Alu and murine B1. Well-defined SINE1 has been found only from Euarchontoglires, a group of mammals, in contrast to the wide distribution of SINE2, which has a tRNA-derived sequence, from animals to plants to protists. Both Alu and B1 are mobilized by L1-type non-LTR retrotransposons, which are the only lineage of autonomous non-LTR retrotransposons active in these mammalian lineages.Short interspersed elements (SINEs) are ubiquitous components of eukaryotic genomes. SINEs are composite transposable elements that are mobilized by non-long terminal repeat (non-LTR) retrotransposons, also called long interspersed elements (LINEs). The 3′ part of SINEs usually originated from that of counterpart non-LTR retrotransposons. The 5′ part of SINEs mostly originated from small RNA genes. SINE1 is a group of SINEs whose 5′ part originated from 7SL RNA, and is represented by primate Eptatretus burgeri genome. This SINE1 family, designated SINE1-1_EBu, is young, and is transposed by RTE-type non-LTR retrotransposon, not L1-type. Comparison with other SINE families from hagfish indicated the birth of SINE1-1_EBu through chimera formation of a 7SL RNA-derived sequence and an older tRNA-derived SINE family. It reveals parallel evolution of SINE1 in two vertebrate lineages with different autonomous non-LTR retrotransposon partners. The comparison between two SINE1 lineages supports that the RNA secondary structure of the Alu domain of 7SL RNA is required for the efficient retrotransposition.Here a new lineage of SINE1 is characterized from the seashore hagfish The hagfish SINE1 is the first evident SINE1 family found outside of Euarchontoglires. Independent evolution of SINE1 with similar RNA secondary structure originated in 7SL RNA indicates the functional importance of 7SL RNA-derived sequence in the proliferation of SINEs. Short interspersed elements (SINEs) are composite mobile elements that can mobilize dependent on the help of counterpart long interspersed elements (LINEs), also called non-long terminal repeat (non-LTR) retrotransposons , 2. SINEAlu from humans and B1 from mice, belong to SINE1. A putative SINE1 family was also found from marsupials [P7SL_MD in Repbase, is composed of a full-length 7SL RNA and a 3’ poly A tail. It is structurally indistinguishable from retrocopies of 7SL RNAs. P7SL_MD was multiplicated into over 12,000 copies in the common ancestor of marsupials [The heads of SINEs typically originated from non-coding RNAs such as 7SL RNA, tRNA, 5S rRNA or small nuclear RNA (snRNA) , 4. The rsupials , but thirsupials . An exparsupials .Alu is the abundant repetitive sequence accounting for 11% of the human genome [Alu sequence is a dimer composed by two 7SL RNA-derived monomer units connected by an A-rich linker [Alu elements are considered to have been originally generated through a fusion between free left Alu monomer (FLAM)-C and free right Alu monomer (FRAM) [Alu monomers, fossil Alu monomer (FAM) and FLAM-A likely predated FLAM-C and FRAM [FLAM-A is nearly identical to the rodent SINE family PB1 [FLAM-A/PB1 was likely born in the common ancestor of Euarchontoglires [B1 [Otolemur garnettii, designated as GarnAlu [Platy-1) were also found from the common marmoset Callithrix jacchus [h linker . Dimericr (FRAM) . Two othand FRAM , 15. FLAmily PB1 , and thutoglires . Most roires [B1 . Tree shires [B1 . SINE fa GarnAlu . Active  jacchus . All SINAlu and B1 are mobilized by the two proteins, L1ORF1p and L1ORF2p, encoded by L1 [L1 is distributed widely in eukaryotes [L1 lineages which are active or have been active recently. Unlike other non-LTR retrotransposons, mammalian L1 does not require the conserved RNA secondary structure in the 3′ UTR for the recognition of the template RNA for reverse transcription [Alu and B1 RNAs as well as any polyadenylated mRNAs, can be mobilized by the L1 machinery. Both Alu and B1 are composed solely by the sequences of 7SL RNA. The relaxed recognition by the L1 machinery is also seen in plants, but it is considered that the machineries of non-mammalian L1 as well as of other non-LTR retrotransposons recognize the RNA secondary structure besides the 3’-polyA tail [ed by L1 . L1 is dkaryotes . Many macription . Due to lyA tail .Alu domain composed by the 5′ and 3′ regions of 7SL RNA. The binding of Alu domain with SRP9/14 is required for the retrotransposition [Alu RNA with SRP9/14 is proposed to be the mechanism of the efficient trans-mobilization by the L1 machinery.SINE1 families from Euarchontoglires share the sequence corresponding to the regions 1-63, 76-83, and 267-299 of the human 7SL RNA . 7SL RNAposition . The binEptatretus burgeri genome. This SINE1 family, designated SINE1-1_EBu, is young, and seems transposed by an RTE-type non-LTR retrotransposon family, not L1-type. It reveals parallel evolution of SINE1 in two vertebrate lineages with different autonomous non-LTR retrotransposon partners. The comparison between two SINE1 lineages indicates that the RNA secondary structure of the Alu domain is required for the efficient retrotransposition.Here, a new SINE1 lineage is characterized from the seashore hagfish Alu families of SINEs. Refinement of repeat consensus sequence revealed that it is a SINE family whose 5′ region shows strong overlap to 7SL RNA genes . The box B is different by 2 nucleotides between human and hagfish, AGTTCTGGGCT and AGCTCTGCGCT (changes are in bold), respectively.The 5′ 96-bp sequence of SINE1-1_EBu is similar to the 3′ terminus of 7SL RNA, while it shows less sequence similarity to the human 7SL RNA gene or SINE1 families in Euarchontoglires . Two families (RTE-2_EBu and RTE-4_EBu) of non-LTR retrotransposons which show > 90% sequence identity to the parts of SINE1-1_EBu, were reconstructed from RTE-related repeats in the RepeatModeler outputs  upon integration, though the lengths of TSDs are not uniform  [CR1 clade of non-LTR retrotransposons, SINE3-1_TC seems mobilized by the I clade of non-LTR retrotransposons, and HaSE3 seems mobilized by the RTE clade of non-LTR retrotransposons [Theoretically, SINE1 can be born multiple times independently, as SINE2 and SINE3. Multiple independent events of birth of SINE2 are well supported by the different origins of tRNA-derived heads and the very wide distribution of SINE2 . The aliamilies) , 34, 35.nsposons .Alu and B1 are mobilized by L1-type non-LTR retrotransposons [L1 is an exception. Mammalian L1 can mobilize any RNAs with poly A tail [Alu and B1 are not similar to the 3′ ends of L1 except poly A tails.nsposons , 21. Regy A tail , includiy A tail –39. The FLAM-A/PB1, which was an internally deleted derivative of 7SL RNA but had a poly A tail. The internal deletion as well as subsequent deletions/duplications distinguishes SINE families with 7SL RNA-derived sequences from retrocopies of 7SL RNA. P7SL_Cpo found from the guinea pig genome corresponds to the full-length 7SL RNA sequence followed by a polyA tail [P7SL_Cpo should have originated independently from other SINE1 families, but the possibility that it corresponds to a set of retrocopies of 7SL RNA cannot be excluded. It is known that various types of small RNAs, such as small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA) generate retrocopies which is composed of the full-length or partial RNA sequence and a 3’-poly A tail [P7SL_Cpo is transposition-competent, is needed to establish the classification of P7SL_Cpo as a SINE1 family. P7SL_MD from marsupials is structurally almost identical to P7SL_Cpo despite the sequence differences due to the divergence between these two groups of mammals [P7SL_Cpo and P7SL_MD to be recognized as SINE1 families.All SINEs with 7SL RNA-derived sequences found in euarchontoglires can be considered to be descendants of a single ancestral SINE1 family, born in the common ancestor of Euarchontoglires [lyA tail . P7SL_Cpy A tail , 38, 40. mammals . There iSINE1-1_EBu was born independently from Alu and B1. SINE1-1_EBu appears mobilized by RTE, based on the sequence similarity of the 3′ tail of SINE1-1_EBu with that of RTE-2_EBu. The internal deletion of 7SL RNA sequence in SINE1-1_EBu clearly excludes the possibility that it is a chimeric retrocopy of 7SL RNA and RTE-related non-LTR retrotransposons. Independent deletion events of the middle region of 7SL RNA, corresponding to the S domain, support no or little functional contribution of the S domain to the SINE proliferation. In contrast, the parallel conservations of base-pairing and the secondary structure of the Alu domain indicate the functional importance of the Alu domain in the SINE proliferation. The conservation of Alu domain is indicated to be linked with the efficient inclusion of Alu RNA in the L1 retrotransposition machinery [Alu domain in the evolution of SINE1-1_EBu.It is obvious that achinery . SimilarSINE1-1_EBu, the first evident SINE1 family outside of Euarchontoglires, reveals the independent, parallel evolution of 7SL RNA-derived SINEs. The conservation of secondary structure of Alu domain in independent SINE1 families indicates the functional importance of ternary structure of Alu domain bound to SRP9/14 in the proliferation of SINEs.The finding of E. burgeri (Eburgeri_3.2) were downloaded from NCBI Assembly database (https://www.ncbi.nlm.nih.gov/assembly) on March 14, 2018. RepeatModeler (http://www.repeatmasker.org/RepeatModeler/) and Repbase [Ciona intestinalis (accession number: HG323729) as a query.The genome sequence of inshore hagfish  Repbase  were use Repbase  with theFLAM-C was predicted based on that of human 7SL RNA and AluY reported in [SINE1-1_EBu was predicted based on the sequence alignment and the secondary structure predicted at Web Servers for RNA Secondary Structure Prediction (https://rna.urmc.rochester.edu/RNAstructureWeb/).Secondary structure of orted in . SecondaAdditional file 1: Figure S1. Target site duplications (TSDs). Figure S2. Sequence alignment of UCON3 domains of SINEs. Figure S3. Sequence alignment of tRNA-derived head regions of hagfish SINEs. Figure S4. Sequence alignments of body domains of hagfish SINEs. Figure S5. Sequence alignments of tails of hagfish SINEs. Figure S6. Age distribution of hagfish SINE families.Additional file 2: Data S1. Consensus sequences of transposable elements characterized in this study.


