yu, 2021 ), and neither of these two variants induces conspicuous symptoms on leaves of the infected mulberry plants (Han et al., 2023 ; Lu et al., 2022 ). MCLV genome encodes nine to ten proteins (5–6 proteins on the virion-sense strand, 4 proteins on the complementary-sense strand) depending on different variants (Fig. 1 A, B). The functions of these proteins have been determined. V1 is presumed to be a coat protein (CP) (Lu et al., 2015 ). V2 (Yin et al., 2024 ), V4 (Han et al., 2024 ), and V6 (Han et al., 2025 ) function to enhance viral genome replication. V3 is a putative movement protein (MP), as well as a post-transcriptional gene silencing suppressor (Lu et al., 2019 ) and a pathogenicity determinant (Lu, 2020). V5, specifically encoded by MCLV vII, is essential for MCLV infection in N. benthamiana (Han et al., 2024 ). The complementary-sense strand contains four ORFs, which encode C1-C4 proteins. C1 and C2 are proteins involved in virus DNA replication (Lu et al., 2015 ; Shakir et al., 2023 ). C3 and C4 function both as pathogenicity determinants and suppressors of post-transcriptional gene silencing (Han, 2025 ; Yin, 2025 ). Like other geminiviruses, the MCLV genome contains an LIR, which is located between V1 and C1 (Fig. 1 A, B). However, whether MCLV LIR also exhibits bidirectional promoter activity, like other geminiviral LIR, has yet to be determined. Notably, the MCLV LIR contains a unique GAAAAA tandem repeat sequence. Although the number of GAAAAA repeats varies between five and eleven depending on different MCLV haplotypes, most MCLV LIRs contain five GAAAAA repeats [(GAAAAA) 5 ] (Yu, 2021 ). BLAST analysis showed that the (GAAAAA) 5 does not exist in any sequences deposited in GenBank. However, whether this special sequence affects the functions of MCLV LIR has not yet been clarified. Here, we confirmed that MCLV LIR, similar to other geminiviral LIRs, has bidirectional promoter activity by constructing transient expression vectors. The deletion of (GAAAAA) 5 did not affect bidirectional promoter activity but inhibited MCLV DNA replication. Based on these findings, a bidirectional promoter suitable for mulberry transgenic research was determined. 2. Materials and methods 2.1 Preparation of materials Nicotiana benthamiana , Arabidopsis thaliana Columbia (Col-0) and mulberry plants used in this study were cultivated in environmentally-controlled rooms with a 16/8-hour light/dark cycle at 25 ˚C, 50–60% relative humidity. An infectious clone of the wild-type MCLV vII (pCA-1.1MCLV WT ), which contains a duplicated LIR and one copy of the sequence flanking the LIR of the MCLV WT genome, was constructed by Han et al (Han et al., 2023 ). The previously constructed pCAMBIA1391z-CaMV-35S (p35S-GUS) was used as a positive control (Smith et al., 2022 ). The plant expression vector pCAMBIA2300 was obtained from Dr. Nan Chao from Jiangsu University of Science and Technology. 2.2 Synthesis of primers The primers used in this study were listed in Table S1 and were synthesized by GenScript Biotech Corporation (Nanjing, China). 2.2 DNA extraction DNA was extracted from plant tissues and N. benthamiana protoplasts using the Ezup Spin Column Plant Genomic DNA Extraction Kit (Sangon, Shanghai, China) following the manufacturer′s protocol. DNA from 0.1 g of leaf tissues was resuspended in 20 µL of double-distilled (dd) water. 2.3 Construction of transient expression vectors To evaluate the bidirectional promoter activity of the MCLV LIR, both virion-sense (P3) (Fig. 2 ) and complementary-sense (rP3) (Fig. 2 ) sequences of the complete LIR were amplified via PCR using the pCA-1.1MCLV WT plasmid as template and primers PF / PR and rPF / rPR (Table S1 ), respectively. To assess the impact of (GAAAAA)₅ on bidirectional promoter activity of MCLV LIR, the (GAAAAA)₅-deleted V-sense LIR sequence (P6) (Fig. 2 ) and (TTTTTC) 5 -deleted C-sense LIR sequence (rP6) (Fig. 2 ) were amplified by overlapping PCR using pCA-1.1MCLV WT plasmid as templates. Firstly, fragments 4 and 1 of P6 and rP6 were PCR-amplified using primers PF / P 4 + 1 R and P 4 + 1 F / PR (Table 1), and rPF / rP 4 + 1 R and rP 4 + 1 F / rPR (Table S1 ), respectively. Then, fragments 4 and 1 of each P6 and rP6 were linked via overlapping-PCR using the respective primer pairs PF/PR and rPF / rPR (Table S1 ) to form P6 and rP6. Based on the obtained results in this study, where the promoter activity of C-sense LIR was higher than that of V-sense and the deletion of (GAAAAA) 5 in the V-sense and (TTTTTC) 5 in the C-sense did not affect the bidirectional promoter activity of LIR, we amplified three rP6-truncated sequences, rP4, rP8 and rP7, and one P6-truncated sequence, P7, to confirm the minimal sequence maintaining bidirectional promoter activity of MCLV LIR. rP4 only contains 232 nts of rP6 5’-end, constructed by deleting 89 nts (including the predicted TSS of rP6) of rP6 3'-end (Fig. 2 ). rP8 contains 282 nts of rP6 5’-end constructed by deleting 34 nts (including the predicted TSS of rP6) at the 3' end of rP6 (Fig. 2 ). rP7 totally includes 232 nts, including 214 nucleotides between the rP6 and P6 TSS, as well as 10 nts downstream of the predicted rP6 TSS and 8 nts downstream of the predicted P6 TSS (Fig. 2 ). P7 is the complementary sequence of rP7 (Fig. 2 ). rP4, rP8, and rP7 were PCR-amplified using the rP6 plasmid as template and the LA PCR™ Kit (TaKaRa, Beijing, China), and primer pairs rPF4 / rPR, rPF1 / rPR, and rPF2 / rPR2 (Table S1 ), respectively. P7 was PCR-amplified using the P6 plasmid as a template and primer pairs PF2 / PR2 (Table S1 ). Amplicons (P3, rP3, P6, rP6, rP7, P7, rP4, rP8) were separated by 1% agarose gel electrophoresis in 1×TAE buffer. The target bands were excised and purified using the SanPrep Column DNA Gel Extraction Kit (Sangon). The obtained fragments were ligated into the pMD-19T vector (TaKaRa). The ligation products were transformed into E. coli DH5α competent cells. The positive clones were verified through sequencing (SUNYA, Hangzhou, China). The recombinant plasmids extracted from each positive clone were digested with BamH I and Hind III. The target fragments were ligated into BamH I/ Hind III-linearized pCAMBIA1391z vector using T4 DNA ligase (TaKaRa). The resulting products were transformed into E. coli DH5α competent cells. The positive clones were confirmed by colony-PCR using corresponding primers. Plasmids were extracted from each positive clone to obtain pCA-P3, pCA-P6, pCA-rP3, pCA-rP6, pCA-rP7, pCA-P7, pCA-rP4 and pCA-rP8. 2.4 Construction of infectious clones To investigate the role of the (GAAAAA) 5 in LIR on viral replication, an MCLV infectious clone lacking the sequence, MCLV mLIR was constructed as previously described (Han et al., 2023 ). Briefly, four fragments (1, 2, 3, 4) were amplified via PCR using the pCA-1.1MCLV WT plasmid as templates (Han et al., 2023 ) and primer pairs MCLV-5'F/R1, F1/R2, F2/R3, and F3/MCLV-3'R (Table S1 ), respectively. Subsequently, fragments 1 and 2 were joined by overlap-PCR using primer pairs MCLV-5'F/R2 (Tables S1) to form fragment 5, and fragments 3 and 4 were fused with primer pairs F2/MCLV-3'R (Table S1 ) to produce fragment 6. Fragments 5 and 6 were separated by 1% agarose gel electrophoresis, respectively. The two target fragments were reclaimed using a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China) and were ligated into Sal I-linearized pCAMBIA2300 using the ClonExpress MultiS One Step cloning kit (Vazyme, Nanjing, China). The resulting construct was transformed into E. coli DH5α, verified by sequencing (SUNYA, Hangzhou, China). The positive clones were confirmed by colony-PCR using primer pairs MCLV-5'F/ MCLV-3'R (Table S1 ). Plasmid was extracted from each positive clone to obtain pCA-MCLV mLIR . 2.5 Agrobacterium transformation and inocula preparation Plasmids (pCA-P3, pCA-P6, pCA-rP3, pCA-rP6, pCA-rP6, pCA-rP7, pCA-P7, pCA-rP4, pCA-rP8, and PCA-MCLV mLIR ) were transformed into Agrobacterium EHA105 competent cells by electroporation via Gene Pulser Xcell System (Bio-Rad, Hercules, CA, USA). Inocula were prepared as previously described (Han et al., 2025 ; Yin et al., 2024 ). 2.6 GUS fluorometric assay GUS fluorometric assays were performed as described previously (Smith et al., 2022 ). Briefly, Agrobacteria containing the constructs pCA-P3, pCA-P6, pCA-rP3, pCA-rP6, pCA-rP6, pCA-rP7, pCA-P7, pCA-rP4, pCA-rP8, p35S-GUS (positive control), and empty pCAMBIA1391z vector (negative control) were infiltrated into N. benthamiana leaves. The infiltrated leaf patches were collected 72 hours post-infiltration (hpi) and the crude lysate was extracted. GUS activity was qualitatively analyzed by measuring fluorescence of reaction products between the crude lysate and GUS substrate MUG (4-methylumbelliferyl-β-D-glucuronide) (Smith et al., 2022 ). Promoter activity is indicated by GUS activity. Tests were performed in triplicate. 2.7 Arabidopsis transformation Arabidopsis plants were transformed via floral dip as previously described(Clough and Bent, 1998 ; Smith et al., 2022 ). 2.8 Histochemical GUS staining Histochemical staining was performed according to the previously described protocol (Smith et al., 2022 ). Briefly, Transgenic seedlings were incubated in GUS staining at 37℃ overnight. Chlorophyll was cleared with 70% ethanol, and staining patterns were visualized under a Leica EZ4 HD stereomicroscope (Leica Microsystems, Germany). 2.9 Promoter element prediction Cis -regulatory elements in the MCLV LIR bidirectional promoter were analyzed using Plant CARE (Rombauts et al., 1999 ), PLACE (Higo et al., 1999 ), and TRANSFAC databases (Wingender et al., 1996 ). Transcription start sites (TSS) were predicted via the NNPP server ( https://www.fruitfly.org/seq_tools/promoter.html ) (Reese, 2001 ). 2.10 Protoplast isolation and transfection N. benthamiana protoplasts were isolated, and transfected with plasmid DNA as previously described (Dai and Wang, 2022 ; Han et al., 2025 ). 2.11 Real-time quantitative PCR (qPCR) analysis DNAs extracted from pCA-1.1MCLV WT - and pCA-MCLV mLIR -transinfected protoplasts at different time points were RNase A-digested and then adjusted to the same concentration. qPCR was performed with the primer pairs qMCLV F1/ qMCLV R1 previously designed (Table S1 ) (Han et al., 2024 ), RNase A-digested DNA, and 2× NovoStartR SYBR qPCR Super Mix Plus Kit (Novoprotein, Shanghai, China) according to the manufacturer's instructions. The standard curve used here was previously established with y = 3.2722x 37.076 (Han et al., 2024 ). The relative growth multiple of the progeny MCLV was calculated as previously designed (Han et al., 2025 ; Yin et al., 2024 ), via, the gene copy number at 24 hours post-transformation (hpt) and 48 hpt divided by the gene copy number at 0 hpt. Three biological replicates for each treatment and three technical replicates per biological replicate were used for all qPCR tests. 3. Results 3.1 Verification of MCLV LIR bidirectional promoter activity To verify where the MCLV LIR exhibited the bidirectional promoter activity similar to other geminiviral LIRs, the constructed pCA-P3 and pCA-rP3 were infiltrated into leaves of N. benthamiana and mulberry plants. GUS activity analysis showed that the promoter activities of P3 andrP3 were significantly higher than those of the negative controls, suggesting that MCLV LIR possesses bidirectional activity. The promoter activity of rP3 was significantly higher than that of P3 in N. benthamiana , but slightly higher in the mulberry plant, indicating the promoter activity of C-sense LIR is consistently higher than that of V-sense. In N. benthamiana plants, the promoter activity of rP3 was slightly lower than that of 35S, while that of P3 was significantly lower than that of 35S. In contrast, in mulberry plants, the promoter activity of rP3 was slightly higher, and that of P3 was marginally lower than 35S. 3.2 (GAAAAA)₅ and its complementary counterpart do not contribute to the bidirectional promoter activity of LIR To determine the influence of (GAAAAA) 5 within V-sense and (TTTTTC) 5 in C-sense of LIR on this bidirectional promoter activity, the constructed deleted-mutants pCA-P6 and pCA-rP6 were infiltrated into leaves of N. benthamiana and mulberry plants. pCA-P3 and pCA-rP3 were used as controls. GUS activity analysis showed that the promoter activities of P6 and rP6 were the same as those of their corresponding counterparts, P3 and rP3, in N. benthamiana and mulberry plants. These results indicated that (GAAAAA)₅ and (TTTTTC) 5 do not affect bidirectional promoter activity. 