Evidence suggestive of Cauliflower mosaic virus transcribing complementary DNA from Cucumber mosaic virus RNA in mixed infection

Evidence suggestive of Cauliflower mosaic virus transcribing complementary DNA from Cucumber mosaic virus RNA in mixed infection | 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 Article Evidence suggestive of Cauliflower mosaic virus transcribing complementary DNA from Cucumber mosaic virus RNA in mixed infection Hakimeh Ighani Mayan, Nemat Sokhandan Bashir, Davoud Koolivand This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7599836/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The aim of this study was to investigate if Cauliflower mosaic virus (CaMV) reverse transcriptase (RT) can make complementary DNA (cDNA) from Cucumber mosaic virus (CMV) genomic RNAs in the mixed infection. First, symptoms of single infection with CaMV or CMV, and dual-infection with CaMV + CMV were investigated in cauliflower ( Brassica oleracea ) plants. CMV symptoms disappeared in the mixed-infection after about 45 days post infection (dpi). The efficiency of the CaMV RT in converting CMV RNA genome to cDNA was investigated by extracting total DNA from the dually-infected plants and subjecting to polymerase chain reaction (PCR) by the use of CMV coat protein (CP)-specific primers which gave the anticipated ~ 675 bp DNA. To further verify the identity of the PCR products, the amplified CMV CP cDNA or CaMV RT DNA were sequenced and the resultant nucleotide data were compared with the counterpart genomic region of other isolates of the viruses available in the Genbank. PCR assays on total DNA from plants with single CMV-infection gave no amplification which ruled out the nonspecific activity of plant endogenous RTs in converting the RNA to cDNA. Also, possible amplification of CMV CP cDNA from traces amount of CMV RNA from the infected plants by a weak reverse transcription activity of Taq DNA polymerase was ruled out by performing PCR on the virus RNA which did not yield any DNA. The results showed CMV-associated symptoms attenuation in the presence of CaMV. This study opens up new insights into the interaction between the viruses. This is the first report, to our knowledge, of cDNA synthesis from an RNA virus by CaMV RT in the mixed-infection. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Plant sciences Cauliflower CaMV Reverse transcriptase cDNA PCR and CMV Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The discovery of reverse transcriptase (RT) enzymes in retroviruses in the 1970s revolutionized molecular biology and laid the foundations for retrovirology and cancer biology [ 5 ]. This discovery challenged the central tenets of molecular biology which assumed that DNA is transcribed into RNA and ultimately into proteins. RTs were described as nucleic acid polymerases that are capable of synthesizing complementary DNA (cDNA) using RNA as a template [ 28 ]. These enzymes require a specific type of RNA oligonucleotide as a in the cells primer to initiate DNA synthesis [ 3 , 5 , 4 ]. Studies have shown that RTs can also facilitate RNA strand cleavage during the formation of RNA/DNA heteroduplexes [ 21 , 28 ]. RT enzymes are found in both plant and animal viruses [ 19 , 20 ]. In addition to Caulimoviridae , RTs play an important role in the replication of members of the families Retroviridae , Pseudoviridae , Metaviridae and Hepadnaviridae [ 28 ]. It has been shown in vitro that RTs of retroviruses and long terminal repeat (LTR) retrotransposons can extend the 3' end of their genomes provided that the oligonucleotide that acts as a primer has a complementary at its 3' end to 1, 2, or 3 overhanging nucleotides at the 3' end of the genome of retroviruses and LTR retrotransposons [ 30 ]. Cauliflower mosaic virus (CaMV) was the first discovered plant pararetrovirus and the first plant dsDNA virus that uses RT to replicate its genome. Reverse transcription in this virus results in the formation of nicked circular DNAs that are converted into episomal (extrachromosomal) DNA in the host cell nucleus [ 28 ]. The genome of CaMV is a circular double-stranded DNA of approximately 8000 bp containing 6–8 open reading frames (ORF). CaMV virions have T = 7 icosahedral symmetry that are enclosed in a polyhedral protein coat of 50 nm in diameter [ 11 ].When a caulimovirus infects a cell, the dsDNA enters the nucleus where the overlapping break sites in the virus genome are removed, repaired and closed, creating a minichromosome with a fully closed circular DNA [ 35 ]. With the help of DNA-dependent RNA polymerase, two RNA fragments are transcribed from the minichromosome. The short RNA fragment (19S) is later translated in large quantities into the 58-kDa protein constituting the viroplasm, and the long fragment (35S), which is translated in turn into all the other proteins. Translation of the 19S mRNA results in the production of the P6 protein which accumulates in the numerous cytoplasmic virus factories where translation of other viral proteins takes place [ 2 , 34 , 35 ]. P6 a transactivator translation enhancer may also play a role in increasing the translation rate of other genes from 35S mRNA [ 1 ]. P1 is a movement protein required for cell-to-cell movement of the virus, P2 plays a role as an auxiliary component in aphid transmission, P3 is considered to be associated with viral components, P4 is a viral coat protein (CP) involved in capsidation of the viral genome, and P5 encodes a reverse transcriptase enzyme. The P7 protein has never been identified in a plant system and can be deleted by mutagenesis without affecting virus infection or transmission [ 2 ]. CaMV mainly infects members of the Brassicaceae family including cauliflower, cabbage, radishes, mustard, turnips, canola and broccoli. Some strains of CaMV (W260 and D4) can also infect Solanaceae species such as members of the genus Datura and tobacco plants (genus Nicotiana ) [ 2 ]. Depending on the virus variant, host plant, symptoms and type of infection (single or mixed), virus transmission occurs in different forms [ 8 , 42 ]. CaMV-infected plants exhibit a diverse range of symptoms including chlorosis, mosaic, vein clearing, vein banding, leaf deformation, and stunting [ 12 ]. Co-infections in plants occur regularly in natural ecosystems. The vulnerability of host plant is often greater in co-infections than in single-infections [ 40 ]. Simultaneous infections with CaMV and Cucumber mosaic virus (CMV) occurs in crops. CMV is one of the most important viruses infecting major agricultural crops including vegetables and it is the type member of the genus Cucumovirus [ 23 ], globally distributed with the widest host range among plant viruses [ 14 ] of over 1300 plant species in over 100 families of monocotyledons and dicotyledons plants including most vegetables, ornamentals, and woody and semi-woody plants [ 9 ]. CMV exhibits a variety of symptoms in different hosts and causes systemic infection in most host plants but may be asymptomatic in a number of crops including alfalfa. Among CMV hosts, cucurbits are the most susceptible to CMV [ 43 ]. Members of Cucumovirus have a tripartite genome and a subgenomic RNA, among which RNA1 and RNA2 are packaged in separate particles, and RNA3 and subgenomic RNA4 in a third particle. The RNA1 and RNA2 collectively code for the RNA polymerase. However, the RNA2-coded protein plays a role in the virus pathogenicity as well, being virus suppressor of RNA silencing (VSR). The RNA3 encodes movement protein and coat protein [ 38 ]. In the present study, in addition to investigating symptoms in single and mixed infections with CMV and/ or CaMV, the ability of the CaMV RT in converting RNA to cDNA was also examined. Results Symptoms caused by viruses in the host plants Symptoms in the cauliflower inoculated with CaMV appeared as chlorotic local lesions after 7 days. Over time, these symptoms became systemic and formed a yellow network. The main vein was widened and the leaf size was reduced. The developed fruit was small, yellowed and shrunk (Fig. 1 - d). However, in CMV- inoculated cauliflower plant, symptoms appeared seven days later than that in CaMV-inoculated cauliflower (14 days after inoculation) and included chlorotic local lesions, vein clearing and vein banding (Fig. 1 - c). In