Monilinia fructigena followed by Sclerotinia borealis, S. sclerotiorum, and Botrytis cinerea in the mummified peach fruits. Moreover, we found a high number of Proteobacteria, including Frateuria aurantia, Neoasaia chiangmaiensis, Robbsia andropogonis, and Ewingella Americana. Furthermore, we identified several viruses and viroids. Bacteriophages were identified by DNA- and RNA-sequencing, while viruses and viroids with RNA genomes were identified by only RNA-sequencing. Moreover, we identified a novel mycovirus referred to as Monilinia umbra-like virus 1 (MULV1) from M. fructigena. Our results revealed the co-inhabitance of fungi and bacteria in the mummified peach fruits, although dominant microorganisms were present. RNA-sequencing revealed that several fungal and bacterial genes were actively transcribed. Comparative analyses suggested that RNA-sequencing provides more detailed information on microbial communities; however, combining DNA- and RNA-sequencing results increased the diversity of microorganisms, suggesting the importance of databases and analysis tools for microbiome studies. Taken together, our study provides a comprehensive overview of microbial communities in mummified peach fruits by DNA shotgun sequencing and RNA-sequencing.The dried peach fruits clinging to peach trees or lying on the ground nearby are known as mummified peach fruits. Here, we examined the microbiome communities of three different mummified peach fruits from the nectarine cultivar “Hahong” by DNA- and RNA-sequencing. We found the dominance of  Prunus persica (L.) Batch) is a member of the genus Prunus in the family Rosaceae. Although a large number of imported fruits have replaced domestic fruits in Korea, the peach fruits cannot be imported due to their short storage period. In Korea, the peach is the third important fruit based on the fruit production and fresh peach fruits are usually produced from July to October. Diverse pathogens and insects are major problems for cultivation of peach trees. Of them, fungal pathogens are regarded as the most severe pathogen, resulting in the loss of quality and quantity of peach production. In particular, it is important to control postharvest fungal diseases in the peach fruit markets [The peach is one of famous stone fruits such as apricot, Japanese mume, plum, and cherry. The peach fruits are widely consumed as fresh or canned peaches. Peach ( markets .Monilinia fructicola and M. laxa) [Botrytis cinerea) [Alternaria spp. [Aspergillus spp. [Cladosporium spp. [Penicillium spp.) [Fusarium species [M. fructicola or M. laxa. Brown rot caused by M. fructicola causes serious damage on the production of peach fruits and can survive the winter in the mummified peach fruits, producing spores that can infect blossoms and young shoots [To date, several fungal pathogens in peach fruits have been identified. For example, brown rot (M. laxa) , grey mocinerea) , Alternaria spp. , Aspergilus spp. , Cladospium spp. , and bluum spp.)  have bee species . Of themg shoots .Monilinia spp. causing brown rot in stone fruit trees [The precise diagnostic tools are important to manage fungal diseases. One of conventional methods to diagnose fungal diseases is the culture dependent approach . Cultivait trees .Dried peach fruits clinging to the peach tree or lying on the ground carrying fungi are called as mummified peach fruits. The mummified fruits are a major source for fungal diseases in peach trees. However, little is known for microbial pathogens in mummified peach fruits. In this study, we revealed fungal and bacterial communities in the mummified peach fruits by DNA shotgun sequencing and RNA sequencing.During summer, we can observe many peach fruits infected by fungi clinging to the peach branch in orchards A,B. We ck-mer-based approach with an efficient memory usage [Pseudomonas spp. were dominantly identified in D1 library  protein database for sequence annotation using DIAMOND program with default parameters [In order to annotate sequenced reads fast, we initially used KRAKEN 2 program which assigns taxonomic labels of sequencing reads using ry usage . The KRA library A while a library B. As comminearum . In addiibraries . In D1 arameters . The BLArameters .In order to observe the taxonomy overview of identified organisms, we combined all annotation results from six libraries according to domain using MEGAN6 program. As we expected, the reads associated with fungi (42.7%) were abundantly present followed by bacteria (30.2%) and plants (19%) A. In addAscomycota were dominantly present in all six libraries (78.2% to 99.08%). In R1 library, Ascomycota (78.24%) was the dominant phylum followed by Mucoromycota (17.61%). Fungi in the phylum Basidomycota were identified in all six libraries. In particular, the proportion of Basidomycota in R1 and R4 was relatively higher than in other four libraries. According to the fungal family, the family Sclerotiniaceae was the most dominant fungal family in all libraries  was the dominant family followed by Pleosporaceae (20.7%), Hypocreaceae (11.8%), Xylariaceae (8.4%), Atheliaceae (2%), and Grifolaceae (1.1%) in D1 library. Based on the genus, Monilinia was the most frequently identified genus in all libraries except R1 (Monilinia in each library ranged from 19.5% (R1) to 58.5% (D4). The Sclerotinia was the second frequently identified genus in most libraries. Interestingly, the proportion for the genus Glomus (42.8%) was the highest in R1 as compared to other libraries. The genus Aspergillus was identified in all six libraries and its proportion was very high in R4 and R7 libraries. In addition, we identified fungi belonging to the genus Penicillium. We identified various fungal species  was the dominant fungal species in RNA libraries  followed by S. sclerotiorum (9.4%), Botrytis cinerea (3.4%), and Talaromyces islandicus (1.9%) , Glomus cerebriforme (2.8%), and Trichoderma atroviride (2.5%)  A. In RNAe (2.5%) B. The prM. fructigena was very high in both DNA (4161 reads) and RNA (2488 reads) (G. cerebriforme was identified from the RNA library (5515 reads) whereas only 38 reads for G. cerebriforme were identified in the DNA library. Similarly, the number of reads associated with Hanseniaspora uvarum (3379 reads) and Lipomyces starkeyi (2384 reads) in the DNA library was much higher than that in the RNA library  C. Intere library C. In bot4 sample D. In S7 rifallax E.M. fructigena, S. borealis, S. sclerotiorum, and B. cinerea were commonly identified in the three DNA libraries. In addition, there were 19 fungal species commonly identified in both D4 and D7 libraries. Each DNA library has unique fungal species such as seven species (D1), six species (D4), and 20 species (D7). A total of seven fungal species including G. cerebriforme, M. fructigena, Stylonychia lemnae, Melampsora larici-populina, S. borealis, S sclerotiorum, and Oxytricha trifallax were commonly identified in three RNA libraries  was the dominant bacterial phylum followed by Bacteroidetes (10.8%), Actinobacteria (9.1%), and Firmicutes (1.5%) after combining all reads associated bacteria (Proteobacteria in each library ranged from 52.7% (R1) to 92.4% (D7). Actinobacteria was the second dominant bacterial phylum in D4 (16.4%), R1 (27.7%), R4 (13.6%), and R7 (7.2%)  and D7 (4.7%). After combining all bacterial reads according to family, Pseudomonadaceae (24.4%) was the dominant family followed by Acetobacteraceae (16.5%), Oxalobacteraceae (12.1%), Erwiniaceae (6.3%), and Flavobacteriaceae (5.3%) (Pseudomonadaceae was the dominant family only in D1 library (34.8%) whereas Acetobacteraceae was the dominant family in D4 (42.5%) and D7 (56.1%) libraries (Microbacteriaceae), R4 (Comamonadaceae), and R7 (Erwiniaceae). Pseudomonas (30.8%), Gluconobacter (9.9%), Janthinobacterium (9.6%), Pantoea (6.6%), and Flavobacterium (4.0%) were major bacterial genera after combining all identified reads associated with bacterial genera (Pseudomonas (42.4%) was the dominant followed by Janthinobacterium (15.5%) , Asaia (27.5%), and Pantoea (22.4%) were major genera. In R7, Pseudomonas (24.5%) and Pantoea (28.2%) were major genera. At the species level, numerous bacterial species were identified. Of them, the representative bacterial species were Frateuria aurantia, Flavobacterium sp. CJ74, uncultured bacterium, Neoasaia chiangmaiensis, and Robbsia andropogonis in all six libraries  B. Bactere (5.3%) C. The Psibraries D. Interel genera E. In D1  (15.5%) F. The geibraries G. In D1,4 and D7 H. UncultPseudomonas virus Andromeda, Pseudomonas virus Bf7, and acinetobacter phage ABPH49 were identified from the D1 library, while two bacteriophages, Pseudomonas phage Phabio and Caudovirales sp., were identified from the D4 library. In the D7 library, Pseudomonas phage OBP was identified. By contrast, DNA viruses as well as RNA viruses were identified from the three RNA libraries. In the R1 library, Sclerotinia sclerotiorum umbra-like virus 3 (SsULV3), a mycovirus infecting fungi with the dsRNA genome, was identified [S. sclerotiorum ourmia-like virus 1 (SsOLV1), S. sclerotiorum ourmia-like virus 2 (SsOLV2), and S. sclerotiorum umbra-like virus 3 (SsULV3). In addition, we identified several plant viruses infecting peach trees, including Asian prunus virus 2 (APV2) and apple chlorotic leaf spot virus (ACLSV). In the R7 library, Pseudomonas phage OBP was the most abundant virus followed by SsOLV1 and SsOLV2.BLASTX against the NR database revealed several virus-associated reads from six libraries . From thentified . In the Sclerotinia sclerotiorum umbra-like virus 3 (SsULV3) from the R1 and R4 libraries. Based on the assembled viral sequences and RT-PCR, we obtained the complete genome sequence for a novel mycovirus referred to as Monilinia umbra-like virus 1 (MULV1) isolate Won 3,444 bp in length. We found that all partial sequences associated with SsOLV1–SsOLV3 were derived from MULV. The genome sequence of MULV1 showed sequence similarity to SsULV3 with 70% coverage and 78.22% nucleotide identity. We found that MULV1 encodes two ORFs, ORF1 (position 154–1407) and ORF2 (1474–3015)  was identified from the R1 and R4 libraries, while peach latent viroid (PLMVd) was identified from all three RNA libraries . Consist74–3015) A. The BL74–3015) B.M. fructigena, we cultured fungi from the mummified peach fruit (S4 sample). Of the recovered fungi on PDA medium, we identified M. fructigena by PCR using internal transcribed spacer (ITS) primers followed by Sanger-sequencing (M. fructigena from the mummified peach fruit (S4 sample) and other peach fruit. RT-PCR and Sanger-sequencing results confirmed the infection of MULV1 in Monilinia species derived from the mummified peach fruit and not the other peach