3.2 Minimal sequence maintaining bidirectional promoter activity of LIR To confirm the minimal sequence maintaining bidirectional promoter activity of LIR, three constructed rP6-truncated mutants (pCA-rP7, pCA-rP4, and pCA-rP8) were infiltrated into leaves of N. benthamiana plants. rP6 was used as a control. GUS activity analysis revealed that, although the promoter activities of rP4 and rP8 were significantly higher than those of the negative control, it is significantly lower than that of rP6. In contrast, the promoter activity of rP7 matched that of rP6, was slightly lower than that of 35S in N. benthamiana plants. Further infiltrating rP7 into mulberry leaves, GUS activity analysis showed that the promoter activity of rP7 was the same as that of rP6 and slightly higher than that of 35S. To confirm the promoter activity of rP7 complementary counterpart, the constructed mutant pCA-P7 were infiltrated into the leaves of N. benthamiana and mulberry plants. pCA-P6 was used as a control. The results revealed that the promoter activity of P7 was comparable to that of P6 in either N. benthamiana or mulberry plants. Thus, the 232 nts fragment (named P232) of P7 and rP7 is the minimal sequence required for maintaining bidirectional promoter activity of LIR (Fig. 5 ). The promoter activities of the 232 nts fragment are slightly lower than that of 35S in the V-sense direction and marginally higher than that of 35S in the C-sense direction in mulberry plants. 3.3 MCLV LIR constitutively drives gene expression in plants To determine the promoter types of MCLV LIR, histochemical GUS staining was performed on different tissues and organs of the pCA-P3- and pCA-P6-positive Arabidopsis seedlings. Intense blue staining was observed in roots (Fig. 6 A and E), leaves (Fig. 6 B and F), flower sepals (Fig. 6 C and G) and stems (Fig. 6 D and H) of both the P3- and P6-positive seedlings. In contrast, no staining was observed in those of the negative controls (wild-type plants) (Fig. 6 I, J, K and L). These results indicated that the MCLV LIR is a constitutive promoter. Additionally, the deletion of (GAAAAA) 5 sequence does not change the promoter type of LIR. 3.4 Prediction of cis -acting elements in MCLV LIR and P232 Since both LIR and P232 exhibit bidirectional promoter characteristics and have nearly identical activities, we predicted their transcription start sites (TSS) and the distribution of elements in their V- and C-sense strands, respectively. Putative transcription start sites (TSS) of the V-sense strand of LIR and P232 was predicted to be 'A', and those of the C-sense strand was predicted to be 'T' using the NNPP software. The predicted cis -acting elements by PLACE software were summarized in Table S2 and Table S3, respectively. A total of 121 cis-acting elements were found in both polarity strands of LIR, with 74 in the V-sense and 47 in the C- sense. P232 has 88 cis -acting elements, with 50 in the V-sense and 38 in the C-sense. Compared with P232, the 33 additional elements in LIR are all located downstream of the TSS of their respective polarity strands, indicating that these elements do not contribute to the activity of the bidirectional promoter. The core promoter elements TATA-box and CAAT-box in the C-sense strand of P232 are located at positions − 30 nt and − 108 nt relative to the transcription start site, respectively (Fig. 7 ). However, the TATA-box and CAAT-box of V-sense strand of P232, further upstream of the TSS, are at -188 bp and − 208 bp region relative to the TSS, respectively (Fig. 7 ). Additionally, we identified other putative cis -regulatory elements, such as G-box, P-box, Box-II, ABRE, a root-specific regulatory element ROOTMOTIFTAPOX1 (ATATT). 3.5 Impact of (GAAAAA)₅ on MCLV replication To determine the impact of (GAAAAA)₅ on MCLV replication, infectious clones of (GAAAAA)₅-deleted MCLV vII (pCA-MCLV mLIR ) and wild-type MCLV vII (pCA-1.1MCLV WT ) were transfected into N. benthamiana protoplasts, respectively. qPCR analysis showed that the growth multiple of MCLV in protoplasts transfected with pCA-MCLV mLIR was approximately. 5.1-fold at 24 hpt, which was significantly lower than the 20.1-fold of MCLV growth in protoplasts transfected with pCA1.1MCLV WT . The growth difference between the two was even more pronounced at 48 hpt. The growth fold of MCLV in protoplasts transfected with pCA-MCLV mLIR was only 5.7-fold at 48 hpt, showing almost no growth compared to 24 hpt. In contrast, the growth fold of MCLV in protoplasts transfected with pCA-MCLV WT reached 52.1-fold at 48 hpt, 2.5 times growth compared to 24 hpt. These results indicated that deletion of the (GAAAAA)₅ sequence substantially weakened MCLV DNA replication in protoplasts, confirming its important role in MCLV replication. 4. Discussion The promoter activities of MCLV LIR and the effect of the specific (GAAAAA)₅ sequence within it on both promoter activity and MCLV replication were analyzed in this study. Like other geminiviral LIR (Ashraf et al., 2014 ; Shivaprasad et al., 2005 ; Usharani et al., 2006 ; Zhan et al., 1991 ), MCLV LIR is characterized by a bidirectional promoter, with a higher activity of C-sense than that of V-sense. The (GAAAAA)₅ embedded in V-sense and (TTTTTC) 5 in C-sense LIR are dispensable for the bidirectional promoter activity and promoter type of MCLV LIR, but are very important for MCLV DNA replication. Based on these findings, a minimal promoter (P232) sequence that maintains bidirectional promoter activity of MCLV LIR was identified. P232 consists of 232 nts, including 214 nts (without 30 nts of (GAAAAA)₅) between two predicted transcription start sites (TSS) and 8 nts downstream of the V-sense TSS and 10 nts downstream of the C-sense TSS. In mulberry plants, the C-sense promoter activity of P232 is higher than that of CaMV 35S commonly used in transgenic plant practice, while the V-sense is only slightly lower than that of CaMV 35S. The fact that P232 is shorter than 35S and exhibits bidirectional promoter activity makes it highly valuable for mulberry genetic engineering. Among LIRs of different geminiviruses, promoter activity varies compared to 35S. For example, C-sense LIRs of cotton leaf curl Multan virus (CLCuMV) (Xie et al., 2003 ) and cotton leaf curl Burewala virus (CLCuBuV) (Khan et al., 2015 ) have promoter activity higher than 35S, while that of Tomato mottle Taíno virus (ToMoTV) is lower (Ramos et al., 2004 ). In the case of MCLV, C-sense LIR showed promoter activity higher than 35S only in its natural host, mulberry, and was slightly lower in its experimental host, N. benthamiana , suggesting that promoters drive gene expression differently across hosts. This may be due to differences in the genetic backgrounds of different hosts, leading to transcription factor- cis -acting element interactions with different specificities and intensities in natural and experimental hosts. Therefore, we suggested that the activity of a promoter in the experimental host does not truly reflect its activity. Although P232 is only two-thirds the full-length sequence of LIR, its V- and C-sense strands almost contain all the effective cis -acting elements from the homologous V- and C-sense strands of LIR, respectively. This may explain why P232 can maintain the bidirectional promoter activity of LIR. Conversely, the fact that P232 can maintain the bidirectional promoter activity of LIR suggests that this 232-nt segment may be the factual sequence through which LIR exerts its promoter function. Elements in the LIR sequence flanking P232 are unlikely to be regulatory elements or to affect promoter activity. Similar to LIRs of other geminiviruses, P232 contains not only the core promoter elements but also some common cis -acting elements in both V-sense and C-sense (Argüello-Astorga et al., 1994 ). ABRE (ABA-responsive element) is a cis -acting element involved in abscisic acid reactivity (Hobo et al., 1999 ) and salt stress-related physiological processes in rice (Liu et al., 2022 ). G-box, I-box and BOX-II are light-responsive elements that are key for transcriptional activity in the Arabidopsis rbcS-1A promoter (Donald and Cashmore, 1990 ). A root-specific regulatory element, ROOTMOTIFTAPOX1 (ATATT), was identified in the C- and V-sense of P232, respectively, inferring that the genes it regulates can be expressed in roots. In eukaryotic gene promoters, the core elements TATA box and CAAT box are typically located around the − 30 bp region and − 70 to -120 bp region upstream of the TSS (Singh et al., 2012 ), respectively. For the C-sense strand of P232, the putative TATA box and CAAT box are located at -30 nts and − 108 nts relative to the TSS, respectively, falling within the corresponding regions of these boxes in common eukaryotic promoters. However, in the V-sense strand of P232, the predicted TATA box and CAAT box are located at -188 bp and − 208 bp relative to the TSS, respectively, far from the corresponding regions of these boxes in common eukaryotic promoters. Thus, whether they play role in driving gene expression remains to be clarified. Declarations CRediT authorship contribution statement Q.-Y.L.: conceptualization; J.-J.Y., Y.-H. and W.-K.S.: writing – review and editing; J.-J.Y., M.-S.Z.: methodology; J.-J.Y. and Q.-Y.L.: writing– original draft preparation; J.-J.Y., Z.-J. W. and K.-Q.L.: software. J.-J.Y., Y.-B. H. and L.-L.S.: data curation. Funding This work was supported by a grant from the National Natural Science Foundation of China (32271885). Data Availability Statement Data related to the findings presented in this paper are available from the corresponding authors upon reasonable request. Conflicts of Interest The authors declare no conflicts of interest. References Argüello-Astorga GR, Guevara-González RG, Herrera-Estrella LR, Rivera-Bustamante RF (1994) Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology 203:90–100 Ashraf MA, Shahid AA, Rao AQ, Bajwa KS, Husnain T (2014) Functional characterization of a bidirectional plant promoter from cotton leaf curl burewala virus using an Agrobacterium -mediated transient assay. Viruses 6:223–242 Choi IR, Stenger DC (1996) The strain-specific cis -acting element of beet curly top geminivirus DNA replication maps to the directly repeated motif of the ori. Virology 226:122–126 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 Czosnek H, Laterrot H (1997) A worldwide survey of tomato yellow leaf curl viruses. Arch Virol 142:1391–1406 Dai Z, Wang A (2022) Isolation and transfection of plant mesophyll protoplasts for virology research. Methods Mol Biol 2400:43–53 Donald RG, Cashmore AR (1990) Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter. Embo J 9:1717–1726 Eagle PA, Hanley-Bowdoin L (1997) Cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication. J Virol 71:6947–6955 Fiallo-Olivé E, Lett JM, Martin DP, Roumagnac P, Varsani A, Zerbini FM, Navas-Castillo J 2021. ICTV Virus Taxonomy Profile: Geminiviridae 2021. J Gen Virol 102. Han PY (2025) Studies on the function of V6 and C4 genes encoded by mulberry crinkle leaf virus. Jiangsu University of Science and Technology, Zhenjiang, Jiangsu province, China Han PY, Yin ZN, Yang JJ, Zhang MS, Huang Y, Zhang J, Lu QY (2025) V6 encoded by mulberry crinkle leaf virus is important for viral DNA replication. Virology 603:110392 Han TT, Tang JX, Fang M, Zhang P, Han PY, Yin ZN, Ma Y, Zhang J, Lu QY (2024) Two genes encoded by mulberry crinkle leaf virus (MCLV): The V4 gene enhances viral replication, and the V5 gene is needed for MCLV infection in Nicotiana benthamiana. Virus Res 339:199288 Han TT, Zhang P, Tang JX, Ma Y, Zhong K, Lu QY (2023) Establishment of infectious clone of mulberry crinkle leaf virus vII variant and identification of its pathogenicity. Acta Sericol Sin 49:8–14 Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D (2000) Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit Rev Biochem Mol Biol 35:105–140 Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis -acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300 Hobo T, Asada M, Kowyama Y, Hattori T (1999) ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Plant J 19:679–689 Khan ZA, Abdin MZ, Khan JA (2015) Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus. PLoS ONE 10:e0121656 Liang P, Navarro B, Zhang Z, Wang H, Lu M, Xiao H, Wu Q, Zhou X, Di Serio F, Li S (2015) Identification and characterization of a novel