the mixed-infection with CaMV + CMV plant growth was severely reduced, widening of the main vein and the reduction in leaf size occurred very severely. Vein banding, vein clearing and chlorotic local lesions appeared, then the entire leaf blade turned yellow and died. The developed fruit was shriveled and dried over time (Fig. 1 . e). Symptoms in infected plants are listed separately in Table 1 . It should be noted that in the mixed-infections, vein clearing and vein banding began to disappear after about 45 days post-inoculation (dpi). Analysis of dsRNA from CMV-inoculated plants DsRNA extraction was successfully performed from five CMV-infected plants (one as a positive control and four inoculated plants) and one healthy control revealed three viral genomic RNA segments with sizes of ~ 3.4, ~ 3.1, and ~ 2.2 kb corresponding to RNA1, RNA2 and RNA3, respectively. In addition, a fourth sub genomic RNA (RNA4) was also present (Fig. 2 ). PCR and RT-PCR Results from PCR using CMV- or CaMV- specific primers from each four-replicative cauliflower plants infected with CaMV + CMV, and infected with CMV or CaMV indicated amplification of the expected fragments from total DNA samples. In total, the expected CaMV RT fragment 2040 bp was amplified from four plants with CaMV-infection and from four plants with CaMV + CMV mixed-infection (Fig. 3 ). CMV CP cDNA with a size of 675 bp was amplified from four plants with CaMV + CMV (Fig. 4 ). PCR on denatured dsRNA samples, and total DNA from four cauliflower plants with single CMV-infection did not give the CMV CP cDNA fragment (see Supplementary Fig. S1 and S2 online). However, when reverse transcription was done on the boiled dsRNA from the CMV-infected plants and followed by PCR, a DNA fragment of roughly 675 bp in length was amplified (see Supplementary Fig. S1 online). The explanations are briefly given in Table 1 . Table 1 Observations and PCR analyses on four replicative cauliflower plants infected with CMV, CaMV, or CMV + CaMV Infection CaMV CMV CMV + CaMV Symptoms observed CLL, WMV, RLS, Y and SF CLL, VC and VB SRPG, RLS, WMV, VB, VC, CLL, Y and SDF PCR with CaMV RT primers on total DNA 2040 bp 2040 bp PCR with CMV CP primers on total DNA NBE 675 bp PCR with CMV CP primers on denatured dsRNA from CMV-infected plants NBE RT-PCR with CMV CP primers on denatured dsRNA from CMV-infected plants 675 bp Abbreviations; CLL: Chlorotic local lesions, WMV: Widened main vein, RLS: Reduced leaf size, Y: Yellowing, SF: shrunk fruit, VC: Vein clearing, VB: vein banding, SRPG: Severe reduction in plant growth, SDF: Shrinkage and drying of fruit, NBE: No band in electrophoresis and -: PCR or RT-PCR was not performed Enzymatic digestion Digestion with the Nco I on the amplified fragment corresponding to CaMV RT gene produced two fragments with sizes of 499 bp and 1535 bp, with the Pst I two fragments with sizes of 282 and 1752 bp, and with the Msp I three fragments of 279, 232, and 1521 bp which all corresponded with predicted restriction map the CaMV RT sequence available in GenBank, demonstrating that the PCR-amplified product belonged to CaMV (Fig. 5 ). Results from sequencing Sequencing of the PCR-amplified CMV CP fragment showed that it was related to the CMV CP gene. Likewise, BLAST analysis of the nucleotide data from sequencing CaMV RT DNA confirmed its identity as CaMV. Discussion Simultaneous infection with two or more viruses in a plant is a common phenomenon that, in addition to affecting the severity of symptoms, may affect the concentration of infecting viruses [ 39 ]. This may be due to the effects on the replication of one virus by the second virus [ 16 ]. There have been numerous reports of the mixed-infection of CaMV with TuMV and CMV in various plants [ 13 , 15 , 22 , 27 , 36 , 37 ]. In the present study, we predicted that in the presence of CaMV, due to the conversion of the CMV RNA genome to cDNA, a decrease in the concentration of the CMV genome would occur and consequently the symptoms caused by CMV in the mixed-infection would be reduced. This prediction was consistent with our observations because the CaMV- related symptoms in the single-infection including severe reduction in leaf size and widening of the main vein were still visible in the mixed-infection with CaMV + CMV whereas the CMV-associated vein clearing and vein banding started to disappear in the mixed-infection after about 45 dpi. These results are consistent with the findings of that in simultaneous infection with CaMV + TuMV in rapeseed, the concentration of TuMV genome decreased after 35 dpi and its symptoms disappeared after a period of time [ 15 , 29 ], therefore suggesting the adverse effect of CaMV on an RNA virus (TuMV). The results of this study indicate that the CaMV somehow overcomes the plant's defense system and continues to generate and assemble its own genome. As a support to this claim, it has been reported that CaMV P6/translational activator/viroplasmin (TAV) neutralizes the plant defense mechanism through suppressing RNA silencing and acts towards the assembly and transmission of CaMV particles [ 25 ]; or according to Hoffmann et al. [ 17 ] the increased severity of symptoms caused by CaMV is explained by the fact that P6 reduces stress granules (SGs) accumulation after stress by suppressing SGs, located in viral factories (VFs), thereby leading to an increase in P6 condensation during the course of infection which ultimately leads to a progressive shift in selected P6 functions including development of the disease symptoms. The reduction in the severity of CMV symptoms in the mixed infections compared to that in single CMV-infections suggests the adverse effect of the second virus. As CaMV disrupts the host's RNA degradation machinery by evading the host cell's mRNA surveillance system, this leads to the accumulation of CaMV mRNA and potentially hinders the replication of co-infecting RNA viruses [ 17 ]. Given the ability of SGs to influence mRNA storage, degradation, translation and protein signaling, CaMV by having P6 in VFs disrupts the efficiency and function of SGs, thereby in addition to interfering with the replication of RNA viruses it prevents the expression of RNA virus genes in the mixed infection. This consequently leads to the attenuation of symptoms (vein clearing and vein banding) associated with RNA viruses in the mixed-infections. However, P6 through physical interactions with translation regulators, especially eIF3g (eukaryotic translation initiation factor 3g), leads to the expression of the CaMV genome and causes disease [ 31 ]. Therefore, CaMV's impact on RNA virus expression and replication can influence the overall disease symptoms observed in plants infected with both viruses [ 24 ]. A similar situation might have occurred in the mixed-infection in the present study. As to the reverse-transcribing activity of CaMV on CMV, PCR on total DNA from the dually-infected plants resulted in amplification of the expected DNA of 675 bp suggesting the reverse transcription of CMV RNA by CaMV. Alongside this, to ascertain that it is not the reverse transcriptase activity of Taq DNA polymerase giving the amplification, PCR was performed directly on denatured dsRNA from CMV-infected plants and, as a result, no DNA were amplified (see Supplementary Fig. S1 online) suggesting that under the PCR conditions applied in this study, the DNA polymerase did not have reverse transcriptase activity. To test the hypothesis that plant endogenous RTs from retrotransposons could act non-specifically under stress and convert RNA to cDNA [ 6 ], PCR was performed with CMV CP primers on total DNA from plants that had single CMV-infection which did not result in any amplification (see Supplementary Fig. S2 online). When the PCR-amplified CMV CP cDNA and CaMV RT DNA were sequenced and the resulting data subjected to BLAST analysis in NCBI, the identities of the amplified fragments were further proven to be of CMV CP and CaMV RT. This also additionally showed the reverse-transcription of CMV RNA by CaMV RT. Because plus-strand CMV RNAs are distributed throughout the plant cytoplasm [ 4 ] just as P5-CaMV being therein [ 18 , 32 ] the subjection of CMV RNAs in the cytosol to the reverse transcription activity of CaMV RT would not be remote from happening especially given the fact that