fruit.To confirm the infection of MULV1 in the quencing C,D. UsinRecently, the rapid development of next-generation sequencing techniques facilitates microbiome associated studies. In general, PCR amplification of marker DNA regions such as 16S ribosomal RNA for bacteria and ITS region for fungi followed by NGS might be the most efficient way to reveal microbial communities in a given sample . AmplicoM. fructigena, S. borealis, S. sclerotiorum, and B. cinerea. As we expected, M. fructigena species were dominantly identified in three peach samples. It has already known that diverse Monilinia species causing brown rot disease leads to the dramatic losses of post harvested peach fruits [M. fructicola is a dominant species in America while M. fructigena and M. laxa are frequently identified in Europe [M. fructicola in different stone fruits in Korea has been reported [Monilinia species in stone fruits has not been reported in our knowledge. This is the first report of M. fructigena in stone fruits including peach in Korea. We only collected peach samples in an orchard, therefore, the occurrence of M. fructigena in different regions in Korea should be further examined. Moreover, the orchard in which we collected samples has many different stone fruits including peach, Japanese plum, apricot, and cherry. Therefore, the infection of M. fructigena in other stone fruits in the same orchard might be very high.In this study, we identified a large number of fungal and bacterial species in the mummified peach fruits by DNA shotgun sequencing and RNA-sequencing. To increase accuracy for microorganism identification, we applied stringent detection limit (the number of reads for the identification of a single species more than 100 reads) as suggested by a recent study  and otheh fruits . Geograpn Europe . Previoureported ; howeverB. cinerea was identified in this study. By contrast, we identified several important fungal species using NR database. This result clearly demonstrates the importance of database for microbiome analyses using next-generation sequencing. Although we identified several fungal species, it is possible that there might be several unknown fungal species from the mummified peach fruits. Fortunately, genome and transcriptome data for several Monilinia species causing brown rot disease in stone fruits and pome are currently available. For instance, genomes of M. fructigena [M. fructicola [Monilinia species [Monilinia species have recently published [Monilinia species have been developed [In addition, we analyzed our sequence data using two different approaches, KRAKEN 2 and DIAMOND. KRAKEN 2 was a fast and efficient program for microbiome study such as bacteria . By contuctigena  and M. fucticola . Moreove species . In addiublished . Furthereveloped ,32.In this study, we analyzed fungal and bacterial communities in the mummified peach fruits by two different approaches including DNA shotgun sequencing (Metagenomics) and RNA-sequencing with poly(A) selection (Metatranscriptomics). Although shotgun metagenomics is an efficient way to identify a wide range of species, but it has not been well used for fungal identification. A previous study has successfully identified fungi in the animal metagenomes containing 71 fungal species from 39 datasets using newly developed FindFungi program . Other sMonilinia species. We obtained the complete genome of MULV1 from the R1 and R4 libraries, which were severely infected by M. fructigena species. The phylogenetic tree and BLAST result indicated that MULV1 is closely related with the known SsULV3 from S. sclerotiorum. Both M. fructigena and S. sclerotiorum belong to the family Sclerotiniaceae. This result suggests that the two virus genomes might have diverged from one fungal host.We identified several viruses and viroids by DNA- and RNA-sequencing. The most abundant viruses in the DNA libraries were bacteriophages with double-stranded DNA genomes; however, bacteriophages were identified by RNA-sequencing, which can reveal phage transcripts. Although our experiment was not designed to capture viral RNA genomes, we identified viruses with RNA genomes, such as plant viruses and viroids, by RNA-sequencing with poly(A) selection. Our previous peach virome study using RNA-sequencing with mRNA library also demonstrated that different types of viral genomes, regardless of the presence of poly-A, can be identified by RNA-sequencing with poly(A) selection . Based oLipomyces starkeyi, Hanseniaspora uvarum, and G. cerebriforme between DNA and RNA sequencing results. It was quite hard for us to find correlation between DNA and RNA sequencing. We suppose that the experimental design and sample size might not be appropriate to reveal correlation between DNA- and RNA-sequencing. In general, DNA-sequencing results provide the presence of microorganisms while RNA-sequencing with poly(A) selection results indicate the expression of genes for identified organisms as described previously [In this study, we used the identical sample for DNA and RNA extraction; however, DNA- and RNA-sequencing results were different from each other. For example, there were some bias such as eviously . Intereseviously . Anothereviously . Thus, iWe collected mummified peach fruits in an orchard located in Hoengseong, Gangwon province in February 2019. Some mummified peach fruits were hanged on the branch of peach trees; however, we randomly sampled the mummified peach fruits on the field. The peach trees for the mummified peach fruits were grown in proximity to each other (2-m × 2-m). All mummified peach fruits in this study were derived from the cultivar “Hahong”, which is a kind of nectarine with smoothy skin. The mummified peach fruits were collected from an area with a radius of 3 m. Only specific peach cultivars producing relatively small size of peach fruits had mummified peach fruits. The collected individual sample was kept in a plastic bag and frozen in the presence of liquid nitrogen.The mummified peach fruits were well dried. The frozen samples were too hard to grind, we crushed samples using a hammer. After that, samples were ground in the presence of liquid nitrogen by using mortar and pestle. The fine powder was used for nucleic acid extraction. Genomic DNA was extracted by using Qiagen DNeasy Plant Mini Kit  according to manufacturer’s instructions. Total RNA was extracted by using Fruit-mate for RNA Purification  and the RNeasy Plant Mini Kit for RNA extraction (Qiagen), following the manufacturers’ instructions. We checked the quality and quantity of extracted nucleic acids by agarose gel electrophoresis and NanoDrop Spectrophotometers . Three samples  showing high quality of DNA and RNA were further used for library preparation.From three samples, we prepared three different DNA libraries using NEBNext Ultra II DNA Library Prep with Sample Purification Beads  following manufacturer’s instruction. The extracted total RNAs were subjected for mRNA extraction based on using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer’s instructions. We used the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) in order to prepare three different RNA libraries using following the manufacturer’s instructions. Individual DNA and RNA libraries were indexed using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (NEB). We measured the quality of the generated libraries by using a 2100 Bioanalyzer . A total of six libraries composed of three DNA and three RNA libraries were paired-end (2 × 100 bp) sequenced by Macrogen Co.  using the HiSeq 2000 platform. We deposited all raw sequences in the SRA database in NCBI with respective accession numbers: SRR11531522 (D1), SRR11531521 (D4), SRR11531520 (D7), SRR11531519 (R1), SRR11531518 (R4), and SRR11531523 (R7) under BioProject PRJNA624910.The obtained raw data were trimmed using the PRINSEQ program with the quality score 20 . We did To obtain complete viral genome sequences, de novo transcriptome assemble was carried out using the Trinity program with default parameters as described previously . The ass®-T Easy Vector . Finally, we determined complete genome sequence of the novel mycovirus by Sanger-sequencing.To confirm the genome of a novel mycovirus, we designed primer-pairs, p1r-4121-273F1 (5′-CAAGCAAGGTAAGCTACTGCCTCA-3′) and p1r-4121-3716R1 (5′-GTCATTCCTACCCTGCGCCC-3′) based on the assembled mycovirus genome sequence. cDNA was synthesized using the SuperScript III Reverse Transcriptase . Using the synthesized cDNA as the template, PCR was conducted using EX-Taq polymerase . The amplified PCR product was cloned to pGEMhttps://github.com/rambaut/figtree/releases).We retrieved top 10 RdRp protein sequences showing sequence similarity to the novel mycovirus. A total of 11 RdRp sequences were aligned by the MAFFT program with the L-INS-I option . We trimMonilinia species by PCR using ITS primers (ITS1 5′-TCCGTAGGTGAACCTGCGG-3′ and ITS4 5′-TCCTCCGCTTATTGATATGC-3′) followed by Sanger-sequencing. Total RNAs were extracted from mycelia after 3 days of incubation on PDA medium for the cultured Monilinia species. Based on RdRp sequence of MULV1, we designed primers (MULV1_forward 5′-AATGTGGGGCCAAAGTGGATG-3′ and MULV1_reverse 5′-GTGGCGCCAGACAACACCTT-3′) for RT-PCR. Using the extracted total RNAs, we carried out RT-PCR using SolGent DiaStar One-Step RT-PCR kit  according to manufacturer’s instruction.To confirm that the identified mycovirus was derived from the fungal host, we cultured fungi from the mummified peach samples on potato dextrose agar (PDA) medium. Of the recovered fungi, we identified M. fructigena followed by S. borealis, S. sclerotiorum, B. cinerea, Glomus cerebriforme, Aspergillus steynii, Cryphonectria parasitica, Trichoderma atroviride, and Talaromyces islandicus. In particular, this is the first report of M. fructigena in peach fruits in Korea. Moreover, we found high amount of proteobacteria including Frateuria aurantia, Neoasaia chiangmaiensis, Robbsia andropogonis, and Ewingella Americana. Our results revealed co-inhabitance of fungi and bacteria in the mummified peach fruits although dominant microorganisms were present. Most important things that much of fungal and bacterial genes are actively transcribed. In addition, we identified several viruses and viroids. Bacteriophages were identified by DNA- and RNA-sequencing while viruses and viroids with RNA genomes were identified by only RNA-sequencing. Moreover, we identified a novel mycovirus referred as Monilinia umbra-like virus 1 (MULV1) infecting Monilinia species. Comparative analyses between DNA and RNA sequencing suggest that RNA sequencing provides better detailed information for microbial communities; however, combining DNA and RNA sequencing results increased the diversity of microorganism including fungi, bacteria, viruses, and viroids. In addition, we demonstrated the importance of databases and analysis tools for microbiome study. Taken together, this is the first study reporting the microbial communities on the mummified peach fruits. Our study provides a comprehensive overview of microbial communities in the mummified peach fruits by DNA shotgun sequencing and RNA sequencing.Here, we carried out DNA shotgun sequencing and RNA sequencing to reveal microbiome associated with the mummified peach fruits. Combining results from both DNA shotgun sequencing and RNA-sequencing revealed the dominance of