Geminivirus with a monopartite genome infecting apple trees. J Gen Virol 96:2411–2420 Liu H, Cui P, Zhang B, Zhu J, Liu C, Li Q (2022) Binding of the transcription factor MYC2-like to the ABRE of the OsCYP2 promoter enhances salt tolerance in Oryza sativa . PLoS ONE 17:e0276075 Loconsole G, Saldarelli P, Doddapaneni H, Savino V, Martelli GP, Saponari M (2012) Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family geminiviridae. Virology 432:162–172 Lu QY, Ma Y, Smith WK, Yu J, Cheng YY, Zhang P, Han TT (2022) The identification of Tautoneura mori as the vector of mulberry crinkle leaf virus and the infectivity of infectious clones in mulberry. Phytopathology 112:435–440 Lu QY, Wu ZJ, Xia ZS, Xie LH (2015) Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L). Arch Virol 160:2135–2138 Lu QY, Yang L, Huang J, Zheng L, Sun X (2019) Identification and subcellular location of an RNA silencing suppressor encoded by mulberry crinkle leaf virus. Virology 526:45–51 Lu QY, Cheng YJ, Sun YY, Yang X, L (2020) V3 protein encoded by mulberry crinkle leaf virus acts as a pathogenicity determinant in Nicotiana benthamiana . Eur J Plant Pathol 157:141–149 Ma Y, Navarro B, Zhang Z, Lu M, Zhou X, Chi S, Di Serio F, Li S (2015) Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease. JGV 96:2421–2434 Moffat A (1999) Plant pathology - Geminiviruses emerge as serious crop threat. Science 1999,286(5446), 1835 Navas-Castillo J, Fiallo-Olivé E, Sánchez-Campos S (2011) Emerging virus diseases transmitted by whiteflies. Annu Rev Phytopathol 49:219–248 Qiu Y, Zhang S, Yu H, Xuan Z, Yang L, Zhan B, Zerbini M, Cao F, M (2020) Identification and characterization of two novel geminiviruses associated with paper mulberry (Broussonetia papyrifera) leaf curl disease. Plant Dis 104:3010–3018 Ramos PL, Fuentes AD, Quintana Q, Castrillo G, Guevara-González RG, Peral R, Rivera-Bustamante RF, Pujol M (2004) Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus replication-associated protein promoter in transgenic plants. Virus Res 102:125–132 Reese MG (2001) Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 26:51–56 Rizvi I, Choudhury NR, Tuteja N (2015) Insights into the functional characteristics of geminivirus rolling-circle replication initiator protein and its interaction with host factors affecting viral DNA replication. Arch Virol 160:375–387 Rombauts S, Déhais P, Van Montagu M, Rouzé P (1999) PlantCARE, a plant cis -acting regulatory element database. Nucleic Acids Res 27:295–296 Roumagnac P, Lett JM, Fiallo-Olivé E, Navas-Castillo J, Zerbini FM, Martin DP, Varsani A (2022) Establishment of five new genera in the family Geminiviridae: Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus. Arch Virol 167:695–710 Shakir S, Mubin M, Nahid N, Serfraz S, Qureshi MA, Lee TK, Liaqat I, Lee S, Nawaz-Ul-Rehman MS (2023) REPercussions: how geminiviruses recruit host factors for replication. Front Microbiol 14:1224221 Shepherd DN, Martin DP, Van Der Walt E, Dent K, Varsani A, Rybicki EP (2010) Maize streak virus: an old and complex 'emerging' pathogen. Mol Plant Pathol 11:1–12 Shivaprasad PV, Akbergenov R, Trinks D, Rajeswaran R, Veluthambi K, Hohn T, Pooggin MM (2005) Promoters, transcripts, and regulatory proteins of mungbean yellow mosaic geminivirus. J Virol 79:8149–8163 Singh N, Sharma R, George A, Singla SK, Palta P, Manik R, Chauhan MS, Singh D (2012) Cloning and characterization of buffalo NANOG gene: alternative transcription start sites, splicing, and polyadenylation in embryonic stem cell-like cells. DNA Cell Biol 31:721–731 Smith WK, Ma Y, Yu J, Cheng YY, Zhang P, Han TT, Lu QY (2022) Characterization of a strong constitutive promoter from paper mulberry vein banding virus. Arch Virol 167:163–170 Usharani KS, Periasamy M, Malathi VG (2006) Studies on the activity of a bidirectional promoter of mungbean yellow mosaic India virus by agroinfiltration. Virus Res 119:154–162 Wingender E, Dietze P, Karas H, Knüppel R (1996) TRANSFAC: a database on transcription factors and their DNA binding sites. Nucleic Acids Res 24:238–241 Xie J, Wang X, Liu Y, Peng Y, Zhou G (2007) First report of the occurrence of wheat dwarf virus in wheat in China. Plant Dis 91:111 Xie Y, Liu Y, Meng M, Chen L, Zhu Z (2003) Isolation and identification of a super strong plant promoter from cotton leaf curl Multan virus. Plant Mol Biol 53:1–14 Yin ZN (2025) Studies on the function of V2 and C3 genes by mulberry crinkle leaf virus. Jiangsu University of Science and Technology, Zhenjiang, Jiangsu province, China Yin ZN, Han PY, Han TT, Huang Y, Yang JJ, Zhang MS, Fang M, Zhong K, Zhang J, Lu QY (2024) V2 protein enhances the replication of genomic DNA of mulberry crinkle leaf virus. Int J Mol Sci 25 Yu J (2021) Study on the genetic diversity of mulberry crinkle leaf virus. Jiangsu University of Science and Technology, Zhenjiang, Jiangsu province, China Zhan XC, Haley A, Richardson K, Morris B (1991) Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the beta-glucuronidase gene. J Gen Virol 72(Pt 11):2849–2852 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Plant Molecular Biology → Version 1 posted Editorial decision: Revision requested 02 Feb, 2026 Reviews received at journal 13 Jan, 2026 Reviewers agreed at journal 01 Jan, 2026 Reviewers agreed at journal 17 Nov, 2025 Reviewers invited by journal 14 Nov, 2025 Editor assigned by journal 06 Nov, 2025 Submission checks completed at journal 05 Nov, 2025 First submitted to journal 05 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8036347","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550020931,"identity":"c9b39748-fb4e-4349-98ea-2750427b4177","order_by":0,"name":"JingJing Yang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"JingJing","middleName":"","lastName":"Yang","suffix":""},{"id":550020932,"identity":"3a677287-a91d-4b43-a5ee-b39e76db709a","order_by":1,"name":"Ying Huang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Huang","suffix":""},{"id":550020933,"identity":"b0ece780-7867-4e24-bca7-433aee6b1833","order_by":2,"name":"MengSi Zhang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"MengSi","middleName":"","lastName":"Zhang","suffix":""},{"id":550020934,"identity":"fb38ba87-13bf-426d-8202-a90d214b8d36","order_by":3,"name":"William Kojo Smith","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"Kojo","lastName":"Smith","suffix":""},{"id":550020935,"identity":"c765e56f-6046-4cb4-b622-0b1f8cccd5b1","order_by":4,"name":"ZiJun Wang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"ZiJun","middleName":"","lastName":"Wang","suffix":""},{"id":550020936,"identity":"42d571ec-0885-491e-8ce7-d3cc5b3667a6","order_by":5,"name":"KeQing Liu","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"KeQing","middleName":"","lastName":"Liu","suffix":""},{"id":550020937,"identity":"098eebee-d0d6-4447-9e7c-3f245a8daade","order_by":6,"name":"YiBo He","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"YiBo","middleName":"","lastName":"He","suffix":""},{"id":550020938,"identity":"a3f7bc2f-76dc-412d-b9cf-1b73e2dc6f89","order_by":7,"name":"LeiLei Shi","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"LeiLei","middleName":"","lastName":"Shi","suffix":""},{"id":550020939,"identity":"ea023c16-6f12-4e2b-911f-45eb7177577c","order_by":8,"name":"QuanYou Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYFACxsYDQFKOjb39ANFaGkBKjfl4ziQQbw9IS+I8CQcD4pTLzz7ccJin7HB6mwRDAsOPim2EtRicSwRqOXc4t0268QBjz5nbRGjhYWw4zNsG1CJzIIGZsY0ILfI9EC3pbBIJBsRpYTgD0ZJAvBYDoJaDc86lG7YBA/kgUX6R72F/+OBNmbW8fHv7wQc/KohxGBiwNYOpA8SqB2mpI0HxKBgFo2AUjDgAAB+APhFPNxOvAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"QuanYou","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-11-05 08:53:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8036347/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8036347/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11103-026-01702-0","type":"published","date":"2026-04-23T15:59:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":97219919,"identity":"eed0a688-f82e-4db0-b6a4-982fa1283be4","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52610,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/43f9cab94bb369d5d4dfc7e1.png"},{"id":97219938,"identity":"c21bdb6f-d7bc-485d-8253-7c6e9ba9b588","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15357942,"visible":true,"origin":"","legend":"","description":"","filename":"AnalysisofbidirectionalpromoteractivityandstructureofthelargeintergenicregionLIRinmulberrycrinkleleafvirus.docx","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/39cc9fbb230b029e1bf9ec2e.docx"},{"id":97219920,"identity":"67333bc5-0019-449a-ac85-9526924d5aab","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20862,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/541c03dae44d411d8c7fce82.png"},{"id":97249742,"identity":"ff615ffa-f8c2-4553-a64b-480ce56f9621","added_by":"auto","created_at":"2025-12-02 13:13:18","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120775,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3A.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/91445a058f3474693df6b30b.png"},{"id":97251397,"identity":"27620810-26c6-4435-a2a5-b888dfd8d254","added_by":"auto","created_at":"2025-12-02 13:17:28","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138275,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3B.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/7c38ae4154851d961527cdb3.png"},{"id":97219927,"identity":"6a8e0e0c-8a09-44e9-a746-e18275dd5a67","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123058,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4A.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/b57438cad8b17769cd1c926a.png"},{"id":97250455,"identity":"7be42444-ba89-4f32-bd51-3891a3ca30c5","added_by":"auto","created_at":"2025-12-02 13:14:33","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113443,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4B.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/f506ad0a3ebf14bcd35676ea.png"},{"id":97251153,"identity":"4460da3a-adbc-4d75-8724-cf8f48ba97c3","added_by":"auto","created_at":"2025-12-02 13:16:14","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15112305,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/9d16dc94c7b604a16fa7c7d5.png"},{"id":97219933,"identity":"555168a2-de2f-4d19-afbe-1b8ad3bfab8f","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22242,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/581b3b340c25b7539d7be878.png"},{"id":97219943,"identity":"7aa0583e-6130-4b17-a75a-6a0af5f9d88b","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95373,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/5407b255504e0786499ddb54.png"},{"id":97219931,"identity":"4bcffa01-6fd9-4c56-9778-bf7bacaf5a06","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"json","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9337,"visible":true,"origin":"","legend":"","description":"","filename":"fbd9315fda0b4edf85462bdfac0fdbdd.json","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/178c57bace5c31efb9cd0b46.json"},{"id":97250275,"identity":"748646f9-c912-4288-b172-5ba2476d86c6","added_by":"auto","created_at":"2025-12-02 13:14:12","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":573695,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/8796b063e35bf080ed266c47.docx"},{"id":97219942,"identity":"349e5e4f-4ee5-4c1a-9ce2-342a88398647","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116685,"visible":true,"origin":"","legend":"","description":"","filename":"fbd9315fda0b4edf85462bdfac0fdbdd1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/552814c9dbff8734985e2147.xml"},{"id":97219936,"identity":"58238c3b-7e88-4347-87de-8a57cb310ae9","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52610,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/519a5b72f9b83b1af996633b.png"},{"id":97251105,"identity":"313e8b5a-e4ca-426b-86a4-36e7ad6a3b25","added_by":"auto","created_at":"2025-12-02 13:16:05","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20862,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/05e5d993166b40a4203862fe.png"},{"id":97250963,"identity":"b49b736b-5546-4458-992d-b8820a377b4f","added_by":"auto","created_at":"2025-12-02 13:15:42","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120775,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3A.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/3f24c32aaed0a6fdad2789be.png"},{"id":97219939,"identity":"45f6ca76-b7fd-4451-8ba5-cb33c7d7a7c0","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138275,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3B.