PCR directly amplified CMV CP cDNA without in vitro reverse transcription. We demonstrated that in co-infection of an RNA virus with CaMV, the RNA virus genome is converted to cDNA by CaMV RT. Because individual CaMV RT molecules are able to perform full polymerase functions to convert ssRNA to dsDNA, this could be related to the flexibility of the loop region in CaMV POL catalytic site that allows the phosphate backbone of the primer DNA, which is the same methionine found in plant cells, to contact the side chain of residue Arg96 from the B0 helix's CaMV POL (flexible loop region in CaMV POL catalytic site that is involved in cDNA synthesis), leading to greater stabilization of the primer strand and bringing it closer to a catalytic location and initiating synthesis of the RNA virus cDNA strand [ 33 ]. This study demonstrates, for the first time, the in vivo activity of CaMV-encoded reverse transcriptase in transcribing CMV RNA within plant cells. An interesting aspect of this finding is that CMV RNA is transcribed by CaMV in the mixed infection which opens a new insight into the interaction between the viruses. At the same time, the RNA virus is attenuated due to formation of RNA + DNA hybrids which reduces significantly the amount of the RNA to go under expression. As this phenomenon takes place in the mixed infection it makes possible to detect the RNA virus directly by PCR without the need for in vitro transcription. Materials and methods Plant host and growth conditions Certified seeds of cauliflower, Brassica oleracea (95% germination rate) sourced from Thailand were used to assess symptoms development following single infections with CaMV, CMV and mixed-infection with CaMV + CMV. Plants were grown under controlled environmental conditions of 22–28°C, 30–40% relative humidity, and a 16 h light/8 h dark photoperiod. Source of virus and glasshouse inoculation CMV isolate PAK-17-2 from previous studies in our Laboratory whose identity was established by serological or molecular tests, was used. About CaMV, the isolate DAR78694 was used as the source virus. To test the efficiency of CaMV RT in converting CMV RNA into complementary DNA in a Cauliflower host plant, at least four biological replicates were used for each treatment: Inoculations with CMV, CaMV or simultaneous infection with CaMV + CMV in Brassica oleracea var. botrytis . The controls included mock inoculated (with buffer) and intact cauliflower. After growing under the greenhouse conditions, the plants were inoculated mechanically at the 2–3 leaf stage with extract of the infected plant material using 0.1 M potassium phosphate buffer (pH 7.2) and kept under greenhouse conditions for symptom observation and subsequent molecular analyses. Infected plant materials were used as viral source and mechanically inoculated on healthy host plants to propagate and maintain the viruses. Following the mechanical inoculations, the plants with single (CaMV or CMV) and mixed (CaMV + CMV) infections as well as control plants were monitored daily to assess symptom development. Upon the appearance of visible symptoms in each of the inoculations, they were recorded. Comparative analysis was then performed to determine differences in symptom expression between the single and mixed-infections. Extraction of double-stranded RNA from CMV-inoculated plants Double-stranded (dsRNA) extraction from the CMV-infected plants was performed according to the method of Delpasand Khabbazi et al. [ 7 ]. A 250 mg plant tissue infected with the virus was extracted in a mortar with 1 ml dsRNA extraction buffer (200 mM Tris, 500 mM NaCl, 10 mM MgCl 2 , 3% SDS, 10% Ethanol, 1% 2-Mercapto ethanol). After vortexing and incubation for 10 minutes at 37°C, chloroform was added in 1:1 ratio to remove plant proteins. After centrifugation and removal of plant tissue, 0.2 ml absolute ethanol and 15 mg CF-11 cellulose were added to the recovered aqueous phase to trap dsRNAs. Next, washing step was performed with 1 ml washing buffer (1X STE [100 mM NaCl, 10 mM Tris-HCL, 1 mM EDTA, pH 8.0]/16% ethanol) and then dsRNA was eluted using 150 µl elution buffer (1X STE), incubated at -20 o C for an hour and subjected to centrifugation at 12000 RPM for 20 minutes. Finally, the precipitate obtained from centrifugation was suspended in 30 µl ddH 2 O. To assess the presence, integrity, and molecular weight of the extracted dsRNA, samples were subjected to horizontal electrophoresis in a 1% agarose gel using TBE (Tris, borate and EDTA) buffer for approximately 90 minutes. Gel was visualized over UV light using a gel documentation system (Qiagen, Tehran, Iran). Total DNA extraction Total DNA was extracted from Cauliflower plants inoculated with CaMV, CaMV + CMV, CMV, and from control plants using the method described by Edwards et al. [ 10 ]. Briefly, 20 mg of infected or control leaf tissue was homogenized in 400 µL of extraction buffer (200 mM Tris-HCl, pH 7.5; 250 mM NaCl; 25 mM EDTA; 0.5% SDS). The homogenate was vortexed thoroughly, and plant debris was removed by centrifugation at high speed. The resulting supernatant was mixed with 300 µL of isopropanol to precipitate DNA, followed by centrifugation. The DNA pellet was air-dried and resuspended in 100 µL of nuclease-free distilled water (ddH₂O). Primer design A pair of specific primers based on the reverse transcriptase gene sequence of CaMV isolate DAR78694 with accession number KX904357, was designed using Perl Primer Version 1.1.21 software [ 26 ] to identify CaMV. Another pair of specific primers but for the amplification of CMV coat protein gene was according to Koolivand et al. [ 23 ]. The primer sequences are shown in Table 2 . Table 2 Sequences of primers used in this study Primers name Sequences Region Reference CaMVRTF 5'- AAGTGATGGATCCTCTACTTCTG-3' RT This study CaMVRTR 5'- TCAATTAGGAGCTCACCTTATTGA-3' RT This study CMVCPF 5'-AGTGGATCCATGGACAAATCTGAATCAACCAG-3' CP [ 23 ] CMVCPR 5'-AACTTCGAATTC(G/T)ACTGGGAGCAC(C/T)CC(A/G)GACGTGGG-3' CP [ 23 ] Reverse transcription polymerase chain reaction (RT-PCR) RT-PCR was performed with CMV CP-specific primers. for the reverse transcription reaction, 1 µl of the forward primer (10 pmol/µl) was mixed with 1 µl of ddH 2 O, and 2 µl out of the 30 µl extracted dsRNA in a microtube and placed at 70°C for 5 min. The samples were immediately transferred to ice, then 6 µl of a mixture containing 2 µl of 10X PCR reaction buffer, 1 mM dNTP, 40 units of RNAsin, and 200 units (0.5 µl) of M-Mulv reverse transcriptase (Kiagene Fanavar Aria, Tehran, Iran) were added into the tube and was placed at room temperature for 15 min before placing in a thermocycler (Q-Sat 24, Hain Lifescience UK Ltd, England) at 42 o C for 60 min. After the completion, the reverse transcriptase was inactivated by placing the tube at 70°C for 10 min. polymerase chain reaction (PCR) PCR was performed separately with each of the CMV and CaMV virus-specific primer sets (Table 2 ) in a final volume of 20 µl, containing 10 µl master mix (Sinaclon, Tehran, Iran), 10 pmol each forward and reverse primer for each virus, 10 ng of template DNA, and 7.6 µl of ddH 2 O. The thermal program applied for PCR-detection of CaMV was 94 o C for 3 min, followed by 30 cycles of 94 o C for 40 s, 45 o C for 60 s and 72 o C for 150 s, and one additional cycle of 72 o C for 7 min; but for CMV the thermal profile included 94 o C for 1 min, followed by 35 cycles of 94 o C for 30 s, 50 o C for 45 s and 72 o C for 60 s, with an additional cycle of 72 o C for 5 min. Four µl of each PCR product was electrophoresed on 1% agarose gel containing ethidium bromide in 0.5x TBE buffer. Electrophoresis was performed at 100 V and 24 mA, and the results were documented by a gel documentation device as mentioned earlier in this paper. Confirmation of the identity of the CaMV RT gene with restriction analysis The PCR-amplified CaMV RT fragment was digested with three different Msp I, Pst I, and Nco I restriction enzymes. Three separate reactions, each containing 5 Units of enzyme, 100 ng of PCR product, and 4 µl of appropriate 10X restriction buffer, were digested by incubation for 1 hour at 37°C. The digested products were then electrophoresed in a 1% agarose gel according to the above-mentioned method. Nucleotide sequence determination The PCR-amplified CMV CP cDNA and CaMV RT