RNA extracted from cell culture of an inactivated RVFV-BJ01 strain was also used as templates, and the detection limit is 1.83 × 103 copies/μl. In addition, there was no cross-reactivity with other viruses that can cause similar fever symptoms. The RVFV-LAMP-VF assay exhibited very high levels of diagnostic sensitivity, which had 100-fold more sensitive than RVFV real-time RT-PCR assay. Accordingly, the RVFV RT-LAMP-VF assay developed in this study is suitable for the rapid and sensitive diagnosis of RVFV without specialized equipment and can rapidly complete detection within 60 min, and the results are visible by vertical flow visualization strip within 5 min.Rift Valley fever (RVF) is a severe infectious disease, which can through mosquito bites, direct contact and aerosol transmission infect sheep, goats, people, camels, cattle, buffaloes, and so on. In this paper, a conserved region of the S RNA segment of Rift Valley fever virus (RVFV) ZH501 strain was used as target sequence. The RVFV RT-LAMP-VF assay was successfully established combined reverse transcription-loop-mediated isothermal amplification with a vertical flow visualization strip. The detection limit is up to 1.94 × 10 Phlebovirus, family Phenuivirudae) that is a significant threat to domestic ruminants and humans  is an arthropod-borne, zoonotic virus  targeting the S RNA segment of RVFV. We compared the sensitivity of the established RVFV RT-LAMP-VF assay with RVFV real-time RT-PCR.The inactivated RVFV-BJ01 strain was gifted by professor Yuhai Bi  from the Key Laboratory of Pathogenic Microbiology and Immunology. The target RNA gene was synthesized by Takara Biotechnology  Co., Ltd., which was selected from a highly conserved region of 270 bp  of the RVFV S segment (accession number DQ380149.1), and 70 bp bases were added before and after the gene to prevent RNA degradation.The mock infected blood sample is made by mixing inactivated RVFV cell culture and fresh volunteer blood at a 1:1 volume ratio. The total RNA was extracted with the TIANamp Virus RNA Kit  from 140 μl mixture and eluted with 60 μl of RNase-free water. Above operation were performed in a biosafety level 2+ laboratory.To establish an RVFV RT-LAMP-VF assay, we designed primers targeting the viral S segment. For primer design, all sequences in the NCBI database  of the full-length RVFV S segment were aligned using MEGA7.0 . FinallyThe RVFV-BJ01 strain was cultured in Vero cells, and the cell culture was collected and inactivated by using beta-propiolactone . The total RNA was extracted with the TIANamp Virus RNA Kit  from 140 μl cell fluid and eluted with 60 μl of RNase-free water. All extraction work was performed in a biosafety level 2+ laboratory.in vitro transcription. First, linearize the plasmid template, use the Takara MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 to cut the gel to recover the above digestion products, use the Takara in vitro Transcription T7 Kit for in vitro transcription reaction, and perform the DNase treatment on the RNA transcripts, using Guide-it IVT RNA Clean-Up Kit for refining, DNase treatment again, refining again. After DNase treatment, the RNA transcripts were purified and quantified. The RNA transcripts were measured with a spectrophotometer, and the copy number was calculated using the following formula: copies/μl = 6.02 × 1023 × 10−9 × concentration/(fragment length × 340). A Nanodrop 2000 ultraviolet visible spectrophotometer was used for quantitative detection of RNA. The concentration of the synthesized RNA transcript was 1,365 ng/μl, which corresponded to 1.94 × 1011 copies/μl. The synthesized RNA transcripts were stored at −80°C after purification.The RVFV S segment target sequence  (accession number DQ380149.1) was synthesized by Takara Biotechnology  Co., Ltd. for Using synthetic RNA of RVFV as a standard template, the isothermal amplification reactions were performed at five different temperatures  for 50 min with a 0.4 μM primer (FIP/BIP) concentration. The amplified products were analyzed with the vertical flow visualization strip  to screen for the optimal temperature. Three replications were performed for each trial. The RNA in the reaction system was replaced with RNase-free water for negative control samples.Based on the optimum reaction temperature, using the synthetic RNA of RVFV as a standard template, the isothermal amplification reactions were amplified for 20, 30, 40, 50, 60 and 70 min with a 0.4 μM primer (FIP/BIP) concentration, respectively, to obtain the optimal time. The amplified products were analyzed with the vertical flow visualization strip. Three replications were performed for each trial. The RNA in the reaction system was replaced with RNase-free water for negative control samples.Under the conditions of optimal amplification temperature and time, using synthetic RNA of RVFV as a standard template, the concentration of FIP/BIP primers was set to 0.2, 0.4, and 0.6 μM  for constant temperature amplification. The amplified products were analyzed with the vertical flow visualization strip, and the optimal amplification primer concentration was determined. Three replications were performed for each trial. The RNA in the reaction system was replaced with RNase-free water for the negative control samples.Synthetic RVFV-RNA was used as a standard template to establish a real-time RT-PCR method. The real-time RT-PCR primer sequences are shown in 11 copies/μl was diluted 10-fold to 1.94 × 10−2 copies/μl. The template was amplified under the optimal reaction conditions to assess the detection limit of the RVFV RT-LAMP assay.The synthetic RVFV-RNA was used to assess the sensitivity of the RT-LAMP assay. The synthetic RVFV-RNA with a concentration of 1.94 × 107 copies/μl was diluted 10-fold to 1.83 × 101 copies/μl. The template was amplified under the optimal reaction conditions to assess the detection limit of the RVFV RT-LAMP-VF assay.The RNA, which was extracted from inactivated RVFV cell culture was used to assess the sensitivity of the RVFV RT-LAMP-VF assay. The RNA content from inactivated RVFV cell culture with a concentration of 1.83 × 10The synthesized RVFV-RNA and total RNA extracted from inactivated RVFV cell culture (RVFV-BJ01 strain), inactivated JEV, inactivated H3N2 influenza virus, and recombinant viruses rNDV-EBOV-GP and rSRV9-MGP (expressing MARV G protein) were amplified under the optimal conditions to evaluate the specificity of the RVFV RT-LAMP-VF assay.Above, the RNase-free water was used to replace the target RNA in the system for negative control samples.The diluted synthesized RVFV-RNA was used as the template. According to the reaction conditions shown above, we compared the analytical sensitivities of the RT-LAMP-VF and real-time RT-PCR with respect to the detection of decreasing numbers of RNA copies under the optimal amplification conditions. Three replications were performed for each trial. The RNA in the reaction system was replaced with RNase-free water for negative control samples.7 copies/μl as tested by real-time RT-PCR. Inactivated RVFV cell culture was mixed with fresh volunteer blood at a 1:1 volume ratio as a mock infected blood sample, and then the total RNA in the mock infected blood sample was extracted by the QIAamp viral RNA minikit. These samples were amplified under the RVFV RT-LAMP-VF optimal amplification conditions, and three replications were performed for each trial. The RNA extracted from the blood sample was used as the template for the negative control. RNase-free water was used to replace the synthetic RNA for blank control samples.The RNA extracted from inactivated RVFV cell culture was present at 1.83 × 102 copies/μl, was detected at 63°C, and so 63°C was considered as the optimum amplification temperature for the RVFV RT-LAMP-VF assay.Synthetic RVFV-RNA samples were used as the template to optimize the amplification conditions for the RVFV RT-LAMP-VF assay. To screen the optimal amplification temperature, we amplified the standard templates at different temperatures  for 50 min. The results revealed that the optimum amplification temperature was achieved at 63°C. Analysis of the results revealed no significant differences in three replications. The lowest standard template copy value, 0.935 × 100 copies/μl, can be detected at both 60 and 70 min amplification. Therefore, 60 min was selected as the optimal amplification time for the RVFV RT-LAMP-VF assay.To determine the optimum amplification time for the RVFV RT-LAMP-VF assay, synthetic RVFV-RNA samples were amplified for different durations  at 63°C, respectively. The results displayed were observed by using the vertical flow visualization strip. The lowest standard template copy value, 1.94 × 10To determine the optimal primer concentration in the amplification, the FIP/BIP concentration was set as 0.2, 0.4, and 0.6 μM  to amplify at 63°C for 60 min. As the results indicate, the same results were obtained at 0.4 and 0.6 μM primer concentrations under equivalent conditions. Thus, 0.4 μM for FIP/BIP, 0.2 μM for LF/LB and 0.1 μM for F3/B3 were selected as the optimal primer concentrations for the RVFV RT-LAMP-VF assay.In summary, the RVFV RT-LAMP-VF assay was established by amplifying for 60 min at 63°C with a FIP/BIP concentration of 0.4 μM.Y = −3.420*log(x) + 41.68, the RNA content of inactivated RVFV-BJ01 was 1.83 × 107 copies/μl.The 10-fold diluted synthesized RVFV-RNA was used as the template to establish the standard curve of the RVFV real-time RT-PCR assay. Then, the RNA content of inactivated RVFV-BJ01 was quantified. As the results show in 4 to 1.94 × 10−2 copies/μl) were used to evaluate the sensitivity of the RVFV RT-LAMP-VF assay. As 0 copies/μl within 60 min.Ten-fold dilutions of synthesized RNA (ranging from 1.94 × 107 to 1.83 × 101 copies/μl) were used to evaluate the sensitivity of the RVFV RT-LAMP-VF assay. As 3 copies/μl within 60 min.Ten-fold dilutions of the RNA extracted from inactivated RVFV cell culture . When reading the results, the agarose gel electrophoresis method is mostly used, presenting the