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/ef41af3a8157c326dd40ca20.png"},{"id":97219945,"identity":"be8bcf24-bfc4-4ec6-8847-e11def521440","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123058,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4A.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/ff9e495467328b197ba281b2.png"},{"id":97251285,"identity":"c97a180d-61f2-493f-bcfc-48a73a647b22","added_by":"auto","created_at":"2025-12-02 13:16:40","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113443,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4B.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/9be7328602a9f3687fe2dccf.png"},{"id":97219954,"identity":"697677b9-276e-4898-ae3e-d0221ad74d7e","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15112305,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/750ec76fa7cb4119e9e13812.png"},{"id":97219953,"identity":"d3e2978e-3fbd-4f6a-92c8-aba3dc0bc7ac","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22242,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/183554c30c694db6c8f29480.png"},{"id":97250744,"identity":"48d37e91-6c3c-4807-947e-5c85797c46ca","added_by":"auto","created_at":"2025-12-02 13:15:09","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95373,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/9a1caa8e2d2157eb9c486212.png"},{"id":97251405,"identity":"625349d4-1820-4e96-9ac1-fc5ac6f032f8","added_by":"auto","created_at":"2025-12-02 13:17:35","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52610,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/2dd6531054b077deb846ef28.png"},{"id":97219950,"identity":"e2542c10-d4de-4cfc-8b94-c73580a39f32","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29892,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/5fb3baa7b0cb96ce39ff0ff5.png"},{"id":97249780,"identity":"7361523e-20d7-40bf-b0ae-818c02c5d607","added_by":"auto","created_at":"2025-12-02 13:13:25","extension":"jpeg","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184058,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/822dc68c645e42e984a40ef9.jpeg"},{"id":97219948,"identity":"9825696d-e02e-4c94-a6a5-2f750ff092d4","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"jpeg","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185259,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/5da26b420a5230524bb606fb.jpeg"},{"id":97219969,"identity":"c31b4f8b-1867-4046-9179-276e1bcd18f2","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15112305,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/3d165560215a2b64d62be0ee.png"},{"id":97219946,"identity":"7a901ca2-abb9-4e60-a4fd-08ae9d039c27","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22937,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/efaef4c8cd2588300c35eb3e.png"},{"id":97219951,"identity":"5cc22554-00c0-4a4c-8420-ac215db89e40","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"emf","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16524,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.emf","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/cd9940f03277ed0383af5e43.emf"},{"id":97219957,"identity":"a7d52f51-a310-4abd-a4e9-c8189f93a3a8","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25528,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/63d47de2ba9beab588b4830e.png"},{"id":97219956,"identity":"0bdb8377-8c20-4e29-b0fa-721a3e55d126","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12284,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/fa9d8e97ae70416a085ce8e7.png"},{"id":97249815,"identity":"62d0a13f-947f-48b3-a149-31354648a56b","added_by":"auto","created_at":"2025-12-02 13:13:29","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55063,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3A.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/e09bc73b106fc434cf575aa6.png"},{"id":97250724,"identity":"c13e7698-a42a-4b2e-8575-b86ec2c081f0","added_by":"auto","created_at":"2025-12-02 13:15:06","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58220,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3B.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/b53b4aea759845dbcfae6707.png"},{"id":97219955,"identity":"d85bf8ec-48aa-40d4-b08f-fa9b878ad863","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55991,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4A.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/032b652507d47b4e55e3757b.png"},{"id":97251290,"identity":"acbc799b-0b5d-4996-ac60-70f85fb0a7da","added_by":"auto","created_at":"2025-12-02 13:16:41","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52651,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4B.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/20e48c69466c4d8cce2ecf96.png"},{"id":97219965,"identity":"41c96301-cae0-43b9-9499-6fda5a08c61b","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1058120,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/0b19317bb7d18d347765d8bb.png"},{"id":97251401,"identity":"8ea68886-1762-4ed1-b50c-d9372dafe6f2","added_by":"auto","created_at":"2025-12-02 13:17:30","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10468,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/869f3141b73fb3a410fe7fa3.png"},{"id":97249694,"identity":"90fb009a-4a3a-4f50-8ef9-c5d12ed8a1e4","added_by":"auto","created_at":"2025-12-02 13:13:16","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45799,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/63445777cbdf0d9b08df85ea.png"},{"id":97219962,"identity":"f824c4cc-a2cd-4f05-a45f-91df451b9cf3","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25528,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/9794e1805c8b1af0f05169da.png"},{"id":97250360,"identity":"5672cefb-bf3b-4245-a73f-ff7d1963ed75","added_by":"auto","created_at":"2025-12-02 13:14:22","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12284,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/fab38a3832243c245633e503.png"},{"id":97251272,"identity":"9a3156d4-d597-404b-a2a0-48b9d2d3a1cd","added_by":"auto","created_at":"2025-12-02 13:16:39","extension":"png","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39203,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/a445c91b24a2b42cbf850f1f.png"},{"id":97219961,"identity":"4b0deca0-5757-4b97-b628-49d2097f9722","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37576,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/059b8ec848f4a41f7d150320.png"},{"id":97219963,"identity":"662faaef-48fb-4bca-9b95-0b0df636b6d4","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1058120,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/6e7c996bb46a9d5ba6aefc95.png"},{"id":97219967,"identity":"122b374d-f883-4cbf-9455-d92470247d46","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10468,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/65f63c1d14a9522347348732.png"},{"id":97219973,"identity":"16d3a47c-60f2-4c9a-8a0d-84780a9d416f","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"png","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42415,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/3f753acef33de1d553ca2747.png"},{"id":97251130,"identity":"94f048a6-1fbb-42ad-841c-d2d9311817a2","added_by":"auto","created_at":"2025-12-02 13:16:09","extension":"xml","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114434,"visible":true,"origin":"","legend":"","description":"","filename":"fbd9315fda0b4edf85462bdfac0fdbdd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/62e9ead6490cb51c1c893c2e.xml"},{"id":97219966,"identity":"e17c3198-4f60-4869-8d5b-bec39927d053","added_by":"auto","created_at":"2025-12-02 07:18:24","extension":"html","order_by":46,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124257,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/85ffbc58f6b7493a49aec91f.html"},{"id":97249735,"identity":"be229c1f-6b38-4918-b8aa-737a5a637509","added_by":"auto","created_at":"2025-12-02 13:13:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132389,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic organization of two MCLV variants, MCLV (A) and MCLV vII (B). LIR, large intergenic region; SIR, short intergenic region; MP, movement protein; CP, coat protein; Rep, replicase; RepA, replication-associated protein.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/9eb55a684abd40eb2c51df6b.png"},{"id":97251282,"identity":"59a395c0-7982-4ec8-a691-ac1a5d765e6d","added_by":"auto","created_at":"2025-12-02 13:16:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118096,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of MCLV LIR and its various length mutans used for recombinant plasmid construct. P3 and rP3 represent the full-length sequence of V- and C-sense of LIR, respectively. The deletion of (GAAAAA)\u003csub\u003e5\u003c/sub\u003e in the V-sense and (TTTTTC)\u003csub\u003e5\u003c/sub\u003e in the C-sense forming truncated mutants P6 and rP6, respectively. Deletion of 34 and 89 nts at the 3'-end of rp6 forming rP8 and rP4, respectively. Based on the predicted transcription start site (TSS) of two orientations of rP6, rP7 was formed by deletion nucleotides after +11 nts of rP6 and after +9 nts of its C-sense forms. The reverse complement of rP7 was named P7.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/d11ebae7ba8428ddca324dc6.png"},{"id":97219922,"identity":"c4e6351a-d2e0-4800-9d75-9638c559a84d","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":667654,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of mean GUS activity in leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e (A) and mulberry plants (B) infiltrated with pCA-P3, pCA-rP3, pCA-P6, pCA- rP6 and p35S-GUS. Promoter activity was estimated from the measured GUS fluorescence in the different samples. EV: empty vector. Data are the means of three independent biological replicates. Error bars represent the mean ± standard deviation (SD). Analysis of significant difference (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) was carried out using the LSD multiple comparison test. Bars with the same letter are not significantly different according to the LSD’s multiple comparison test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/943a731cc121bd661100268b.png"},{"id":97250010,"identity":"fab7004f-d730-498e-9e45-f75969124cec","added_by":"auto","created_at":"2025-12-02 13:13:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":601280,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of mean GUS activity in leaves of\u003cem\u003e N. benthamiana\u003c/em\u003e (A) and mulberry plants (B) infiltrated with pCA-rP6, pCA-rP7, pCA-P6, pCA-P7, pCA-rP8, pCA-rP4 and p35S-GUS Promoter activity was estimated from the measured GUS fluorescence in the different samples. EV: empty vector. Data are the means of three independent biological replicates. Error bars represent the mean ± standard deviation (SD). Analysis of significant difference (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) was carried out using the LSD multiple comparison test. Bars with the same letter are not significantly different according to the LSD multiple comparison test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/22a5784acf9eee61ba6e0aaf.png"},{"id":97219924,"identity":"6dc461a8-86c2-4ca6-9413-9a1b8916564b","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1315064,"visible":true,"origin":"","legend":"\u003cp\u003eHistochemical assessment of GUS expression of different tissues in transgenic Arabidopsis plants transformed with pCA-P3 (A, B, C and D), pCA-P6 (E, F, G and H). \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003ethaliana \u003c/em\u003eColumbia (Col-0) was used as negative control (I, J, K and L). Different tissues were incubated overnight in GUS staining at 37℃. Staining was visualized by a microscope.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/8256f1c86cd4e3b3dbdef842.png"},{"id":97251187,"identity":"b81a5110-f241-48c0-9aff-96086710709f","added_by":"auto","created_at":"2025-12-02 13:16:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46617,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide sequence, putative core elements TATA-box and CAAT-box, and transcription start sites (TSS) in virion- (V-) and complementary- (C-) sense of the directional promoter P232. The predicted TATA-box and CAAT-box in the V- and C-sense are highlighted with shaded backgrounds and boxes, respectively. The bigger italic letters and the solid arrows on the top of them indicate the predicted TSS in V- and C-sense direction. The two hollow arrows pointing left and right indicate C-sense V-sense of P232, respectively.