DNA were subjected to purification and sequencing with the forward primer. The sequencing was done by Pishgam Company (Tehran, Iran). The generated sequences were compared with the counterpart sequences data available in Genbank (NCBI). Declarations CRediT authorship contribution statement Hakimeh Ighani Mayan: Performed the lab work, preparing first draft of the manuscript and data curation; Nemat Sokhandan Bashir acted as the chief investigator: Writing, Editing and conceptualization; Davod Koolivand acted as the associate supervisor: Reviewing and editing. Competing Interests Statement The authors declare no competing interests. Funding This research was funded by the Research Management Office of the University of Tabriz. Author Contribution H.I.M. Performed the lab work, preparing first draft of the manuscript and data curationN.S.B. Acted as the chief investigator: Writing, Editing and conceptualizationD.K. Acted as the associate supervisor: Reviewing and editing Acknowledgements We express our gratitude to the Research Management Office of the University of Tabriz for their financial support. Data Availability The extracted genomes DNAs and dsRNAs from all plants with different viral infections in this study are maintained in 1.5 ml microtubes with respective extraction codes in the Virology and Genetic Engineering Laboratory the University of Tabriz. The resulting PCR products are likewise stored at the same location. Plant samples inoculated with different viruses are stored in microtubes after drying in CaCl2. 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Characterization of synergy between Cucumber mosaic virus and Alternaria alternata in Nicotiana tabacum. Physiol. Mol. Plant. Path . 108 , 101404 (2019). Verma, I. M., Meuth, N. L., Bromfeld, E., Manly, K. F. & Baltimore, D. Covalently linked RNA-DNA molecule as initial product of RNA tumour virus DNA polymerase. Nat. New Biol. 233, 131 – 34 (1971). Yasaka, R. et al. The temporal evolution and global spread of Cauliflower mosaic virus, a plant pararetrovirus. j. pone . 0085641 10.1371/ (2014). Zitter, T. A. & Murphy, J. F. The ecology of Cucucmber mosaic virus and sustainable agriculture. Virus Res. 71 , 9–21 (2009). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. 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1","display":"","copyAsset":false,"role":"figure","size":937329,"visible":true,"origin":"","legend":"\u003cp\u003eSymptoms observed in cauliflower plants inoculated with CaMV, CMV or CMV + CaMV after 40 dpi; (a) Healthy intact plant (no symptoms), (b) Mock inoculation with buffer (no symptoms), (c) Infection with CMV appeared as chlorotic local lesions, vein clearing and vein banding, (d) Infection with CaMV shown up as chlorotic local lesions, reduced leaf size and widening of the main vein, (e) Mixed-infection with CaMV + CMV shown up as chlorotic local lesions, vein clearing, vein banding, reduced leaf size and widening of the main vein\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/51179f84a360930b01f164e3.png"},{"id":94870454,"identity":"7990b12a-b79a-46ec-8d43-1763e20d1591","added_by":"auto","created_at":"2025-10-31 14:50:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146594,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoresis on 1% agarose gel of dsRNA samples extraction from CMV-infected plants; Lane M: 500 ng Lambda DNA \u003cem\u003eEco\u003c/em\u003eRI + \u003cem\u003eHind\u003c/em\u003eIII, Lanes 1, 2, 3, and 4: CMV-inoculated cauliflower plants, Lane 5: A positive control, CMV-infected tobacco plant, Lane 6: Extraction from healthy cauliflower\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/cebab56a06fd11a11f10f28d.png"},{"id":94985951,"identity":"a30e6539-0b3b-4df1-9748-fe2764fcd723","added_by":"auto","created_at":"2025-11-03 06:59:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94848,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoresis of products from PCR with \u003cem\u003eCauliflower mosaic virus\u003c/em\u003e (CaMV) reverse transcriptase-specific primers in 1% agarose gel; Lane M: 1Kb Plus DNA Ladder, Lanes 1, 2, 3, and 4: four cauliflower plants with CaMV-infection, Lane 5: Negative control (no template), Lane 6: Positive control (Dried plant sample containing CaMV), Lanes 7, 8, 9, and 10: four cauliflower plants with CaMV + CMV mixed infections\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/eabd119bc35e895b22a6b7d5.png"},{"id":94986375,"identity":"fc0bda88-9e13-46d5-b3d2-ffa69e40f8ac","added_by":"auto","created_at":"2025-11-03 07:00:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222577,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoresis in 1% agarose gel of products from PCR with CMV CP specific primers on plants with mixed infections; Lane M: 500 ng Lambda DNA \u003cem\u003eEco\u003c/em\u003eRI + \u003cem\u003eHind\u003c/em\u003eIII, Lane 1: Positive control (plasmid containing CMV CP gene), Lane 2: Negative control (no template), Lanes 3, 4, 5, and 6: four cauliflower plants with CaMV + CMV mixed infections\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/e4b8ab4b74d66442d6e9e7bb.png"},{"id":94986567,"identity":"8cc80c14-eb88-44c7-892e-2c745bd38ca3","added_by":"auto","created_at":"2025-11-03 07:00:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174869,"visible":true,"origin":"","legend":"\u003cp\u003eRestriction analysis on 1% agarose gel of CaMV reverse transcriptase coding DNA amplified by PCR; Lane M: 1Kb Plus DNA Ladder, Lane 1: CaMV RT PCR product (without digestion), Lane 2: CaMV RT digested with \u003cem\u003eNco\u003c/em\u003eI, Lane 3: CaMV RT digested with \u003cem\u003ePst\u003c/em\u003eI, Lane 4: CaMV RT digested with \u003cem\u003eMsp\u003c/em\u003eI\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/a02d75524dbbcdb07911136a.png"},{"id":95524943,"identity":"9a3ed7de-4b1f-4591-bc8c-013b129318d4","added_by":"auto","created_at":"2025-11-10 10:03:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2624607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/1426cec9-6a24-4719-8c4d-266219ccd5a6.pdf"},{"id":94985570,"identity":"141d0fbe-769f-4059-b0d5-52d8811887f5","added_by":"auto","created_at":"2025-11-03 06:58:26","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":85700,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7599836/v1/6c2e6439a82b8bcf5022d96b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evidence suggestive of Cauliflower mosaic virus transcribing complementary DNA from Cucumber mosaic virus RNA in mixed infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe discovery of reverse transcriptase (RT) enzymes in retroviruses in the 1970s revolutionized molecular biology and laid the foundations for retrovirology and cancer biology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This discovery challenged the central tenets of molecular biology which assumed that DNA is transcribed into RNA and ultimately into proteins. RTs were described as nucleic acid polymerases that are capable of synthesizing complementary DNA (cDNA) using RNA as a template [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These enzymes require a specific type of RNA oligonucleotide as a in the cells primer to initiate DNA synthesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Studies have shown that RTs can also facilitate RNA strand cleavage during the formation of RNA/DNA heteroduplexes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. RT enzymes are found in both plant and animal viruses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition to \u003cem\u003eCaulimoviridae\u003c/em\u003e, RTs play an important role in the replication of members of the families \u003cem\u003eRetroviridae\u003c/em\u003e, \u003cem\u003ePseudoviridae\u003c/em\u003e, \u003cem\u003eMetaviridae\u003c/em\u003e and \u003cem\u003eHepadnaviridae\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It has been shown \u003cem\u003ein vitro\u003c/em\u003e that RTs of retroviruses and long terminal repeat (LTR) retrotransposons can extend the 3' end of their genomes provided that the oligonucleotide that acts as a primer has a complementary at its 3' end to 1, 2, or 3 overhanging nucleotides at the 3' end of the genome of retroviruses and LTR retrotransposons [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eCauliflower