problem of false positives . In addiThe synthesized RVFV-RNA was used as the template to determine the optimal amplifications, including temperature and time and the concentration of primers. Among the results demonstrated in this paper, 63°C was considered as the optimum amplification temperature and 60 min was selected as the optimal amplification time; 0.4 μM FIP/BIP, 0.2 μM LF/LB, and 0.1 μM F3/B3 were selected as the optimal primer concentrations.0 copies/μl) was 100-fold more sensitive than the RVFV real-time RT-PCR assay (1.94 × 102 copies/μl). RVFV RT-LAMP-VF was subjected to specific evaluation by detecting viruses with similar clinical symptoms . The results indicated no cross-reaction with inactivated JEV, inactivated H3N2 influenza virus, and recombinant virus rNDV-EBOV-GP and rSRV9-MGP.In this study, we compared the sensitivity of the RVFV RT-LAMP-RV assay and RVFV real-time RT-PCR assay. The results of the study showed that the RVFV RT-LAMP-VF assay (1.94 × 10Because the collected clinical RVFV samples were mostly blood, we mixed volunteer blood with inactivated RVFV cell culture at a 1:1 volume ratio to simulate a clinical sample, which was subjected to the RVFV RT-LAMP-VF assay. The results indicated that the sensitivity and specificity of RVFV RT-LAMP-VF were not interfered with by other components in the blood sample.RT-LAMP is an inexpensive and sensitive detection method for viral RNA and is suitable for field surveys, where specialized equipment is often unavailable. RT-LAMP assays for Ebola virus , Zika vi0 copies/μl RNA transcripts and 1.83 × 103 copies/μl viral RNA. This method offers the advantages of high sensitivity, strong specificity, visual reading, and easy operation. Hence, the RVFV RT-LAMP-VF assay has the potential for rapid detection on point-care-testing. Since the entire amplification process is carried out under constant temperature conditions, only a simple water bath or metal bath is needed. In addition, after the reaction solution is added to the PCR tube, there is no need to open the cap to read the result, which avoids false positives caused by aerosol contamination, and the vertical flow visualization strip has the advantages of simple operation and easy portability. This makes this method highly practical, especially for insufficient equipment areas. Based on the above experimental results, from the perspective of epidemiological investigation, epidemic prevention, and control and technology reserve of Rift Valley fever, the establishment of the RVFV RT-LAMP-VF assay is of great significance. With the development of technology, a digital diagnostic method with rapid execution, high accuracy, sensitivity and specificity, low cost, and suitability for on-site diagnosis will become a trend.One specific conclusion is that the established RVFV RT-LAMP-VF assay has a detection limit of 1.94 × 10The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Use Committee of the Chinese People’s Liberation Army. The patients/participants provided their written informed consent to participate in this study.YZ, YG, and XX designed the experiments. QH, SZ, PH, FY, JB, ZC, and HC performed the experimentation. QH, DL, HW, NF, and HJ analyzed the data. QH and SZ wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found “pnuC motif” RNAs might function as aptamers for a novel class of NAD+-sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD+, nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD+ binding.A bacterial noncoding RNA motif almost exclusively associated with  Most known riboswitch classes sense compounds that are derived from RNA monomers or their precursors . This finadA motif RNAs  and limited to the phylum Acidobacteria . This name was chosen because representatives commonly reside in the 5′ untranslated regions of open reading frames annotated as pnuC. These genes code for membrane proteins that presumably transport the nucleoside derivative nicotinamide riboside (NR)  . Thus, tpnuC motif RNAs function as riboswitches selective for NAD+ and its NR-containing fragments. RNA constructs carrying the consensus pnuC motif undergo substantial folding changes that are induced only by their cognate ligands. Biochemical analysis of analogs indicates that the aptamer selectively recognizes functional groups on the nicotinamide-derived nucleobase. This latter finding demonstrates that natural RNAs directly recognize the redox-active moiety of this coenzyme and its immediate precursors. These findings add to the number and functional diversity of bacterial RNAs that monitor the levels of the ubiquitous coenzyme NAD+.In this report, we present bioinformatic and biochemical data indicating that pnuC motif . Given the small number of highly similar examples, most nucleotide positions appear to be highly conserved. Although we predict that three base-paired substructures are formed, called P1, P1a, and a possible pseudoknot (pk), only stem P1a is supported by evidence of nucleotide covariation consistent with base-pairing. This stem also appears to carry a variable loop region whose sequence is not important for the function of the RNA. Although the proposed P1 and pseudoknot substructures lack evidence of covariation, these structures are consistent with structural probing data as described in detail in a later section.The consensus model for the uC motif A was derpnuC genes associated with the newly discovered RNA motif are annotated as transporters for NR    B. Also, n (PnuT)  and ribon (PnuT) , or haven (PnuT) . The exiStreptococcus sp. HMSC070B10) was most similar to a protein proven to selectively transport NR .To assess this possibility, we conducted an analysis of the amino acid sequences of representative transporter proteins in this collection. Multiple sequence analysis using Clustal Omega  revealedsport NR  comparedsport NR C. Simila+. Therefore, we focused our biochemical analyses on assessing the ligand binding function of NAD+ and its derivatives with constructs carrying the pnuC motif consensus.Indeed, some of the more distantly related PnuT and PnuX proteins are the products of genes occasionally found associated with riboswitches for TPP and FMN, respectively (data not shown). Thus, the specificities of the PnuC-like transporters can be predicted by the riboswitch class regulating their respective genes, and therefore it is logical that genes coding for NR transporters would be associated with a riboswitch class for NADpnuC motif present in Streptococcus parasanguinis. RNA constructs called 85 pnuC or 65 pnuC . These results are consistent with the hypothesis that PnuC proteins associated with pnuC motif representatives are most likely NR transporters and that gene expression should be regulated by a compound indicative of the status of cellular NAD+ concentration.In-line probing reactions conducted with 85 scission B, which + and NMN only begin with nucleotides forming the junction between P1 and P1a of the 85 pnuC construct  . Both constructs exhibit a pattern of bands from in-line probing reactions that are consistent with the predicted base-pairing interactions, including the pk substructure. These results suggest that the aptamer domain is confined to the most highly conserved part of the RNA. Both the nicotinamide and ribose moieties appear to carry the most important contacts for recognition by the aptamer. Moreover, the structure formed in the presence of NAD+ is consistent with a riboswitch mechanism wherein the ribosome binding site (RBS) is sequestered to suppress gene expression when ligand is bound  value for the RNA-cofactor interaction , suggesting again that the shorter construct carries the complete aptamer domain. There is no indication from this data that the riboswitch aptamer senses the adenosyl moiety of NAD+.We subjected the 65 eraction C. The fry 100 µM D. Simila+, which is the predominant biochemical form of the nicotinamide moiety, has been measured at ∼2.6 mM in Escherichia coli cells  for this coenzyme also has been published recently  and M2 (A60U) fail to respond to 1 mM NMN . The 65 pnuC RNA construct strongly discriminates against all compounds tested that carry a modification to the nicotinamide ring   B. The RNnd NaMN) . As expeSupplemental Figs. S3. S4) indicate that the aptamer of NAD+-II riboswitches selectively recognizes the nicotinamide moiety joined to a ribose via a β-N-glycosidic linkage. In-line probing assays conducted at various concentrations of ligand were used to establish KD values for the three compounds that exhibit binding activity  and NMN moieties appear to be bound by separate but similar aptamer domains arranged in tandem  were reduced to 43 unique representatives that were bounded by the nucleotides beginning near the left shoulder of P1 and the start codon. Alignments were manually prepared and analyzed to establish the consensus sequence and structural model. Given the small number of unique representatives, base-pair annotations were made if any evidence of covariation was observed, without statistical analysis.The collection of 130 pnuC riboswitch candidate (Streptococcus sp. HMSC070B10) in comparison to transporters for NR (PnuC (Nicotinamide Riboside); Haemophilus influenzae), deoxynucleosides , thiamin , and riboflavin . An unrooted phylogenetic tree was established based on the resulting sequence alignments.Clustal Omega was used to conduct comparative sequence analyses of a PnuC protein associated with the pnuC motif RNA served as the query, which was compared pairwise with all other protein sequences .Pairwise analyses of the sequences of the same proteins were also conducted by using BLASTP . A FASTASupplemental Fig. S6). Template DNAs were hybridized to single-stranded T7 RNA polymerase promoter strands in transcription reactions, which were incubated at 37°C for 2–3 h. The resulting RNA transcripts were separated by using denaturing (8 M urea) 10% polyacrylamide gel electrophoresis (PAGE). RNAs were recovered from the gel, dephosphorylated using rAPid alkaline phosphatase (Roche Applied Science), and subsequently 5′ 32P-labeled using [γ-32P]-ATP and T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's protocols.RNA oligonucleotides were prepared as previously described  using th32P-labeled RNAs (trace) were incubated in the absence or presence of ligand candidates as indicated in the presence of 20 mM MgCl2, 100 mM KCl, and 50 mM Tris-HCl (pH 8.3 at ∼23°C). The reaction products were separated by denaturing (8 M urea) 10% PAGE and were visualized by using a Typhoon phosphorimager . As described previously (KD.In-line probing reactions were performed as previously described . 32P-labeviously , band inSupplemental material is available for this article.


Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with  Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection.Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated  Varicella-zoster virus (VZV) is a ubiquitous human alphaherpesvirus and causative agent of both varicella (chickenpox) and herpes zoster (HZ or shingles) . Varicelin silico ORF predictions and homologous ORFs in the closely related human alphaherpesvirus herpes simplex virus 1 (HSV-1)  (The 125-kb double-stranded DNA (dsDNA) genome of VZV, first sequenced in 1986, encodes at least 71 unique open reading frames (ORFs) that are expressed during lytic infection  of viral proteins. Here, a single point mutation or frameshift may impact multiple distinct RNAs at the same time, a factor that may be further confounded by inaccurate or missing transcript annotations. The reannotation of the human cytomegalovirus (HCMV) transcriptional and translational landscape and subsequent refinements of HSV-1, Kaposi’s sarcoma-associated herpesvirus (KSHV), and human herpesvirus 6 (HHV6) transcriptome architectures have all demonstrated that herpesviruses exhibit a complex transcriptional pattern of alternative splicing, opposing transcription, read-through transcription, fusion transcripts, 5′ untranslated region (5′ UTR) and 3′ UTR variations, and previously unidentified noncoding RNAs (ncRNAs) –17. Inde–IE), early (E), and late (L). Expression of E and L transcripts is considered dependent on viral proteins of the preceding kinetic classes, while expression of IE transcripts occurs in the absence of viral protein synthesis  data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures. The last is particularly challenging to infer using conventional short-read sequencing approaches . In conthttps://github.com/DepledgeLab/vzv-2.0) using IGV  RT-PCR was performed on RNA extracted from ARPE-19 cells infected with cell-free VZV EMC-1 for 72 h. RT+/RT-, reverse transcriptase added or omitted during cDNA synthesis. All numbered bands were confirmed by Sanger sequencing of the RT-PCR product. Primer sequences are indicated in Table S5. (G) RNA 9A/9 variants: pair A, ORF9var_Fw–ORF9var_Rv; #1, unspliced; #2, RNA 9-2; #3, RNA 9A-2; pair B, ORF9var_Fw–ORF9var1_spliceRv; #4, RNA 9-2; pair C, ORF9var_Fw–ORF9var2_splice_Rv; #5, RNA 9A-2. (H) RNA 48 variants: pair A, ORF48_Fw–ORF48_Rv; #6, RNA 48-1/48-2; #7, RNA 48-3; pair B, ORF48_Fw–ORF48_spliceRv; #8, RNA 48-3. (I) RNA 12-13 fusion RNA: ORF12-13_Fw–ORF12-13_Rv; #9, RNA 12-3; #10, RNA 12-2. (J) RNA 31-32 fusion RNA: pair A, ORF31-32_Fw–ORF31-32_Rv_#1; #11, RNA 31-3; pair B, ORF31-32_Fw–ORF31-32_Rv_#2; #12, RNA 31-3. (K) RNA 13.5-1: ORF13-5_Fw–ORF13-5_Rv, #13. (L) RNA 43 variants: VZV_ORF43A_Fw–ORF43.5_Rv, RNA 43-2; #14, unspliced RNA 43-1/RNA 43-2; #15, spliced RNA 43-1/RNA 43-2. (M) Northern blot analysis was performed on RNA extracted from ARPE-19 cells infected with cell-associated VZV EMC-1 or mock infected for 72 h to detect RNA 64 variants. Band #16 indicates the polycistronic RNA 63-64 precursor RNA, and bands #17 and #18 indicate RNA 64-1 and a low-abundant alternative RNA 64 isoform, respectively. Download FIG S1, TIF file, 1.0 MB.Confirmation of selected novel viral RNAs expressed during lytic VZV infection. The structure and location of selected VZV transcripts are shown alongside gel images confirming their expression. (A to F) Illumina RNA-Seq (light blue) and Nanopore dRNA-Seq  coverage plots are derived from 96-h cell-associated infections of ARPE-19 epithelial cells. Transcript colors denote their status as canonical (gray), variant (orange), fusion (blue), or putatively noncoding (red). Copyright © 2020 Braspenning et al.2020Braspenning et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01568-20.7TABLE S3Table S3, XLSX file, 0.04 MB.The complete VZV reannotation. Download Copyright © 2020 Braspenning et al.2020Braspenning et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01568-20.2FIG S2P = 0.34 and P = 0.24 by unpaired Student’s t test. (D) Relative plaque size  at 72 hpi. P = 0.490 by unpaired Student’s t test. (E) Relative expression of RNA 43-1 and RNA 62-1 at 24 hpi. P = 0.085 and P = 0.449 by unpaired Student’s t test. (F) Relative expression levels of RNA 43-1 and RNA 43-2 in cell-cycle-synchronized VZV-infected cells after 12-h cycloheximide, 24-h mock, or 24-h phosphonoacetic acid (PAA) treatment. (G) Relative expression levels of RNA 43-1 and RNA 43-2 in cell-cycle-synchronized VZV-infected cells at the indicated time points postinfection. All expression levels were normalized to β-actin transcripts. Download FIG S2, TIF file, 1.2 MB.Functional analysis of VZV RNA 43-2. (A to E) ARPE-19 cell lines stably expressing empty pcDNA3.1  or RNA 43-2 (light blue) were generated. Squares, circles, and triangles represent the 3 independently generated stable ARPE-19 cell lines. (A) PCR confirmation of DNA encoding RNA 43-2 in empty pcDNA3.1 or RNA 43-2 stable cell lines. (B) RT-PCR confirmation of RNA 43-2 expression from RNA 43-2 stable cell lines, RT+/RT-, reverse transcriptase added or omitted during cDNA synthesis. (C to E) Cells were infected with cell-free VZV EMC-1. (C) Percentage of VZV-infected cells (glycoprotein E [gE] positive) at 48 hpi or 72 hpi determined by flow cytometry. Copyright © 2020 Braspenning et al.2020Braspenning et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Fusion transcripts combine sequences from two or more distinct canonical viral transcripts and most likely result from transcription termination occurring at an alternative CPAS downstream of the canonical CPAS, followed by internal splicing . The resAdditionally, we discovered two novel polyadenylated VZV transcripts: RNA 13.5-1 and RNA 43-2. RNA 13.5-1 is 634 nt long, encodes two putative CDS domains (88 and 55 amino acids [aa]), and is positioned antisense to the RNA 14-1 (encoding pORF14). Like RNA 14-1, RNA 13.5-1 stretches across the R2 reiterative region, a short-repeat region that exhibits length variations between viral strains and within viral populations and thus leads to length variations in the encoded transcripts  Fig. 2 . We alsoin silico approach to predict the coding potential of all 136 polyadenylated VZV RNAs -derived neurons , 42. AgaP = 0.08 by Student’s t test) . RNA 43-1 encodes pORF43 and has previously been designated as an essential gene , which p t test) .IE RNAs is not dependent on de novo viral protein production, whereas transcription of E RNAs depends on IE proteins. L RNAs are further subclassified into two kinetic classes, Leaky-Late (LL) and True-Late (TL); LL RNAs are expressed at very low levels before, and TL RNAs exclusively after, viral DNA replication has commenced. To rule out the possibility of contamination introduced through cell-free infections, we also sequenced samples treated with ActD to control for the potential presence of residual background transcripts in the virus preparations and confirmed only minimal amounts of VZV transcripts to be present , cycloheximide , or phosphonoacetic acid , and viral RNAs were subsequently profiled using dRNA-Seq and RNA-Seq . Transcr present .10.1128/mBio.01568-20.3FIG S3y axis denotes coverage range. Transcripts annotated are colored according to kinetic class: IE, green; TE, yellow; EL, dark gold; LL, red; TL, dark red; TA/TL, purple; unclassified, gray. Canonical CDS regions are indicated by wide boxes with UTRs shown as thin boxes. Absence of CDS region indicates that an RNA has an uncertain coding potential. Reiterative repeat regions R1 to R5 and both copies of the OriS are shown as black boxes embedded in the genome track. (B) The inset black hatched box presents a closeup view of the RNA 9 locus. Download FIG S3, TIF file, 0.6 MB.Kinetic class of viral RNAs expressed during lytic VZV infection of ARPE-19 cells. (A) Coverage plots derived from Illumina RNA-Seq of ARPE-19 cells infected with cell-free VZV EMC-1 and treated with actinomycin D , cycloheximide , or phosphonoacetic acid  or untreated . Copyright © 2020 Braspenning et al.2020Braspenning et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the IE and 53 were classified as E RNAs, including the experimentally validated transcripts encoding pORF28 and pORF29  and VLT (pVLT), both of which have been experimentally confirmed previously (Transactivated/True-Late (TA/TL)—a putative new designation applied to transcripts that show detectable yet limited expression in CHX-treated samples, no or minimal expression in PAA-treated samples, and high expression in untreated samples at 24 hpi. Three VZV transcripts clustered closely with IE RNAs but showed variable expression patterns. One of these, RNA 0-1 (encoding pORF0), shares features with both IE and E transcripts and was provisionally classed as IE based on its relatively high abundance in CHX-treated samples (lytVLT63-3 (encoding pORF63) appears to be transactivated during infection and then expressed with E kinetics (TA/E), and RNA 29-1 showed an expression pattern similar to E transcripts. Finally, we also note that E transcripts could be classified into two distinct subgroups termed True-Early and Early-Late, dependent on whether their expression was reduced or consistent in untreated samples compared to PAA-treated samples.Given the very high sensitivity of our Illumina RNA-Seq and Nanopore dRNA-Seq analyses, many VZV transcripts were detected under all experimental conditions albeit at vastly different abundancies . To clasd pORF29 . A furtheviously . Importa samples , whereasde novo protein production and, in agreement with prior studies  were expressed at very high levels  in the  studies , 25, wernscripts  and 7. Snscripts , 25, 48.TL) shows a 3′ truncation that disrupts the CDS through the absence of a stop codon. While this transcript is predicted to encode several putative micro-ORFs, it is not known whether any of these are translated. Surprisingly, our data indicate that following very limited transactivation during initial infection, expression of these transcripts is dependent on de novo  protein synthesis and viral DNA replication, thereby classifying these RNA 62 transcripts as TA/TL. This contradicts the current classification of RNA62-1 (encoding pORF62) as an IE transcript, although we note that this classification was obtained by analogy to the function of its HSV-1 orthologue infected cell polypeptide 4 (ICP4)  region. The gene 62 duplications (62a and 62b) are considered perfect in that even full-length sequence reads align against both copies with an equal match. VZV transcripts RNA 62-1 and RNA 62-2 encode, respectively, the major viral transcriptional activator protein, pORF62, and a predicted N-terminal truncated pORF62 variant. A