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/aa867901f32b214171e574b9.png"},{"id":97250627,"identity":"1a4fd5fd-e4fa-456d-a0cc-6ab860f6065b","added_by":"auto","created_at":"2025-12-02 13:14:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":302915,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR analysis of MCLV replication in protoplasts transfected with pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e and pCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e. The growth multiple was calculated using the formula of the copy number at 24 h/0 h and 48 h/0 h post-transfection in different samples. The data are the means of three independent biological replicates. Error bars represent the standard deviation (SD). Asterisks indicate a statistically significant difference according to the unpaired Student’s t-test (two-tailed), **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/c00f6be9859e23812c1962ed.png"},{"id":107928020,"identity":"f25ec271-b2e6-4c6a-a50d-a51534eca6cc","added_by":"auto","created_at":"2026-04-27 16:06:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3497171,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/b607ea7b-d14f-401c-a5ba-ed1b7280aafa.pdf"},{"id":97219930,"identity":"49ac215b-1ded-4d56-8c4d-f9d1d497f1af","added_by":"auto","created_at":"2025-12-02 07:18:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":573695,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8036347/v1/636d109201e3c57db7f7a05c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of bidirectional promoter activity and structure of the large intergenic region (LIR) in mulberry crinkle leaf virus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGeminiviruses are a class of economically important DNA viruses that infect a wide range of plants. Their genomes consist of single-component or two-component circular single-stranded DNA molecules ranging in size from 2.5 to 5.2 kb (Fiallo-Oliv\u0026eacute; et al., 2021). More than 700 geminiviral species reported to date were classified into 14 genera established by the International Committee on Taxonomy of Viruses (ICTV): \u003cem\u003eBecurtovirus\u003c/em\u003e, \u003cem\u003eBegomovirus\u003c/em\u003e, \u003cem\u003eCapulavirus\u003c/em\u003e, \u003cem\u003eCitlodavirus\u003c/em\u003e, \u003cem\u003eCurtovirus\u003c/em\u003e, \u003cem\u003eEragrovirus\u003c/em\u003e, \u003cem\u003eGrablovirus\u003c/em\u003e, \u003cem\u003eMaldovirus\u003c/em\u003e, \u003cem\u003eMastrevirus\u003c/em\u003e, \u003cem\u003eMulcrilevirus\u003c/em\u003e, \u003cem\u003eOpunvirus\u003c/em\u003e, \u003cem\u003eTopocuvirus\u003c/em\u003e, \u003cem\u003eTopilevirus\u003c/em\u003e, and \u003cem\u003eTurncurtovirus\u003c/em\u003e (Fiallo-Oliv\u0026eacute; et al., 2021; Roumagnac et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Geminiviruses can infect various plants including economically important crops, such as wheat (Xie et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), maize (Shepherd et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and tomato (Czosnek and Laterrot, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), as well as fruit trees and woody plants, such as citrus (Loconsole et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), apple (Liang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), mulberry (Lu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and paper mulberry (Qiu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Their infection not only impairs crop growth and reduces yield but also compromises product quality, thereby significantly undermining agricultural productivity and economic development (Moffat, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Navas-Castillo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to several genes encoding proteins associated with viral replication, movement, virion packaging, and pathogenicity, the genomes of all reported geminiviruses also contain a large intergenic region (LIR) that lies between the complementary-sense (C-sense) and virion-sense (V-sense) strands. LIRs contain the origin (v\u003cem\u003e-ori\u003c/em\u003e) required for the replication of geminiviral genomes, which locates in the putative stem-loop with nonanucleotide motif 5\u0026prime;-TAAT(G)ATT\u0026darr;A(C)C-3\u0026prime; (the arrow indicates the location of the DNA origin of replication). The v\u003cem\u003e-ori\u003c/em\u003e serves as the recognition and nicking site for the viral replication-associated protein (Rep) to initiate rolling-circle replication (RCR) (Rizvi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, both V-sense and C-sense sequences of LIR contain several of \u003cem\u003ecis\u003c/em\u003e-acting elements that maintain promoter activity, such as the core promoter elements TATA box and CAAT box, as well as elements related to light, biotic and abiotic stress response, and tissue-specific elements (Eagle and Hanley-Bowdoin, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Structural and sequence properties of geminiviral LIRs determine their importance for viral replication and bidirectional transcription (Ashraf et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Choi and Stenger, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Hanley-Bowdoin et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMulberry crinkle leaf virus (MCLV), identified from mulberry plants, is a representative species of the genus \u003cem\u003eMulcrilevirus\u003c/em\u003e in the family \u003cem\u003eGeminiviridae\u003c/em\u003e (Lu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ma et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Roumagnac et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). MCLV is primarily transmitted by leafhoppers and the infected vegetative propagation materials (Lu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yu, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There are two MCLV variants (MCLV and MCLV vII) present in naturally infected mulberry plants (Yu, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and neither of these two variants induces conspicuous symptoms on leaves of the infected mulberry plants (Han et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). MCLV genome encodes nine to ten proteins (5\u0026ndash;6 proteins on the virion-sense strand, 4 proteins on the complementary-sense strand) depending on different variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). The functions of these proteins have been determined. V1 is presumed to be a coat protein (CP) (Lu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). V2 (Yin et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), V4 (Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and V6 (Han et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) function to enhance viral genome replication. V3 is a putative movement protein (MP), as well as a post-transcriptional gene silencing suppressor (Lu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and a pathogenicity determinant (Lu, 2020). V5, specifically encoded by MCLV vII, is essential for MCLV infection in \u003cem\u003eN. benthamiana\u003c/em\u003e (Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The complementary-sense strand contains four ORFs, which encode C1-C4 proteins. C1 and C2 are proteins involved in virus DNA replication (Lu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shakir et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). C3 and C4 function both as pathogenicity determinants and suppressors of post-transcriptional gene silencing (Han, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yin, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLike other geminiviruses, the MCLV genome contains an LIR, which is located between V1 and C1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). However, whether MCLV LIR also exhibits bidirectional promoter activity, like other geminiviral LIR, has yet to be determined. Notably, the MCLV LIR contains a unique GAAAAA tandem repeat sequence. Although the number of GAAAAA repeats varies between five and eleven depending on different MCLV haplotypes, most MCLV LIRs contain five GAAAAA repeats [(GAAAAA)\u003csub\u003e5\u003c/sub\u003e] (Yu, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). BLAST analysis showed that the (GAAAAA)\u003csub\u003e5\u003c/sub\u003e does not exist in any sequences deposited in GenBank. However, whether this special sequence affects the functions of MCLV LIR has not yet been clarified.\u003c/p\u003e\u003cp\u003eHere, we confirmed that MCLV LIR, similar to other geminiviral LIRs, has bidirectional promoter activity by constructing transient expression vectors. The deletion of (GAAAAA)\u003csub\u003e5\u003c/sub\u003e did not affect bidirectional promoter activity but inhibited MCLV DNA replication. Based on these findings, a bidirectional promoter suitable for mulberry transgenic research was determined.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Preparation of materials\u003c/h2\u003e\u003cp\u003e\u003cem\u003eNicotiana benthamiana\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e Columbia (Col-0) and mulberry plants used in this study were cultivated in environmentally-controlled rooms with a 16/8-hour light/dark cycle at 25 ˚C, 50\u0026ndash;60% relative humidity. An infectious clone of the wild-type MCLV vII (pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e), which contains a duplicated LIR and one copy of the sequence flanking the LIR of the MCLV\u003csup\u003eWT\u003c/sup\u003e genome, was constructed by Han et al (Han et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The previously constructed pCAMBIA1391z-CaMV-35S (p35S-GUS) was used as a positive control (Smith et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The plant expression vector pCAMBIA2300 was obtained from Dr. Nan Chao from Jiangsu University of Science and Technology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Synthesis of primers\u003c/h2\u003e\u003cp\u003eThe primers used in this study were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and were synthesized by GenScript Biotech Corporation (Nanjing, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2 DNA extraction\u003c/h2\u003e\u003cp\u003eDNA was extracted from plant tissues and \u003cem\u003eN. benthamiana\u003c/em\u003e protoplasts using the Ezup Spin Column Plant Genomic DNA Extraction Kit (Sangon, Shanghai, China) following the manufacturer\u0026prime;s protocol. DNA from 0.1 g of leaf tissues was resuspended in 20 \u0026micro;L of double-distilled (dd) water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Construction of transient expression vectors\u003c/h2\u003e\u003cp\u003eTo evaluate the bidirectional promoter activity of the MCLV LIR, both virion-sense (P3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and complementary-sense (rP3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) sequences of the complete LIR were amplified via PCR using the pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e plasmid as template and primers PF / PR and rPF / rPR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively. To assess the impact of (GAAAAA)₅ on bidirectional promoter activity of MCLV LIR, the (GAAAAA)₅-deleted V-sense LIR sequence (P6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and (TTTTTC)\u003csub\u003e5\u003c/sub\u003e-deleted C-sense LIR sequence (rP6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were amplified by overlapping PCR using pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e plasmid as templates. Firstly, fragments 4 and 1 of P6 and rP6 were PCR-amplified using primers PF / P\u003csub\u003e4\u0026thinsp;+\u0026thinsp;1\u003c/sub\u003eR and P\u003csub\u003e4\u0026thinsp;+\u0026thinsp;1\u003c/sub\u003eF / PR (Table\u0026nbsp;1), and rPF / rP\u003csub\u003e4\u0026thinsp;+\u0026thinsp;1\u003c/sub\u003eR and rP\u003csub\u003e4\u0026thinsp;+\u0026thinsp;1\u003c/sub\u003eF / rPR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively. Then, fragments 4 and 1 of each P6 and rP6 were linked via overlapping-PCR using the respective primer pairs PF/PR and rPF / rPR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) to form P6 and rP6.