mosaic virus\u003c/em\u003e (CaMV) was the first discovered plant pararetrovirus and the first plant dsDNA virus that uses RT to replicate its genome. Reverse transcription in this virus results in the formation of nicked circular DNAs that are converted into episomal (extrachromosomal) DNA in the host cell nucleus [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe genome of CaMV is a circular double-stranded DNA of approximately 8000 bp containing 6\u0026ndash;8 open reading frames (ORF). CaMV virions have T\u0026thinsp;=\u0026thinsp;7 icosahedral symmetry that are enclosed in a polyhedral protein coat of 50 nm in diameter [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].When a \u003cem\u003ecaulimovirus\u003c/em\u003e infects a cell, the dsDNA enters the nucleus where the overlapping break sites in the virus genome are removed, repaired and closed, creating a minichromosome with a fully closed circular DNA [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. With the help of DNA-dependent RNA polymerase, two RNA fragments are transcribed from the minichromosome. The short RNA fragment (19S) is later translated in large quantities into the 58-kDa protein constituting the viroplasm, and the long fragment (35S), which is translated in turn into all the other proteins. Translation of the 19S mRNA results in the production of the P6 protein which accumulates in the numerous cytoplasmic virus factories where translation of other viral proteins takes place [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. P6 a transactivator translation enhancer may also play a role in increasing the translation rate of other genes from 35S mRNA [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. P1 is a movement protein required for cell-to-cell movement of the virus, P2 plays a role as an auxiliary component in aphid transmission, P3 is considered to be associated with viral components, P4 is a viral coat protein (CP) involved in capsidation of the viral genome, and P5 encodes a reverse transcriptase enzyme. The P7 protein has never been identified in a plant system and can be deleted by mutagenesis without affecting virus infection or transmission [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCaMV mainly infects members of the \u003cem\u003eBrassicaceae\u003c/em\u003e family including cauliflower, cabbage, radishes, mustard, turnips, canola and broccoli. Some strains of CaMV (W260 and D4) can also infect \u003cem\u003eSolanaceae\u003c/em\u003e species such as members of the genus Datura and tobacco plants (genus \u003cem\u003eNicotiana\u003c/em\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Depending on the virus variant, host plant, symptoms and type of infection (single or mixed), virus transmission occurs in different forms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. CaMV-infected plants exhibit a diverse range of symptoms including chlorosis, mosaic, vein clearing, vein banding, leaf deformation, and stunting [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCo-infections in plants occur regularly in natural ecosystems. The vulnerability of host plant is often greater in co-infections than in single-infections [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Simultaneous infections with CaMV and \u003cem\u003eCucumber mosaic virus\u003c/em\u003e (CMV) occurs in crops. CMV is one of the most important viruses infecting major agricultural crops including vegetables and it is the type member of the genus \u003cem\u003eCucumovirus\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], globally distributed with the widest host range among plant viruses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] of over 1300 plant species in over 100 families of monocotyledons and dicotyledons plants including most vegetables, ornamentals, and woody and semi-woody plants [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CMV exhibits a variety of symptoms in different hosts and causes systemic infection in most host plants but may be asymptomatic in a number of crops including alfalfa. Among CMV hosts, cucurbits are the most susceptible to CMV [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMembers of \u003cem\u003eCucumovirus\u003c/em\u003e have a tripartite genome and a subgenomic RNA, among which RNA1 and RNA2 are packaged in separate particles, and RNA3 and subgenomic RNA4 in a third particle. The RNA1 and RNA2 collectively code for the RNA polymerase. However, the RNA2-coded protein plays a role in the virus pathogenicity as well, being virus suppressor of RNA silencing (VSR). The RNA3 encodes movement protein and coat protein [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, in addition to investigating symptoms in single and mixed infections with CMV and/ or CaMV, the ability of the CaMV RT in converting RNA to cDNA was also examined.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSymptoms caused by viruses in the host plants\u003c/p\u003e\u003cp\u003eSymptoms in the cauliflower inoculated with CaMV appeared as chlorotic local lesions after 7 days. Over time, these symptoms became systemic and formed a yellow network. The main vein was widened and the leaf size was reduced. The developed fruit was small, yellowed and shrunk (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e- d). However, in CMV- inoculated cauliflower plant, symptoms appeared seven days later than that in CaMV-inoculated cauliflower (14 days after inoculation) and included chlorotic local lesions, vein clearing and vein banding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e- c). In the mixed-infection with CaMV\u0026thinsp;+\u0026thinsp;CMV plant growth was severely reduced, widening of the main vein and the reduction in leaf size occurred very severely. Vein banding, vein clearing and chlorotic local lesions appeared, then the entire leaf blade turned yellow and died. The developed fruit was shriveled and dried over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. e). Symptoms in infected plants are listed separately in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It should be noted that in the mixed-infections, vein clearing and vein banding began to disappear after about 45 days post-inoculation (dpi).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of dsRNA from CMV-inoculated plants\u003c/p\u003e\u003cp\u003eDsRNA extraction was successfully performed from five CMV-infected plants (one as a positive control and four inoculated plants) and one healthy control revealed three viral genomic RNA segments with sizes of ~\u0026thinsp;3.4, ~\u0026thinsp;3.1, and ~\u0026thinsp;2.2 kb corresponding to RNA1, RNA2 and RNA3, respectively. In addition, a fourth sub genomic RNA (RNA4) was also present (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePCR and RT-PCR\u003c/p\u003e\u003cp\u003eResults from PCR using CMV- or CaMV- specific primers from each four-replicative cauliflower plants infected with CaMV\u0026thinsp;+\u0026thinsp;CMV, and infected with CMV or CaMV indicated amplification of the expected fragments from total DNA samples.