third transcript  . To confA lytVLT . Notably-5 cells  to C.P4 . To c10.1128/mBio.01568-20.4FIG S4Late kinetics during VZV infection. (A to C) ARPE-19 cells, hESC-derived neurons, and MRC-5 cells were infected with cell-free VZV strain pOka for 20 to 24 h in the presence or absence of phosphonoformic acid. Fold change in transcript expression in phosphonoformic acid-treated samples relative to untreated samples  and normalized to β-actin using the 2−ΔΔCT method. (D and E) Representative images of VZV strain EMC-1-infected MRC-5 (D) and MeWo (E) cells immunofluorescently stained for pORF61 (green) and pORF62 (red). Nuclei were counterstained with Hoechst stain (blue). Top row, ×400 magnification; bottom rows, ×1,000 magnification. Download FIG S4, TIF file, 1.5 MB.ORF62 transcripts are expressed with Copyright © 2020 Braspenning et al.2020Braspenning et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the LL but not IE transcripts.Similarly, PAA treatment severely reduced the abundance and affected the cellular localization of pORF62 in VZV-infected ARPE-19 cells . In the Understanding the full coding capacity of a given virus is crucial to understanding its biology. With the advent of new RNA-sequencing methodologies, it has become clear that transcription of herpesvirus genomes is much more complex than previously anticipated. Here, we demonstrate that VZV is no exception and provide a comprehensive reannotation of the VZV transcriptome during lytic infection of human retinal pigment epithelial cells and hESC-derived neurons, incorporating data from two distinct VZV strains. By integrating RNA-Seq, CAGE-Seq, and dRNA-Seq, we have resolved the architecture of the lytic VZV transcriptome. Specifically, we report the TSS and CPAS for all annotated VZV transcripts—including refinement of the 5′ UTRs and 3′ UTRs in RNAs encoding canonical ORFs—and show that VZV further diversifies its transcription through the use of (i) additional TSS and CPAS, (ii) disruption of transcription termination, and (iii) alternative splicing. As a result, several transcript isoforms are expressed from the same locus and fusion RNAs that modify UTRs and CDS of multiple transcripts are produced. Given that 5′ UTR sequences influence the translational efficiency of the downstream CDS , alternaAlthough the VZV genome is considered relatively stable, multiple strains currently cocirculate and recombine , 51, potIE kinetics) and at higher abundance than RNA 43-1 (E kinetics) (Many herpesviruses express ncRNAs during lytic and latent infections . Previouinetics) , delinealytVLT63-3, all spliced VZV RNAs used the canonical splice donor (GT) site. The splice donor sites were highly enriched for C/A (−3 position), A (−2), G (−1), and A (+1). All spliced VZV RNAs used the canonical splice acceptor site (AG), often flanked by C (−1) and G/A (+1). Consistent with the use of the cellular splicing machinery to process viral pre-mRNAs, VZV consensus splice donor and acceptor sites closely resemble those of the human transcriptome (Twenty-eight of 136 VZV transcripts are (multiply) spliced. Strikingly, the majority of splicing events occur in a hypercomplex region of the VZV genome encoding both pVLT and pORF61. We have previously shown this locus to be characterized by extensive alternative splicing  and herecriptome .de novo protein synthesis or viral DNA replication. Here, we provide an unbiased transcriptome-wide classification of VZV transcripts during lytic infection of epithelial cells and observed novel patterns of viral RNA expression, designated TE, EL, and TA/TL, in addition to traditional IE, LL, and TL kinetic classes. The data suggest that a subset of the relative expression of TE transcripts is reduced following viral DNA replication, whereas this is not the case for EL transcripts. Notably, TE VZV RNAs are enriched for transcripts encoding proteins involved in viral DNA replication , whereas EL RNAs encode a more diverse panel of VZV proteins also involved in virion formation and egress . TA/TL RNAs were detectable at low levels in the absence of de novo protein production, but their abundant expression was highly dependent on viral DNA replication. We hypothesize that transcription of TA/TL transcripts in CHX-treated samples is due to low-level transactivation by viral tegument proteins delivered from incoming virions. Additionally, our kinetic classification identified that multiple transcripts originating from the same locus could either share the same kinetic class  or be of different kinetic classes   and the putative ncRNA 43-2 (IE) diverge in CPAS usage and expression kinetics  . While tkinetics . Similarand 63-3 .IE transcripts and the role of pORF62 during lytic VZV infection. The most abundantly expressed VZV IE transcripts, produced in the absence of new protein synthesis, encode pORF4 and pORF61, which are also the earliest proteins detected during lytic VZV infection (TL and TA/TL kinetics), suggesting that tegument-derived pORF62 but not de novo pORF62 transactivates RNA 4-1 and RNA 61-1 expression, at least during establishment of the lytic infection cycle. Classification of RNA 62-1 (pORF62) as TA/TL is also supported by prior observations that a marked increase in pORF62 abundance occurs only after DNA replication has commenced  or in DMEM/F-12 + GlutaMAX-I (Thermo Fisher Scientific) supplemented with heat-inactivated 8% FBS (Sigma-Aldrich). Human neuroblastoma SH-SY5Y cells were grown in a 1:1 (vol/vol) mixture of Eagle’s Minimum Essential Medium (EMEM) with Earle’s balanced salt solution (EBSS) (Lonza) and Ham’s F-12 (Gibco) medium supplemented with 15% FBS (Lonza), l-sodium glutamate, penicillin-streptomycin, nonessential amino acids , and natrium-bicarbonate (Lonza). MRC-5 human embryonic lung fibroblasts and human melanoma MeWo cells were cultured in DMEM (Lonza), supplemented with 10% FBS, l-sodium glutamate, and penicillin-streptomycin. Human embryonic stem cell -derived neural stem cells (NSC) (Thermo Fisher Scientific) were cultured, propagated, and differentiated into neurons as described previously  was maintained in, and the cell-free virus was prepared from, ARPE-19 cells as described previously for MRC-5 cells  for 30 min at 37°C prior to infection.Human retinal pigmented epithelium ARPE-19 cells (American Type Culture Collection [ATCC] CRL-2302) were grown in a 1:1 (vol/vol) mixture of Dulbecco’s Modified Eagle's medium (DMEM) (Lonza) and Ham’s F-12 (Gibco) medium supplemented with 10% heat-inactivated fetal bovine serum  and 0.6 mg/ml eviously . Cell cu-5 cells . VZV str-5 cells , 64. Celg at 4°C. RNA was isolated from the aqueous phase using the RNeasy minikit (Qiagen) according to manufacturer’s instructions, including on-column DNase I treatment, as described previously (+). As control, the same reaction was performed without reverse transcriptase (RT−). Alternatively, RNA was isolated using the FavorPrep blood/cultured cell total RNA minikit (Favorgen Biotech) in combination with the NucleoSpin RNA/DNA buffer set (Macherey-Nagel). DNA was first eluted from the column in 100 μl DNA elution buffer, subsequently the column was treated with recombinant DNase I  for 30 min at 37°C, and finally RNA was eluted in 50 μl nuclease-free water. RNA was directly treated with Baseline-Zero DNase  for 30 min at 37°C. cDNA was synthesized with 12 μl of RNA and anchored oligo(dT)18 primer in a 20-μl reaction mixture using the Transcriptor first-strand cDNA synthesis kit at 55°C for 30 min for reverse transcriptase reaction (Roche Diagnostics).ARPE-19 cells were infected with cell-associated VZV EMC-1 by cocultivation of uninfected and VZV EMC-1-infected ARPE-19 cells at an 8:1 cell ratio for 96 h. Alternatively, ARPE-19, SH-SY5Y, MRC-5, and MeWo cells were infected with cell-free VZV EMC-1 for indicated times. Cells were harvested in 1 ml TRIzol (Thermo Fisher Scientific), mixed with 200 μl chloroform, and centrifuged for 15 min at 12,000 × eviously . RNA con+ and RT− cDNA reaction mixtures using AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific) and primer pairs corresponding to each newly identified VZV transcript  was amplified using cDNA from VZV EMC-1-infected ARPE-19 cells and primers NheI_ORF43-5_Fw and XhoI_ORF43.5_Rv , harvested at 48 hpi or 72 hpi, fixed and permeabilized with BD Cytofix/Cytoperm, stained for VZV glycoprotein E (gE)  in BD PermWash, and labeled with secondary allophycocyanin (APC)-conjugated goat anti-mouse Ig antibody (BD Biosciences). The frequency of VZV-infected  cells was measured on a BD FACSLyric flow cytometer and analyzed using FlowJo software (BD Biosciences).Confluent monolayers of stable pcDNA3.1-empty or pcDNA3.1-RNA 43-2 cells grown in a 12-well plate were infected with 2,000 PFU/well VZV EMC-1. At 72 h postinfection, plates were washed and fixed with 4% paraformaldehyde (PFA) in PBS. Subsequently plates were permeabilized using 0.1% Triton X-100 in PBS for 10 min, blocked with 5% normal goat serum in PBS-0.05% Tween 20 (PBS-T) for 30 min, and incubated with mouse anti-VZV gE antibody (MAB8612) diluted in PBS-T containing 0.1% bovine serum albumin (BSA) for 1 h at room temperature. Cells were washed with PBS-T, incubated for 1 h with polyclonal rabbit anti-mouse Ig antibody (Dako) in PBS-T + 0.1% BSA, washed, and stained with Alexa Fluor 488 (AF488)-conjugated goat anti-rabbit Ig (H+L) antibody (Thermo Fisher) in PBS-T. Plates were measured using the Immunospot S6 Ultimate UV image analyzer, and plaque size was determined using Immunospot software (Cellular Technology Limited).g (MOI after spin-inoculation = 0.1 to 0.2). Cells were incubated for 45 min at 37°C, after which the inoculum was replaced with fresh medium, medium with 10 μg/ml actinomycin D , medium with 50 μg/ml cycloheximide , or medium with 400 μg/ml phosphonoacetic acid . Infected cells were harvested in 500 μl TRIzol at 12 hpi (ActD and CHX) or 24 hpi (untreated and PAA) for RNA extraction. Alternatively, ARPE-19 cells, MRC-5 cells (1 × 105 cells/well on a 24-well plate), and hESC-derived neurons (1 × 105 cells/well on a 24-well plate as NSC and differentiated for 18 days) were infected with cell-free VZV pOka (20 μl of 4 × 104 PFU/ml) in the presence or absence of phosphonoformic acid (200 μg/ml) (Sigma-Aldrich) for 1 h, the inoculum was replaced with fresh medium with phosphonoformic acid (200 μg/ml), and cultures were maintained for 24 h.ARPE-19 cells were synchronized in the cell cycle using a double thymidine block approach . Briefly− and RT+ cDNA using 4× TaqMan Fast Advanced master mix (Applied Biosystems) on a 7500 TaqMan PCR system. Primer-probe sets directed to CDS regions of RNAs 61, 62, and 63 and VLT have been described previously (6 copies (10-fold dilution) of pOka-BAC genome or VLT plasmid .Quantitative TaqMan real-time PCR (qPCR) was performed in duplicate on RTeviously , 68, andeviously  were fir plasmid  and the NC_001348.1) was generated using the MAXIscript kit  according to the manufacturer’s instructions. Northern blot analysis was performed using the NorthernMax-Gly kit (Ambion). In brief, 7.5 μg total RNA extracted from mock- and VZV EMC-1-infected ARPE-19 cells was separated by electrophoresis on a 1% agarose gel and transferred to a nylon membrane . Membranes were incubated for 15 min at 80°C to cross-link RNA, prehybridized in ULTRAhyb buffer for 30 min at 68°C, and hybridized with biotinylated ORF64-specific probe (0.1 nM diluted in ULTRAhyb buffer) overnight at 68°C. Probe binding was visualized using the AP chemiluminescent blotting kit (KPL) and ChemiDoc MP imaging system (Bio-Rad).Biotinylated probe directed to VZV ORF64  and monoclonal mouse anti-pORF62 antibody 1:200) (0 (20). Chttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)  and aligned against the VZV reference genome  using BBMAP (https://sourceforge.net/projects/bbmap/) with postalignment processing performed using SAMtools (Stranded RNA libraries were prepared from poly(A)-selected RNA using the NEBNext Ultra II Directional RNA library prep kit for Illumina (New England Biolabs) and sequenced using a NextSeq 550. Sequence reads were trimmed using TrimGalore  and subsequently sequenced using an Illumina NextSeq 550, as previously described . ResultiFor each biological sample, up to 1,000 ng of poly(A) RNA was isolated from up to 50 μg of total RNA using the Dynabeads mRNA purification kit . Isolated poly(A) RNA was subsequently spiked with 0.3 μl of a synthetic Enolase 2 (ENO2) calibration RNA (Oxford Nanopore Technologies Ltd.), and dRNA-Seq libraries were prepared as described previously . Raw fasIllumina-assisted correction of splice junctions in RNA-Seq data was performed using FLAIR v1.3  in a strTSS and CPAS were identified by parsing SAM files to BED12 files in a strand-specific manner using BEDTools and then truncating each aligned sequence read to its 5′ or 3′ termini for TSS and CPAS identification, respectively. Peak regions containing TSS and CPAS were identified using the HOMER findpeaks module (-o auto -style tss) using a –localSize of 100 and 500 and –size of 15 and 50 for TSS and CPAS, respectively. TSS peaks were compared against Illumina annotated splice sites to identify and remove peak artifacts derived from local alignment errors around splice junctions. Thirty-eight putative TSS identified in the dRNA-Seq data set alone were flagged as artifacts and removed. Each of these TSS mapped precisely to a splice acceptor within a spliced RNA, and closer inspection of the reads showed these to result from local alignment processes (https://github.com/itsvenu/UCSC-Utils-Download) and subsequently extracting a fasta sequence for each annotated RNA using the getfasta function within BEDTools. dRNA-Seq reads were then aligned against the transcriptome database using parameters optimized for transcriptome-level alignment (minimap2 -ax map-ont -p 0.99). RNA abundance counts were generated by counting alignments against a given RNA only if the alignment 5′ end was located within the first 50 nt of the RNA and the alignment was not marked as supplementary. Transcript per million (TPM) counts (Using the updated VZV strain Dumas annotation presented here, we generated a transcriptome database by parsing our GFF3 file to a BED12 file using the gff3ToGenePred and genePredtoBED functions within UCSCutils  counts  were genCPC 2.0  was usedFigures associated with this study were generated using the R packages Gviz  and GenoStatistical analyses were performed where appropriate using GraphPad Prism 5 software with specific tests indicated in the figure legends.PRJEB38829. Analyzed data sets generated as part of this study, including a database of transcripts, BED12 alignment files, and GFF3 files describing our VZV annotation, are freely available at https://github.com/DepledgeLab/vzv-2.0.All sequencing data sets associated with this study are available via the European Nucleotide Archive under the accession number


Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230–270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0–SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction.To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic  RNA silencing is an antiviral immune mechanism in a variety of eukaryotes including fungi, plants, and invertebrates. Antiviral RNA silencing is initiated when virus double-stranded RNA (dsRNA) structures are recognized and are processed into virus-derived small interfering RNAs (vsiRNAs) by RNaseIII-like enzymes Dicer-like proteins (DCLs) ,2. Then,To counteract host antiviral RNA silencing, plant viruses have evolved numerous viral suppressors of RNA silencing (VSRs). Although VSRs have been identified in almost all plant virus genera, there is clear diversity in VSR sequence and structure, indicating that they have evolved independently . Recent 10–13P] in the N-terminal region and conserved residues [(K/R)IYGEDGX3FWR] in the C-terminal region  from residues 62 to 79  and LPP . Then, LP or LPP mutants were transiently co-infiltrated with GFP in N. benthamiana. At 5 dpi, GFP fluorescence showed that the LP mutant did not affect its VSR activity; however, the LPP mutant abolished its VSR activity  IYGEDGX3FWR affect P0 silencing suppressor activity [RCYGKALTSDIWE) of P0PM displayed only 40% identity with the consensus sequence (PM mutant IWRR (amino acids 225 and 226 from IW to RR) destroyed P0PM VSR activity. Mutation of amino acid 225 or 226  alone also suppressed RNA silencing  IYGEDGXactivity ,43,46. Asequence B, we fouilencing C. Westerilencing D. These PL-IM [PM has a similar F-box like motif; therefore, we examined the interaction between P0PM and SKP1 using a yeast two-hybrid (Y2H) system. The ORFs of P0PM and N. benthamiana SKP1 (NbSKP1) were cloned into the GAL4 DNA binding domain (BD) and GAL4 activation domain (AD), respectively. The interaction of P0 from brassica yellows virus P0 (P0Br) and NbSKP1 was used as a positive control [Br and AD-NbSKP1 grew on SD/–Leu/–Trp/–Ade/–His plates  was transiently co-expressed with 3×Flag-tagged P0PM or P0PL in N. benthamiana leaves through agroinfiltration. P0PL-induced AtAGO1 degradation was used as a positive control [TBSV was added in this assay to ensure expression of 6Myc-AtAGO1, and protein samples were analyzed at 5 dpi. As shown in PL compared with the empty vector pGD. When 6Myc-AtAGO1 was co-expressed with P0PM, 6Myc-AtAGO1 protein was hardly detected, indicating that P0PM triggered AGO1 degradation.Previous studies showed that poleroviruses’ P0s target AGO1 protein for degradation ,32,43,46PM VSR activity, the P0PM mutants LP, LPP, I225R, and IWRR were transiently co-expressed with 6Myc- AtAGO1. Western blot analysis of 6Myc-AtAGO1 protein revealed that LP and I225R with VSR activity were able to trigger AGO1 degradation, whereas LPP and IWRR, without VSR activity, did not affect AGO1 accumulation, which was similar to the empty vector pGD  is a VSR that suppresses both local and systemic RNA silencing [BrA abolishes its local and systemic RNA silencing suppression, while deletion of 25 amino acid residues in the C-terminus (225–249 aa) still allows for suppression of local RNA silencing but not systemic RNA silencing [PM abolished its VSR activity, and deletion of 41 C-terminal amino acid residues (230–270 aa) did not have any effect. This could be explained by our prediction of the secondary structure of P0PM, in which we found that the C-terminal domain did not form any structure. Taken together, these results indicate that N-terminal residues in the P0 proteins are essential for VSR activity, but C-terminal residues are not required for its local VSR activity; only those P0 with systemic VSR activity need its C-terminal residues. The reason for the difference in systemic RNA silencing suppression between these P0s and role of the C-terminal residues should be further investigated. Thus, obtaining and analyzing the crystal structure of P0 may provide more explanation as to how these amino acids affect the suppression of RNA silencing.Recently, we demonstrated that BrYV-A P0  transgenic N. benthamiana 16c plants were grown at 24 °C with a 16 h light/8 h dark cycle.All of the primers are listed in PM and its mutants with stop codon were cloned into pGD for the GFP agroinfiltration assay. In the AGO1 degradation experiment, P0PM and corresponding P0PM mutants without stop codon were cloned into pGD–3Flag, a modified version of vector pGD that has a C-terminal-fused 3×Flag tag.The vectors pGD and pGDG  were usePM was cloned into pGDBKT7 containing a binding domain (BD) to generate BD–P0PM. Construction of pGDBKT7–P0BrA and pGAD–NbSKP1 was described previously [For the Y2H assay, the full length of P0eviously ,57.Agrobacterium tumefaciens strain EHA105 using the freeze–thaw method [2, 10 mM MES, and 100 μM acetosyringone); and incubated at room temperature for at least 3 h before infiltration. The A. tumefaciens cultures were infiltrated into N. benthamiana leaves, and then the infiltrated leaves were detached for the corresponding assays. Final dilutions of the cultures used in co-infiltration assays were 0.5 OD600 for each construct.Plasmids were transformed into the w method . Co-infiw method . The recLeaves were illuminated under a BLAK-RAY non-UV semiconductor inspection lamp , and images were taken with a digital camera  at 5 and 14 days post-infiltration (dpi). All experiments shown in the results were repeated at least three times.PM, and BD–P0BrA were transformed into the yeast host strain Y187. AD and AD–NbSKP1 were transformed into the yeast host strain AH109. Protein interactions were tested using the yeast mating assay. The mated transformants were plated onto synthetic dropout (SD) media lacking Trp and Leu (SD/−WL) and SD media lacking Ade, His, Trp, and Leu (SD/−AHLW). The plates were incubated at 30 °C for 3–5 days.For the Y2H assay, the Clontech Matchmaker GAL4 Two-Hybrid System 3  was used. BD, BD–P0N. benthamiana leaves using 2× sodium dodecyl sulfate (SDS) sample buffer . Proteins were separated with SDS polyacrylamide gel electrophoresis. Western blots were performed with the primary anti-Flag antibody , anti-GFP antibody (1:3000), or anti-anti-c-myc antibody  and then incubated with anti-rabbit goat HRP secondary antibody . Finally, the membrane was detected with an enhanced chemiluminescence detection kit  according to the manufacturer’s instructions. Total proteins were extracted from infiltrated