\u003c/p\u003e\u003cp\u003eBased on the obtained results in this study, where the promoter activity of C-sense LIR was higher than that of V-sense and the deletion of (GAAAAA)\u003csub\u003e5\u003c/sub\u003e in the V-sense and (TTTTTC)\u003csub\u003e5\u003c/sub\u003e in the C-sense did not affect the bidirectional promoter activity of LIR, we amplified three rP6-truncated sequences, rP4, rP8 and rP7, and one P6-truncated sequence, P7, to confirm the minimal sequence maintaining bidirectional promoter activity of MCLV LIR. rP4 only contains 232 nts of rP6 5\u0026rsquo;-end, constructed by deleting 89 nts (including the predicted TSS of rP6) of rP6 3'-end (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). rP8 contains 282 nts of rP6 5\u0026rsquo;-end constructed by deleting 34 nts (including the predicted TSS of rP6) at the 3' end of rP6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). rP7 totally includes 232 nts, including 214 nucleotides between the rP6 and P6 TSS, as well as 10 nts downstream of the predicted rP6 TSS and 8 nts downstream of the predicted P6 TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). P7 is the complementary sequence of rP7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). rP4, rP8, and rP7 were PCR-amplified using the rP6 plasmid as template and the LA PCR\u0026trade; Kit (TaKaRa, Beijing, China), and primer pairs rPF4 / rPR, rPF1 / rPR, and rPF2 / rPR2 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively. P7 was PCR-amplified using the P6 plasmid as a template and primer pairs PF2 / PR2 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmplicons (P3, rP3, P6, rP6, rP7, P7, rP4, rP8) were separated by 1% agarose gel electrophoresis in 1\u0026times;TAE buffer. The target bands were excised and purified using the SanPrep Column DNA Gel Extraction Kit (Sangon). The obtained fragments were ligated into the pMD-19T vector (TaKaRa). The ligation products were transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α competent cells. The positive clones were verified through sequencing (SUNYA, Hangzhou, China). The recombinant plasmids extracted from each positive clone were digested with \u003cem\u003eBamH\u003c/em\u003e I and \u003cem\u003eHind\u003c/em\u003e III. The target fragments were ligated into \u003cem\u003eBamH\u003c/em\u003e I/\u003cem\u003eHind\u003c/em\u003e III-linearized pCAMBIA1391z vector using T4 DNA ligase (TaKaRa). The resulting products were transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α competent cells. The positive clones were confirmed by colony-PCR using corresponding primers. Plasmids were extracted from each positive clone to obtain pCA-P3, pCA-P6, pCA-rP3, pCA-rP6, pCA-rP7, pCA-P7, pCA-rP4 and pCA-rP8.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Construction of infectious clones\u003c/h2\u003e\u003cp\u003eTo investigate the role of the (GAAAAA)\u003csub\u003e5\u003c/sub\u003e in LIR on viral replication, an MCLV infectious clone lacking the sequence, MCLV\u003csup\u003emLIR\u003c/sup\u003e was constructed as previously described (Han et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Briefly, four fragments (1, 2, 3, 4) were amplified via PCR using the pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e plasmid as templates (Han et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and primer pairs MCLV-5'F/R1, F1/R2, F2/R3, and F3/MCLV-3'R (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively. Subsequently, fragments 1 and 2 were joined by overlap-PCR using primer pairs MCLV-5'F/R2 (Tables S1) to form fragment 5, and fragments 3 and 4 were fused with primer pairs F2/MCLV-3'R (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) to produce fragment 6. Fragments 5 and 6 were separated by 1% agarose gel electrophoresis, respectively. The two target fragments were reclaimed using a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China) and were ligated into \u003cem\u003eSal\u003c/em\u003e I-linearized pCAMBIA2300 using the ClonExpress MultiS One Step cloning kit (Vazyme, Nanjing, China). The resulting construct was transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α, verified by sequencing (SUNYA, Hangzhou, China). The positive clones were confirmed by colony-PCR using primer pairs MCLV-5'F/ MCLV-3'R (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Plasmid was extracted from each positive clone to obtain pCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.5 \u003cem\u003eAgrobacterium\u003c/em\u003e transformation and inocula preparation\u003c/h2\u003e\u003cp\u003ePlasmids (pCA-P3, pCA-P6, pCA-rP3, pCA-rP6, pCA-rP6, pCA-rP7, pCA-P7, pCA-rP4, pCA-rP8, and PCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e) were transformed into Agrobacterium EHA105 competent cells by electroporation via Gene Pulser Xcell System (Bio-Rad, Hercules, CA, USA). Inocula were prepared as previously described (Han et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.6 GUS fluorometric assay\u003c/h2\u003e\u003cp\u003eGUS fluorometric assays were performed as described previously (Smith et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, Agrobacteria containing the constructs pCA-P3, pCA-P6, pCA-rP3, pCA-rP6, pCA-rP6, pCA-rP7, pCA-P7, pCA-rP4, pCA-rP8, p35S-GUS (positive control), and empty pCAMBIA1391z vector (negative control) were infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The infiltrated leaf patches were collected 72 hours post-infiltration (hpi) and the crude lysate was extracted. GUS activity was qualitatively analyzed by measuring fluorescence of reaction products between the crude lysate and GUS substrate MUG (4-methylumbelliferyl-β-D-glucuronide) (Smith et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Promoter activity is indicated by GUS activity. Tests were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.7 \u003cem\u003eArabidopsis\u003c/em\u003e transformation\u003c/h2\u003e\u003cp\u003e\u003cem\u003eArabidopsis\u003c/em\u003e plants were transformed via floral dip as previously described(Clough and Bent, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Smith et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Histochemical GUS staining\u003c/h2\u003e\u003cp\u003eHistochemical staining was performed according to the previously described protocol (Smith et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, Transgenic seedlings were incubated in GUS staining at 37℃ overnight. Chlorophyll was cleared with 70% ethanol, and staining patterns were visualized under a Leica EZ4 HD stereomicroscope (Leica Microsystems, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Promoter element prediction\u003c/h2\u003e\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e-regulatory elements in the MCLV LIR bidirectional promoter were analyzed using Plant CARE (Rombauts et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), PLACE (Higo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), and TRANSFAC databases (Wingender et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Transcription start sites (TSS) were predicted via the NNPP server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fruitfly.org/seq_tools/promoter.html\u003c/span\u003e\u003cspan address=\"https://www.fruitfly.org/seq_tools/promoter.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Reese, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Protoplast isolation and transfection\u003c/h2\u003e\u003cp\u003e\u003cem\u003eN. benthamiana\u003c/em\u003e protoplasts were isolated, and transfected with plasmid DNA as previously described (Dai and Wang, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Real-time quantitative PCR (qPCR) analysis\u003c/h2\u003e\u003cp\u003eDNAs extracted from pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e- and pCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e-transinfected protoplasts at different time points were RNase A-digested and then adjusted to the same concentration. qPCR was performed with the primer pairs qMCLV F1/ qMCLV R1 previously designed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) (Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), RNase A-digested DNA, and 2\u0026times; NovoStartR SYBR qPCR Super Mix Plus Kit (Novoprotein, Shanghai, China) according to the manufacturer's instructions. The standard curve used here was previously established with y\u0026thinsp;=\u0026thinsp;3.2722x 37.076 (Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The relative growth multiple of the progeny MCLV was calculated as previously designed (Han et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), via, the gene copy number at 24 hours post-transformation (hpt) and 48 hpt divided by the gene copy number at 0 hpt. Three biological replicates for each treatment and three technical replicates per biological replicate were used for all qPCR tests.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Verification of MCLV LIR bidirectional promoter activity\u003c/h2\u003e\u003cp\u003eTo verify where the MCLV LIR exhibited the bidirectional promoter activity similar to other geminiviral LIRs, the constructed pCA-P3 and pCA-rP3 were infiltrated into leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e and mulberry plants. GUS activity analysis showed that the promoter activities of P3 andrP3 were significantly higher than those of the negative controls, suggesting that MCLV LIR possesses bidirectional activity. The promoter activity of rP3 was significantly higher than that of P3 in \u003cem\u003eN. benthamiana\u003c/em\u003e, but slightly higher in the mulberry plant, indicating the promoter activity of C-sense LIR is consistently higher than that of V-sense. In \u003cem\u003eN. benthamiana\u003c/em\u003e plants, the promoter activity of rP3 was slightly lower than that of 35S, while that of P3 was significantly lower than that of 35S. In contrast, in mulberry plants, the promoter activity of rP3 was slightly higher, and that of P3 was marginally lower than 35S.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 (GAAAAA)₅ and its complementary counterpart do not contribute to the bidirectional promoter activity of LIR\u003c/h2\u003e\u003cp\u003eTo determine the influence of (GAAAAA)\u003csub\u003e5\u003c/sub\u003e within V-sense and (TTTTTC)\u003csub\u003e5\u003c/sub\u003e in C-sense of LIR on this bidirectional promoter activity, the constructed deleted-mutants pCA-P6 and pCA-rP6 were infiltrated into leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e and mulberry plants. pCA-P3 and pCA-rP3 were used as controls. GUS activity analysis showed that the promoter activities of P6 and rP6 were the same as those of their corresponding counterparts, P3 and rP3, in \u003cem\u003eN. benthamiana\u003c/em\u003e and mulberry plants. These results indicated that (GAAAAA)₅ and (TTTTTC)\u003csub\u003e5\u003c/sub\u003e do not affect bidirectional promoter activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Minimal sequence maintaining bidirectional promoter activity of LIR\u003c/h2\u003e\u003cp\u003eTo confirm the minimal sequence maintaining bidirectional promoter activity of LIR, three constructed rP6-truncated mutants (pCA-rP7, pCA-rP4, and pCA-rP8) were infiltrated into leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e plants. rP6 was used as a control. GUS activity analysis revealed that, although the promoter activities of rP4 and rP8 were significantly higher than those of the negative control, it is significantly lower than that of rP6. In contrast, the promoter activity of rP7 matched that of rP6, was slightly lower than that of 35S in \u003cem\u003eN. benthamiana\u003c/em\u003e plants. Further infiltrating rP7 into mulberry leaves, GUS activity analysis showed that the promoter activity of rP7 was the same as that of rP6 and slightly higher than that of 35S.\u003c/p\u003e\u003cp\u003eTo confirm the promoter activity of rP7 complementary counterpart, the constructed mutant pCA-P7 were infiltrated into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e and mulberry plants. pCA-P6 was used as a control. The results revealed that the promoter activity of P7 was comparable to that of P6 in either \u003cem\u003eN. benthamiana\u003c/em\u003e or mulberry plants.