\u003c/p\u003e\u003cp\u003eIn total, the expected CaMV RT fragment 2040 bp was amplified from four plants with CaMV-infection and from four plants with CaMV\u0026thinsp;+\u0026thinsp;CMV mixed-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). CMV CP cDNA with a size of 675 bp was amplified from four plants with CaMV\u0026thinsp;+\u0026thinsp;CMV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). PCR on denatured dsRNA samples, and total DNA from four cauliflower plants with single CMV-infection did not give the CMV CP cDNA fragment (see Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2 online). However, when reverse transcription was done on the boiled dsRNA from the CMV-infected plants and followed by PCR, a DNA fragment of roughly 675 bp in length was amplified (see Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online). The explanations are briefly given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eObservations and PCR analyses on four replicative cauliflower plants infected with CMV, CaMV, or CMV\u0026thinsp;+\u0026thinsp;CaMV\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInfection\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCaMV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCMV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCMV\u0026thinsp;+\u0026thinsp;CaMV\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSymptoms observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCLL, WMV, RLS,\u003c/p\u003e\u003cp\u003eY and SF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCLL, VC and VB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSRPG, RLS, WMV, VB, VC, CLL, Y and SDF\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCR with CaMV RT\u003c/p\u003e\u003cp\u003eprimers on total DNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2040 bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2040 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCR with CMV CP\u003c/p\u003e\u003cp\u003eprimers on total DNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNBE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e675 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCR with CMV CP primers\u003c/p\u003e\u003cp\u003eon denatured dsRNA from\u003c/p\u003e\u003cp\u003eCMV-infected plants\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNBE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRT-PCR with CMV CP primers\u003c/p\u003e\u003cp\u003eon denatured dsRNA from\u003c/p\u003e\u003cp\u003eCMV-infected plants\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e675 bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAbbreviations; CLL: Chlorotic local lesions, WMV: Widened main vein, RLS: Reduced leaf size, Y: Yellowing, SF: shrunk fruit, VC: Vein clearing, VB: vein banding, SRPG: Severe reduction in plant growth, SDF: Shrinkage and drying of fruit, NBE: No band in electrophoresis and -: PCR or RT-PCR was not performed\u003c/p\u003e\u003cp\u003eEnzymatic digestion\u003c/p\u003e\u003cp\u003eDigestion with the \u003cem\u003eNco\u003c/em\u003eI on the amplified fragment corresponding to CaMV RT gene produced two fragments with sizes of 499 bp and 1535 bp, with the \u003cem\u003ePst\u003c/em\u003eI two fragments with sizes of 282 and 1752 bp, and with the \u003cem\u003eMsp\u003c/em\u003eI three fragments of 279, 232, and 1521 bp which all corresponded with predicted restriction map the CaMV RT sequence available in GenBank, demonstrating that the PCR-amplified product belonged to CaMV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eResults from sequencing\u003c/p\u003e\u003cp\u003eSequencing of the PCR-amplified CMV CP fragment showed that it was related to the CMV CP gene. Likewise, BLAST analysis of the nucleotide data from sequencing CaMV RT DNA confirmed its identity as CaMV.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSimultaneous infection with two or more viruses in a plant is a common phenomenon that, in addition to affecting the severity of symptoms, may affect the concentration of infecting viruses [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This may be due to the effects on the replication of one virus by the second virus [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. There have been numerous reports of the mixed-infection of CaMV with TuMV and CMV in various plants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, we predicted that in the presence of CaMV, due to the conversion of the CMV RNA genome to cDNA, a decrease in the concentration of the CMV genome would occur and consequently the symptoms caused by CMV in the mixed-infection would be reduced. This prediction was consistent with our observations because the CaMV- related symptoms in the single-infection including severe reduction in leaf size and widening of the main vein were still visible in the mixed-infection with CaMV\u0026thinsp;+\u0026thinsp;CMV whereas the CMV-associated vein clearing and vein banding started to disappear in the mixed-infection after about 45 dpi. These results are consistent with the findings of that in simultaneous infection with CaMV\u0026thinsp;+\u0026thinsp;TuMV in rapeseed, the concentration of TuMV genome decreased after 35 dpi and its symptoms disappeared after a period of time [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], therefore suggesting the adverse effect of CaMV on an RNA virus (TuMV).\u003c/p\u003e\u003cp\u003eThe results of this study indicate that the CaMV somehow overcomes the plant's defense system and continues to generate and assemble its own genome. As a support to this claim, it has been reported that CaMV P6/translational activator/viroplasmin (TAV) neutralizes the plant defense mechanism through suppressing RNA silencing and acts towards the assembly and transmission of CaMV particles [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; or according to Hoffmann et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] the increased severity of symptoms caused by CaMV is explained by the fact that P6 reduces stress granules (SGs) accumulation after stress by suppressing SGs, located in viral factories (VFs), thereby leading to an increase in P6 condensation during the course of infection which ultimately leads to a progressive shift in selected P6 functions including development of the disease symptoms.\u003c/p\u003e\u003cp\u003eThe reduction in the severity of CMV symptoms in the mixed infections compared to that in single CMV-infections suggests the adverse effect of the second virus. As CaMV disrupts the host's RNA degradation machinery by evading the host cell's mRNA surveillance system, this leads to the accumulation of CaMV mRNA and potentially hinders the replication of co-infecting RNA viruses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Given the ability of SGs to influence mRNA storage, degradation, translation and protein signaling, CaMV by having P6 in VFs disrupts the efficiency and function of SGs, thereby in addition to interfering with the replication of RNA viruses it prevents the expression of RNA virus genes in the mixed infection. This consequently leads to the attenuation of symptoms (vein clearing and vein banding) associated with RNA viruses in the mixed-infections. However, P6 through physical interactions with translation regulators, especially eIF3g (eukaryotic translation initiation factor 3g), leads to the expression of the CaMV genome and causes disease [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, CaMV's impact on RNA virus expression and replication can influence the overall disease symptoms observed in plants infected with both viruses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A similar situation might have occurred in the mixed-infection in the present study.\u003c/p\u003e\u003cp\u003eAs to the reverse-transcribing activity of CaMV on CMV, PCR on total DNA from the dually-infected plants resulted in amplification of the expected DNA of 675 bp suggesting the reverse transcription of CMV RNA by CaMV. Alongside this, to ascertain that it is not the reverse transcriptase activity of Taq DNA polymerase giving the amplification, PCR was performed directly on denatured dsRNA from CMV-infected plants and, as a result, no DNA were amplified (see Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online) suggesting that under the PCR conditions applied in this study, the DNA polymerase did not have reverse transcriptase activity. To test the hypothesis that plant endogenous RTs from retrotransposons could act non-specifically under stress and convert RNA to cDNA [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], PCR was performed with CMV CP primers on total DNA from plants that had single CMV-infection which did not result in any amplification (see Supplementary Fig. S2 online).\u003c/p\u003e\u003cp\u003eWhen the PCR-amplified CMV CP cDNA and CaMV RT DNA were sequenced and the resulting data subjected to BLAST analysis in NCBI, the identities of the amplified fragments were further proven to be of CMV CP and CaMV RT. This also additionally showed the reverse-transcription of CMV RNA by CaMV RT.\u003c/p\u003e\u003cp\u003eBecause plus-strand CMV RNAs are distributed throughout the plant cytoplasm [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] just as P5-CaMV being therein [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] the subjection of CMV RNAs in the cytosol to the reverse transcription activity of CaMV RT would not be remote from happening especially given the fact that PCR directly amplified CMV CP cDNA without \u003cem\u003ein vitro\u003c/em\u003e reverse transcription.\u003c/p\u003e\u003cp\u003eWe demonstrated that in co-infection of an RNA virus with CaMV, the RNA virus genome is converted to cDNA by CaMV RT. Because individual CaMV RT molecules are able to perform full polymerase functions to convert ssRNA to dsDNA, this could be related to the flexibility of the loop region in CaMV POL catalytic site that allows the phosphate backbone of the primer DNA, which is the same methionine found in plant cells, to contact the side chain of residue Arg96 from the B0 helix's CaMV POL (flexible loop region in CaMV POL catalytic site that is involved in cDNA synthesis), leading to greater stabilization of the primer strand and bringing it closer to a catalytic location and initiating synthesis of the RNA virus cDNA strand [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study demonstrates, for the first time, the \u003cem\u003ein vivo\u003c/em\u003e activity of CaMV-encoded reverse transcriptase in transcribing CMV RNA within plant cells. An interesting aspect of this finding is that CMV RNA is transcribed by CaMV in the mixed infection which opens a new insight into the interaction between the viruses. At the same time, the RNA virus is attenuated due to formation of RNA\u0026thinsp;+\u0026thinsp;DNA hybrids which reduces significantly the amount of the RNA to go under expression. As this phenomenon takes place in the mixed infection it makes possible to detect the RNA virus directly by PCR without the need for \u003cem\u003ein vitro\u003c/em\u003e transcription.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePlant host and growth conditions\u003c/p\u003e\u003cp\u003eCertified seeds of cauliflower, \u003cem\u003eBrassica oleracea\u003c/em\u003e (95% germination rate) sourced from Thailand were used to assess symptoms development following single infections with CaMV, CMV and mixed-infection with CaMV\u0026thinsp;+\u0026thinsp;CMV. Plants were grown under controlled environmental conditions of 22\u0026ndash;28\u0026deg;C, 30\u0026ndash;40% relative humidity, and a 16 h light/8 h dark photoperiod.\u003c/p\u003e\u003cp\u003eSource of virus and glasshouse inoculation\u003c/p\u003e\u003cp\u003eCMV isolate PAK-17-2 from previous studies in our Laboratory whose identity was established by serological or molecular tests, was used. About CaMV, the isolate DAR78694 was used as the source virus.\u003c/p\u003e\u003cp\u003eTo test the efficiency of CaMV RT in converting CMV RNA into complementary DNA in a Cauliflower host plant, at least four biological replicates were used for each treatment: Inoculations with CMV, CaMV or simultaneous infection with CaMV\u0026thinsp;+\u0026thinsp;CMV in \u003cem\u003eBrassica oleracea var. botrytis\u003c/em\u003e. The controls included mock inoculated (with buffer) and intact cauliflower. After growing under the greenhouse conditions, the plants were inoculated mechanically at the 2\u0026ndash;3 leaf stage with extract of the infected plant material using 0.1 M potassium phosphate buffer (pH 7.2) and kept under greenhouse conditions for symptom observation and subsequent molecular analyses. Infected plant materials were used as viral source and mechanically inoculated on healthy host plants to propagate and maintain the viruses.\u003c/p\u003e\u003cp\u003eFollowing the mechanical inoculations, the plants with single (CaMV or CMV) and mixed (CaMV\u0026thinsp;+\u0026thinsp;CMV) infections as well as control plants were monitored daily to assess symptom development. Upon the appearance of visible symptoms in each of the inoculations, they were recorded. Comparative analysis was then performed to determine differences in symptom expression between the single and mixed-infections.\u003c/p\u003e\u003cp\u003eExtraction of double-stranded RNA from CMV-inoculated plants\u003c/p\u003e\u003cp\u003e Double-stranded (dsRNA) extraction from the CMV-infected plants was performed according to the method of Delpasand Khabbazi et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A 250 mg plant tissue infected with the virus was extracted in a mortar with 1 ml dsRNA extraction buffer (200 mM Tris, 500 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 3% SDS, 10% Ethanol, 1% 2-Mercapto ethanol). After vortexing and incubation for 10 minutes at 37\u0026deg;C, chloroform was added in 1:1 ratio to remove plant proteins. After centrifugation and removal of plant tissue, 0.2 ml absolute ethanol and 15 mg CF-11 cellulose were added to the recovered aqueous phase to trap dsRNAs. Next, washing step was performed with 1 ml washing buffer (1X STE [100 mM NaCl, 10 mM Tris-HCL, 1 mM EDTA, pH 8.0]/16% ethanol) and then dsRNA was eluted using 150 \u0026micro;l elution buffer (1X STE), incubated at -20 \u003csup\u003eo\u003c/sup\u003eC for an hour and subjected to centrifugation at 12000 RPM for 20 minutes. Finally, the precipitate obtained from centrifugation was suspended in 30 \u0026micro;l ddH\u003csub\u003e2\u003c/sub\u003eO. To assess the presence, integrity, and molecular weight of the extracted dsRNA, samples were subjected to horizontal electrophoresis in a 1% agarose gel using TBE (Tris, borate and EDTA) buffer for approximately 90 minutes. Gel was visualized over UV light using a gel documentation system (Qiagen, Tehran, Iran).\u003c/p\u003e\u003cp\u003eTotal DNA extraction\u003c/p\u003e\u003cp\u003eTotal DNA was extracted from Cauliflower plants inoculated with CaMV, CaMV\u0026thinsp;+\u0026thinsp;CMV, CMV, and from control plants using the method described by Edwards et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Briefly, 20 mg of infected or control leaf tissue was homogenized in 400 \u0026micro;L of extraction buffer (200 mM Tris-HCl, pH 7.5; 250 mM NaCl; 25 mM EDTA; 0.5% SDS). The homogenate was vortexed thoroughly, and plant debris was removed by centrifugation at high speed. The resulting supernatant was mixed with 300 \u0026micro;L of isopropanol to precipitate DNA, followed by centrifugation. The DNA pellet was air-dried and resuspended in 100 \u0026micro;L of nuclease-free distilled water (ddH₂O).\u003c/p\u003e\u003cp\u003ePrimer design\u003c/p\u003e\u003cp\u003eA pair of specific primers based on the reverse transcriptase gene sequence of CaMV isolate DAR78694 with accession number KX904357, was designed using Perl Primer Version 1.1.21 software [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] to identify CaMV. Another pair of specific primers but for the amplification of CMV coat protein gene was according to Koolivand et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The primer sequences are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequences of primers used in this study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimers name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequences\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRegion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCaMVRTF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'- AAGTGATGGATCCTCTACTTCTG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCaMVRTR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'- TCAATTAGGAGCTCACCTTATTGA-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCMVCPF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-AGTGGATCCATGGACAAATCTGAATCAACCAG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCMVCPR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-AACTTCGAATTC(G/T)ACTGGGAGCAC(C/T)CC(A/G)GACGTGGG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eReverse transcription polymerase chain reaction (RT-PCR)\u003c/p\u003e\u003cp\u003eRT-PCR was performed with CMV CP-specific primers. for the reverse transcription reaction, 1 \u0026micro;l of the forward primer (10 pmol/\u0026micro;l) was mixed with 1 \u0026micro;l of ddH\u003csub\u003e2\u003c/sub\u003eO, and 2 \u0026micro;l out of the 30 \u0026micro;l extracted dsRNA in a microtube and placed at 70\u0026deg;C for 5 min. The samples were immediately transferred to ice, then 6 \u0026micro;l of a mixture containing 2 \u0026micro;l of 10X PCR reaction buffer, 1 mM dNTP, 40 units of RNAsin, and 200 units (0.5 \u0026micro;l) of M-Mulv reverse transcriptase (Kiagene Fanavar Aria, Tehran, Iran) were added into the tube and was placed at room temperature for 15 min before placing in a thermocycler (Q-Sat 24, Hain Lifescience UK Ltd, England) at 42\u003csup\u003eo\u003c/sup\u003eC for 60 min. After the completion, the reverse transcriptase was inactivated by placing the tube at 70\u0026deg;C for 10 min.