\u003c/p\u003e\u003cp\u003eThus, the 232 nts fragment (named P232) of P7 and rP7 is the minimal sequence required for maintaining bidirectional promoter activity of LIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The promoter activities of the 232 nts fragment are slightly lower than that of 35S in the V-sense direction and marginally higher than that of 35S in the C-sense direction in mulberry plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 MCLV LIR constitutively drives gene expression in plants\u003c/h2\u003e\u003cp\u003eTo determine the promoter types of MCLV LIR, histochemical GUS staining was performed on different tissues and organs of the pCA-P3- and pCA-P6-positive \u003cem\u003eArabidopsis\u003c/em\u003e seedlings. Intense blue staining was observed in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and E), leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and F), flower sepals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and G) and stems (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and H) of both the P3- and P6-positive seedlings. In contrast, no staining was observed in those of the negative controls (wild-type plants) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, J, K and L). These results indicated that the MCLV LIR is a constitutive promoter. Additionally, the deletion of (GAAAAA)\u003csub\u003e5\u003c/sub\u003e sequence does not change the promoter type of LIR.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Prediction of \u003cem\u003ecis\u003c/em\u003e-acting elements in MCLV LIR and P232\u003c/h2\u003e\u003cp\u003eSince both LIR and P232 exhibit bidirectional promoter characteristics and have nearly identical activities, we predicted their transcription start sites (TSS) and the distribution of elements in their V- and C-sense strands, respectively. Putative transcription start sites (TSS) of the V-sense strand of LIR and P232 was predicted to be 'A', and those of the C-sense strand was predicted to be 'T' using the NNPP software. The predicted \u003cem\u003ecis\u003c/em\u003e-acting elements by PLACE software were summarized in Table S2 and Table S3, respectively. A total of 121 cis-acting elements were found in both polarity strands of LIR, with 74 in the V-sense and 47 in the C- sense. P232 has 88 \u003cem\u003ecis\u003c/em\u003e-acting elements, with 50 in the V-sense and 38 in the C-sense. Compared with P232, the 33 additional elements in LIR are all located downstream of the TSS of their respective polarity strands, indicating that these elements do not contribute to the activity of the bidirectional promoter. The core promoter elements TATA-box and CAAT-box in the C-sense strand of P232 are located at positions \u0026minus;\u0026thinsp;30 nt and \u0026minus;\u0026thinsp;108 nt relative to the transcription start site, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, the TATA-box and CAAT-box of V-sense strand of P232, further upstream of the TSS, are at -188 bp and \u0026minus;\u0026thinsp;208 bp region relative to the TSS, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, we identified other putative \u003cem\u003ecis\u003c/em\u003e-regulatory elements, such as G-box, P-box, Box-II, ABRE, a root-specific regulatory element ROOTMOTIFTAPOX1 (ATATT).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Impact of (GAAAAA)₅ on MCLV replication\u003c/h2\u003e\u003cp\u003eTo determine the impact of (GAAAAA)₅ on MCLV replication, infectious clones of (GAAAAA)₅-deleted MCLV vII (pCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e) and wild-type MCLV vII (pCA-1.1MCLV\u003csup\u003eWT\u003c/sup\u003e) were transfected into \u003cem\u003eN. benthamiana\u003c/em\u003e protoplasts, respectively. qPCR analysis showed that the growth multiple of MCLV in protoplasts transfected with pCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e was approximately. 5.1-fold at 24 hpt, which was significantly lower than the 20.1-fold of MCLV growth in protoplasts transfected with pCA1.1MCLV\u003csup\u003eWT\u003c/sup\u003e. The growth difference between the two was even more pronounced at 48 hpt. The growth fold of MCLV in protoplasts transfected with pCA-MCLV\u003csup\u003emLIR\u003c/sup\u003e was only 5.7-fold at 48 hpt, showing almost no growth compared to 24 hpt. In contrast, the growth fold of MCLV in protoplasts transfected with pCA-MCLV\u003csup\u003eWT\u003c/sup\u003e reached 52.1-fold at 48 hpt, 2.5 times growth compared to 24 hpt. These results indicated that deletion of the (GAAAAA)₅ sequence substantially weakened MCLV DNA replication in protoplasts, confirming its important role in MCLV replication.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe promoter activities of MCLV LIR and the effect of the specific (GAAAAA)₅ sequence within it on both promoter activity and MCLV replication were analyzed in this study. Like other geminiviral LIR (Ashraf et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Shivaprasad et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Usharani et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zhan et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), MCLV LIR is characterized by a bidirectional promoter, with a higher activity of C-sense than that of V-sense. The (GAAAAA)₅ embedded in V-sense and (TTTTTC)\u003csub\u003e5\u003c/sub\u003e in C-sense LIR are dispensable for the bidirectional promoter activity and promoter type of MCLV LIR, but are very important for MCLV DNA replication. Based on these findings, a minimal promoter (P232) sequence that maintains bidirectional promoter activity of MCLV LIR was identified. P232 consists of 232 nts, including 214 nts (without 30 nts of (GAAAAA)₅) between two predicted transcription start sites (TSS) and 8 nts downstream of the V-sense TSS and 10 nts downstream of the C-sense TSS. In mulberry plants, the C-sense promoter activity of P232 is higher than that of CaMV 35S commonly used in transgenic plant practice, while the V-sense is only slightly lower than that of CaMV 35S. The fact that P232 is shorter than 35S and exhibits bidirectional promoter activity makes it highly valuable for mulberry genetic engineering.\u003c/p\u003e\u003cp\u003eAmong LIRs of different geminiviruses, promoter activity varies compared to 35S. For example, C-sense LIRs of cotton leaf curl Multan virus (CLCuMV) (Xie et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and cotton leaf curl Burewala virus (CLCuBuV) (Khan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) have promoter activity higher than 35S, while that of Tomato mottle Ta\u0026iacute;no virus (ToMoTV) is lower (Ramos et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the case of MCLV, C-sense LIR showed promoter activity higher than 35S only in its natural host, mulberry, and was slightly lower in its experimental host, \u003cem\u003eN. benthamiana\u003c/em\u003e, suggesting that promoters drive gene expression differently across hosts. This may be due to differences in the genetic backgrounds of different hosts, leading to transcription factor-\u003cem\u003ecis\u003c/em\u003e-acting element interactions with different specificities and intensities in natural and experimental hosts. Therefore, we suggested that the activity of a promoter in the experimental host does not truly reflect its activity.\u003c/p\u003e\u003cp\u003eAlthough P232 is only two-thirds the full-length sequence of LIR, its V- and C-sense strands almost contain all the effective \u003cem\u003ecis\u003c/em\u003e-acting elements from the homologous V- and C-sense strands of LIR, respectively. This may explain why P232 can maintain the bidirectional promoter activity of LIR. Conversely, the fact that P232 can maintain the bidirectional promoter activity of LIR suggests that this 232-nt segment may be the factual sequence through which LIR exerts its promoter function. Elements in the LIR sequence flanking P232 are unlikely to be regulatory elements or to affect promoter activity. Similar to LIRs of other geminiviruses, P232 contains not only the core promoter elements but also some common \u003cem\u003ecis\u003c/em\u003e-acting elements in both V-sense and C-sense (Arg\u0026uuml;ello-Astorga et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). ABRE (ABA-responsive element) is a \u003cem\u003ecis\u003c/em\u003e-acting element involved in abscisic acid reactivity (Hobo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and salt stress-related physiological processes in rice (Liu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). G-box, I-box and BOX-II are light-responsive elements that are key for transcriptional activity in the Arabidopsis \u003cem\u003erbcS-1A\u003c/em\u003e promoter (Donald and Cashmore, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). A root-specific regulatory element, ROOTMOTIFTAPOX1 (ATATT), was identified in the C- and V-sense of P232, respectively, inferring that the genes it regulates can be expressed in roots.\u003c/p\u003e\u003cp\u003eIn eukaryotic gene promoters, the core elements TATA box and CAAT box are typically located around the \u0026minus;\u0026thinsp;30 bp region and \u0026minus;\u0026thinsp;70 to -120 bp region upstream of the TSS (Singh et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), respectively. For the C-sense strand of P232, the putative TATA box and CAAT box are located at -30 nts and \u0026minus;\u0026thinsp;108 nts relative to the TSS, respectively, falling within the corresponding regions of these boxes in common eukaryotic promoters. However, in the V-sense strand of P232, the predicted TATA box and CAAT box are located at -188 bp and \u0026minus;\u0026thinsp;208 bp relative to the TSS, respectively, far from the corresponding regions of these boxes in common eukaryotic promoters. Thus, whether they play role in driving gene expression remains to be clarified.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e\n\u003cp\u003eQ.-Y.L.: conceptualization; J.-J.Y., Y.-H. and W.-K.S.: writing \u0026ndash; review and editing; J.-J.Y., M.-S.Z.: methodology; J.-J.Y. and Q.-Y.L.: writing\u0026ndash; original draft preparation; J.-J.Y., Z.-J. W. and K.-Q.L.: software. J.-J.Y., Y.-B. H. and L.-L.S.: data curation.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Natural Science Foundation of China (32271885).\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eData related to the findings presented in this paper are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArg\u0026uuml;ello-Astorga GR, Guevara-Gonz\u0026aacute;lez RG, Herrera-Estrella LR, Rivera-Bustamante RF (1994) Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology 203:90\u0026ndash;100\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAshraf MA, Shahid AA, Rao AQ, Bajwa KS, Husnain T (2014) Functional characterization of a bidirectional plant promoter from cotton leaf curl burewala virus using an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transient assay. Viruses 6:223\u0026ndash;242\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi IR, Stenger DC (1996) The strain-specific \u003cem\u003ecis\u003c/em\u003e-acting element of beet curly top geminivirus DNA replication maps to the directly repeated motif of the ori. Virology 226:122\u0026ndash;126\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClough SJ, Bent AF (1998) Floral dip: a simplified method for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of Arabidopsis thaliana. Plant J 16:735\u0026ndash;743\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzosnek H, Laterrot H (1997) A worldwide survey of tomato yellow leaf curl viruses. Arch Virol 142:1391\u0026ndash;1406\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai Z, Wang A (2022) Isolation and transfection of plant mesophyll protoplasts for virology research. Methods Mol Biol 2400:43\u0026ndash;53\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonald RG, Cashmore AR (1990) Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis \u003cem\u003erbcS-1A\u003c/em\u003e promoter. Embo J 9:1717\u0026ndash;1726\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEagle PA, Hanley-Bowdoin L (1997) \u003cem\u003eCis\u003c/em\u003e elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication. J Virol 71:6947\u0026ndash;6955\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFiallo-Oliv\u0026eacute; E, Lett JM, Martin DP, Roumagnac P, Varsani A, Zerbini FM, Navas-Castillo J 2021. ICTV Virus Taxonomy Profile: Geminiviridae 2021. J Gen Virol 102.