\u003c/p\u003e\u003cp\u003epolymerase chain reaction (PCR)\u003c/p\u003e\u003cp\u003ePCR was performed separately with each of the CMV and CaMV virus-specific primer sets (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in a final volume of 20 \u0026micro;l, containing 10 \u0026micro;l master mix (Sinaclon, Tehran, Iran), 10 pmol each forward and reverse primer for each virus, 10 ng of template DNA, and 7.6 \u0026micro;l of ddH\u003csub\u003e2\u003c/sub\u003eO. The thermal program applied for PCR-detection of CaMV was 94\u003csup\u003eo\u003c/sup\u003eC for 3 min, followed by 30 cycles of 94\u003csup\u003eo\u003c/sup\u003eC for 40 s, 45\u003csup\u003eo\u003c/sup\u003eC for 60 s and 72\u003csup\u003eo\u003c/sup\u003eC for 150 s, and one additional cycle of 72\u003csup\u003eo\u003c/sup\u003eC for 7 min; but for CMV the thermal profile included 94\u003csup\u003eo\u003c/sup\u003eC for 1 min, followed by 35 cycles of 94\u003csup\u003eo\u003c/sup\u003eC for 30 s, 50\u003csup\u003eo\u003c/sup\u003eC for 45 s and 72\u003csup\u003eo\u003c/sup\u003eC for 60 s, with an additional cycle of 72\u003csup\u003eo\u003c/sup\u003eC for 5 min. Four \u0026micro;l of each PCR product was electrophoresed on 1% agarose gel containing ethidium bromide in 0.5x TBE buffer. Electrophoresis was performed at 100 V and 24 mA, and the results were documented by a gel documentation device as mentioned earlier in this paper.\u003c/p\u003e\u003cp\u003eConfirmation of the identity of the CaMV RT gene with restriction analysis\u003c/p\u003e\u003cp\u003eThe PCR-amplified CaMV RT fragment was digested with three different \u003cem\u003eMsp\u003c/em\u003eI, \u003cem\u003ePst\u003c/em\u003eI, and \u003cem\u003eNco\u003c/em\u003eI restriction enzymes. Three separate reactions, each containing 5 Units of enzyme, 100 ng of PCR product, and 4 \u0026micro;l of appropriate 10X restriction buffer, were digested by incubation for 1 hour at 37\u0026deg;C. The digested products were then electrophoresed in a 1% agarose gel according to the above-mentioned method.\u003c/p\u003e\u003cp\u003eNucleotide sequence determination\u003c/p\u003e\u003cp\u003eThe PCR-amplified CMV CP cDNA and CaMV RT DNA were subjected to purification and sequencing with the forward primer. The sequencing was done by Pishgam Company (Tehran, Iran). The generated sequences were compared with the counterpart sequences data available in Genbank (NCBI).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCRediT authorship contribution statement\u003c/h2\u003e\u003cp\u003eHakimeh Ighani Mayan: Performed the lab work, preparing first draft of the manuscript and data curation; Nemat Sokhandan Bashir acted as the chief investigator: Writing, Editing and conceptualization; Davod Koolivand acted as the associate supervisor: Reviewing and editing.\u003c/p\u003e\u003ch2\u003eCompeting Interests Statement\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was funded by the Research Management Office of the University of Tabriz.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.I.M. Performed the lab work, preparing first draft of the manuscript and data curationN.S.B. Acted as the chief investigator: Writing, Editing and conceptualizationD.K. Acted as the associate supervisor: Reviewing and editing\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe express our gratitude to the Research Management Office of the University of Tabriz for their financial support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe extracted genomes DNAs and dsRNAs from all plants with different viral infections in this study are maintained in 1.5 ml microtubes with respective extraction codes in the Virology and Genetic Engineering Laboratory the University of Tabriz. The resulting PCR products are likewise stored at the same location. Plant samples inoculated with different viruses are stored in microtubes after drying in CaCl2.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmack, S. C. \u0026amp; Antunes, M. S. CaMV35S promoter-A plant biology and biotechnology workhorse in the era of synthetic biology. \u003cem\u003eCur. Plant Biol.\u003c/em\u003e 24, 100179. (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cpb.2020.100179\u003c/span\u003e\u003cspan address=\"10.1016/j.cpb.2020.100179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBak, A. \u0026amp; Emerson, J. B. Cauliflower mosaic virus (CaMV) Biology, Management, and Relevance to GM Plant Detection for Sustainable Organic Agriculture. \u003cem\u003eFront. Sustain. Food Syst.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 1\u0026ndash;8 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaltimore, D. \u0026amp; Smoler, D. Primer requirement and template specificity of the DNA polymerase of RNA tumor viruses. \u003cem\u003eProc. Nat. 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The ecology of Cucucmber mosaic virus and sustainable agriculture. \u003cem\u003eVirus Res.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 9\u0026ndash;21 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cauliflower, CaMV, Reverse transcriptase, cDNA, PCR and CMV","lastPublishedDoi":"10.21203/rs.3.rs-7599836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7599836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to investigate if \u003cem\u003eCauliflower mosaic virus\u003c/em\u003e (CaMV) reverse transcriptase (RT) can make complementary DNA (cDNA) from \u003cem\u003eCucumber mosaic virus\u003c/em\u003e (CMV) genomic RNAs in the mixed infection. First, symptoms of single infection with CaMV or CMV, and dual-infection with CaMV\u0026thinsp;+\u0026thinsp;CMV were investigated in cauliflower (\u003cem\u003eBrassica oleracea\u003c/em\u003e) plants. CMV symptoms disappeared in the mixed-infection after about 45 days post infection (dpi). The efficiency of the CaMV RT in converting CMV RNA genome to cDNA was investigated by extracting total DNA from the dually-infected plants and subjecting to polymerase chain reaction (PCR) by the use of CMV coat protein (CP)-specific primers which gave the anticipated\u0026thinsp;~\u0026thinsp;675 bp DNA. To further verify the identity of the PCR products, the amplified CMV CP cDNA or CaMV RT DNA were sequenced and the resultant nucleotide data were compared with the counterpart genomic region of other isolates of the viruses available in the Genbank. PCR assays on total DNA from plants with single CMV-infection gave no amplification which ruled out the nonspecific activity of plant endogenous RTs in converting the RNA to cDNA. Also, possible amplification of CMV CP cDNA from traces amount of CMV RNA from the infected plants by a weak reverse transcription activity of \u003cem\u003eTaq\u003c/em\u003e DNA polymerase was ruled out by performing PCR on the virus RNA which did not yield any DNA. The results showed CMV-associated symptoms attenuation in the presence of CaMV. This study opens up new insights into the interaction between the viruses. This is the first report, to our knowledge, of cDNA synthesis from an RNA virus by CaMV RT in the mixed-infection.\u003c/p\u003e","manuscriptTitle":"Evidence suggestive of Cauliflower mosaic virus transcribing complementary DNA from Cucumber mosaic virus RNA in mixed infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 14:50:29","doi":"10.21203/rs.3.rs-7599836/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e1431aa-5e3d-4935-9ea8-63da35e651f5","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57090492,"name":"Biological sciences/Biological techniques"},{"id":57090493,"name":"Biological sciences/Biotechnology"},{"id":57090494,"name":"Biological sciences/Genetics"},{"id":57090495,"name":"Biological sciences/Microbiology"},{"id":57090496,"name":"Biological sciences/Molecular biology"},{"id":57090497,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-11-07T05:53:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 14:50:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7599836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7599836","identity":"rs-7599836","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}