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan PY (2025) Studies on the function of \u003cem\u003eV6\u003c/em\u003e and \u003cem\u003eC4\u003c/em\u003e genes encoded by mulberry crinkle leaf virus. Jiangsu University of Science and Technology, Zhenjiang, Jiangsu province, China\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan PY, Yin ZN, Yang JJ, Zhang MS, Huang Y, Zhang J, Lu QY (2025) \u003cem\u003eV6\u003c/em\u003e encoded by mulberry crinkle leaf virus is important for viral DNA replication. Virology 603:110392\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan TT, Tang JX, Fang M, Zhang P, Han PY, Yin ZN, Ma Y, Zhang J, Lu QY (2024) Two genes encoded by mulberry crinkle leaf virus (MCLV): The \u003cem\u003eV4\u003c/em\u003e gene enhances viral replication, and the V5 gene is needed for MCLV infection in Nicotiana benthamiana. Virus Res 339:199288\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan TT, Zhang P, Tang JX, Ma Y, Zhong K, Lu QY (2023) Establishment of infectious clone of mulberry crinkle leaf virus vII variant and identification of its pathogenicity. Acta Sericol Sin 49:8\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D (2000) Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit Rev Biochem Mol Biol 35:105\u0026ndash;140\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHigo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant \u003cem\u003ecis\u003c/em\u003e-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297\u0026ndash;300\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHobo T, Asada M, Kowyama Y, Hattori T (1999) ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Plant J 19:679\u0026ndash;689\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan ZA, Abdin MZ, Khan JA (2015) Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus. PLoS ONE 10:e0121656\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang P, Navarro B, Zhang Z, Wang H, Lu M, Xiao H, Wu Q, Zhou X, Di Serio F, Li S (2015) Identification and characterization of a novel \u003cem\u003eGeminivirus\u003c/em\u003e with a monopartite genome infecting apple trees. J Gen Virol 96:2411\u0026ndash;2420\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu H, Cui P, Zhang B, Zhu J, Liu C, Li Q (2022) Binding of the transcription factor MYC2-like to the ABRE of the OsCYP2 promoter enhances salt tolerance in \u003cem\u003eOryza sativa\u003c/em\u003e. PLoS ONE 17:e0276075\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoconsole G, Saldarelli P, Doddapaneni H, Savino V, Martelli GP, Saponari M (2012) Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family geminiviridae. Virology 432:162\u0026ndash;172\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu QY, Ma Y, Smith WK, Yu J, Cheng YY, Zhang P, Han TT (2022) The identification of \u003cem\u003eTautoneura mori\u003c/em\u003e as the vector of mulberry crinkle leaf virus and the infectivity of infectious clones in mulberry. Phytopathology 112:435\u0026ndash;440\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu QY, Wu ZJ, Xia ZS, Xie LH (2015) Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L). Arch Virol 160:2135\u0026ndash;2138\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu QY, Yang L, Huang J, Zheng L, Sun X (2019) Identification and subcellular location of an RNA silencing suppressor encoded by mulberry crinkle leaf virus. Virology 526:45\u0026ndash;51\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu QY, Cheng YJ, Sun YY, Yang X, L (2020) \u003cem\u003eV3\u003c/em\u003e protein encoded by mulberry crinkle leaf virus acts as a pathogenicity determinant in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. Eur J Plant Pathol 157:141\u0026ndash;149\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa Y, Navarro B, Zhang Z, Lu M, Zhou X, Chi S, Di Serio F, Li S (2015) Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease. JGV 96:2421\u0026ndash;2434\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoffat A (1999) Plant pathology - Geminiviruses emerge as serious crop threat. Science 1999,286(5446), 1835\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNavas-Castillo J, Fiallo-Oliv\u0026eacute; E, S\u0026aacute;nchez-Campos S (2011) Emerging virus diseases transmitted by whiteflies. Annu Rev Phytopathol 49:219\u0026ndash;248\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQiu Y, Zhang S, Yu H, Xuan Z, Yang L, Zhan B, Zerbini M, Cao F, M (2020) Identification and characterization of two novel geminiviruses associated with paper mulberry (Broussonetia papyrifera) leaf curl disease. Plant Dis 104:3010\u0026ndash;3018\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRamos PL, Fuentes AD, Quintana Q, Castrillo G, Guevara-Gonz\u0026aacute;lez RG, Peral R, Rivera-Bustamante RF, Pujol M (2004) Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus replication-associated protein promoter in transgenic plants. Virus Res 102:125\u0026ndash;132\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReese MG (2001) Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 26:51\u0026ndash;56\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRizvi I, Choudhury NR, Tuteja N (2015) Insights into the functional characteristics of geminivirus rolling-circle replication initiator protein and its interaction with host factors affecting viral DNA replication. Arch Virol 160:375\u0026ndash;387\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRombauts S, D\u0026eacute;hais P, Van Montagu M, Rouz\u0026eacute; P (1999) PlantCARE, a plant \u003cem\u003ecis\u003c/em\u003e-acting regulatory element database. Nucleic Acids Res 27:295\u0026ndash;296\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoumagnac P, Lett JM, Fiallo-Oliv\u0026eacute; E, Navas-Castillo J, Zerbini FM, Martin DP, Varsani A (2022) Establishment of five new genera in the family Geminiviridae: Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus. Arch Virol 167:695\u0026ndash;710\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShakir S, Mubin M, Nahid N, Serfraz S, Qureshi MA, Lee TK, Liaqat I, Lee S, Nawaz-Ul-Rehman MS (2023) REPercussions: how geminiviruses recruit host factors for replication. Front Microbiol 14:1224221\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShepherd DN, Martin DP, Van Der Walt E, Dent K, Varsani A, Rybicki EP (2010) Maize streak virus: an old and complex 'emerging' pathogen. Mol Plant Pathol 11:1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShivaprasad PV, Akbergenov R, Trinks D, Rajeswaran R, Veluthambi K, Hohn T, Pooggin MM (2005) Promoters, transcripts, and regulatory proteins of mungbean yellow mosaic geminivirus. J Virol 79:8149\u0026ndash;8163\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh N, Sharma R, George A, Singla SK, Palta P, Manik R, Chauhan MS, Singh D (2012) Cloning and characterization of buffalo NANOG gene: alternative transcription start sites, splicing, and polyadenylation in embryonic stem cell-like cells. DNA Cell Biol 31:721\u0026ndash;731\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmith WK, Ma Y, Yu J, Cheng YY, Zhang P, Han TT, Lu QY (2022) Characterization of a strong constitutive promoter from paper mulberry vein banding virus. Arch Virol 167:163\u0026ndash;170\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUsharani KS, Periasamy M, Malathi VG (2006) Studies on the activity of a bidirectional promoter of mungbean yellow mosaic India virus by agroinfiltration. Virus Res 119:154\u0026ndash;162\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWingender E, Dietze P, Karas H, Kn\u0026uuml;ppel R (1996) TRANSFAC: a database on transcription factors and their DNA binding sites. Nucleic Acids Res 24:238\u0026ndash;241\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie J, Wang X, Liu Y, Peng Y, Zhou G (2007) First report of the occurrence of wheat dwarf virus in wheat in China. Plant Dis 91:111\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie Y, Liu Y, Meng M, Chen L, Zhu Z (2003) Isolation and identification of a super strong plant promoter from cotton leaf curl Multan virus. Plant Mol Biol 53:1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin ZN (2025) Studies on the function of \u003cem\u003eV2\u003c/em\u003e and \u003cem\u003eC3\u003c/em\u003e genes by mulberry crinkle leaf virus. Jiangsu University of Science and Technology, Zhenjiang, Jiangsu province, China\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin ZN, Han PY, Han TT, Huang Y, Yang JJ, Zhang MS, Fang M, Zhong K, Zhang J, Lu QY (2024) \u003cem\u003eV2\u003c/em\u003e protein enhances the replication of genomic DNA of mulberry crinkle leaf virus. Int J Mol Sci 25\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu J (2021) Study on the genetic diversity of mulberry crinkle leaf virus. Jiangsu University of Science and Technology, Zhenjiang, Jiangsu province, China\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhan XC, Haley A, Richardson K, Morris B (1991) Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the beta-glucuronidase gene. J Gen Virol 72(Pt 11):2849\u0026ndash;2852\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Geminiviruses, Mulberry crinkle leaf virus, Large intergenic region, Bidirectional promoter, (GAAAAA)5 sequence, Replication","lastPublishedDoi":"10.21203/rs.3.rs-8036347/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8036347/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mulberry crinkle leaf virus (MCLV) is a representative member of the genus \u003cem\u003eMulcrilevirus\u003c/em\u003e of the family \u003cem\u003eGeminiviridae\u003c/em\u003e. Here, the bidirectional promoter activity of the long intergenic region (LIR) of MCLV and the roles of a specific element consisting of five GAAAAA repeats [(GAAAAA)\u003csub\u003e5\u003c/sub\u003e] in LIR were investigated using Agrobacterium-mediated transient expression and transfection of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e protoplasts. Transient expression results demonstrated that, similar to other geminiviruses, MCLV's LIR also exhibits bidirectional promoter activity. The promoter activity of the C-sense is significantly higher in the experimental host, \u003cem\u003eN. benthamiana\u003c/em\u003e, while slightly higher than that of the V-sense in the natural host, mulberry (\u003cem\u003eMorus spp\u003c/em\u003e.). However, the promoter activity of LIR is inconsistent between the experimental and natural hosts, especially its C-sense promoter activity, which is slightly lower in the experimental host but higher in the natural host than that of the cauliflower mosaic virus (CaMV) 35S. The (GAAAAA)\u003csub\u003e5\u003c/sub\u003e element does not affect the activity and type of LIR promoter, but its absence significantly reduces the replication of the MCLV genome in protoplasts. Based on the constructed truncated mutants, a fragment of 232 nts (named P232) lacking (GAAAAA)\u003csub\u003e5\u003c/sub\u003e was deduced to be the minimal sequence required for sustaining LIR promoter activity. These findings lay the foundation for further research on MCLV and transgenic breeding of mulberry plants.\u003c/p\u003e","manuscriptTitle":"Analysis of bidirectional promoter activity and structure of the large intergenic region (LIR) in mulberry crinkle leaf virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 07:18:18","doi":"10.21203/rs.3.rs-8036347/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-02T11:03:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T23:14:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136687248714451345927995278887907966172","date":"2026-01-01T23:08:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233113796741316652255028498719349974862","date":"2025-11-17T09:32:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T14:58:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T05:01:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-06T04:58:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology","date":"2025-11-05T08:44:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5edb1b2e-07a8-4589-9e22-02ba07bfd8da","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:04:21+00:00","versionOfRecord":{"articleIdentity":"rs-8036347","link":"https://doi.org/10.1007/s11103-026-01702-0","journal":{"identity":"plant-molecular-biology","isVorOnly":false,"title":"Plant Molecular Biology"},"publishedOn":"2026-04-23 15:59:15","publishedOnDateReadable":"April 23rd, 2026"},"versionCreatedAt":"2025-12-02 07:18:18","video":"","vorDoi":"10.1007/s11103-026-01702-0","vorDoiUrl":"https://doi.org/10.1007/s11103-026-01702-0","workflowStages":[]},"version":"v1","identity":"rs-8036347","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8036347","identity":"rs-8036347","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}