The enormous diversity of RNA viruses in insects is continuously validated. Parasitoid wasps, as biocontrol insects which are widely used against insect pests in agroecosystems, may also carry many “good” RNA viruses. Anisopteromalus calandrae (8), Lariophagus distinguendus (3), and Theocolax elegans (18), which can parasitize and control rice weevil Sitophilus oryzae, a serious insect pest of farm-stored grains. By further bioinformatic analysis and sequencing, we identified six novel RNA viruses with complete genomes and named them WWPSRV-1, WWPSRV-2, AcPSRV-1, AcNSRV-1, AcNSRV-2, and LdNSRV-1. PCR-based detection revealed that WWPSRV-1 and WWPSRV-2 had the possibility of interspecies virus transmission, especially WWPSRV-2, which was also present in the rice weevil adults. Phylogenetically, three out of these six viruses appeared to be members of order Picornavirales: WWPSRV-1 belonged to unassigned virus families of this order, whereas WWPSRV-2 and AcPSRV-1 belonged to families Iflaviridae and Dicistroviridae, respectively. The conserved picornavirus-typical domains helicase, protease, and RNA-dependent RNA polymerase could be found in the nonstructural protein encoded by the three viruses, whose genomes consisted of the different numbers of open reading frames (ORFs). The other three RNA viruses could be classified to order Mononegavirales: AcNSRV-1 and AcNSRV-2 belonged to family Lispiviridae, whereas LdNSRV-1 belonged to a big family Rhabdoviridae. The genomes of the three viruses contained at least five ORFs, encoding deduced proteins in the following order: 3′-N-P-M-G-L-5′. All the ORFs were separated by conserved intergenic sequences which likely regulated the transcription termination and initiation. Our findings enhance the understanding of RNA viruses in weevil wasps and set the foundation for the future study of the association among weevils, weevil wasps, and RNA viruses.In this study, many virus-like fragments were obtained from transcriptomes of three wasp species, including IMPORTANCE The enormous diversity of RNA viruses in insects is continuously validated. Parasitoid wasps, as biocontrol insects which are widely used against insect pests in agroecosystems, may also carry many “good” RNA viruses. Some RNA viruses in parasitoid wasps have been reported to affect the host wasps or the wasps’ host. Here, six novel RNA viruses with complete genomes were identified in three parasitoid wasps of the rice weevil. One of these viruses was also detected in the rice weevil adults. Phylogenetically, WWPSRV-1 was the first unambiguous detection of Nora-like virus in insect parasitoids. WWPSRV-2 and AcPSRV-1 belong to families Iflaviridae and Dicistroviridae, some viruses of which can result in lethal infections in silkworms and honeybees. The other three RNA viruses belong to order Mononegavirales, which comprises many well-known insect-associated viruses. Dicistroviridae, Parvoviridae, and Circoviridae, are identified in an important stingless bee Melipona quadrifasciata which have the largest copy numbers in unhealthy bees and may be pathogenic  are widely used as biocontrol agents against insect (Arthropoda: Hexapoda: Insecta) pests in agroecosystems. They are frequently associated with viruses or virion-like particles (VLPs). In the past decade, RNA viruses have been reported in some parasitoid wasps, as well as in virus-host interactions. Several viruses have been found to affect the host wasps or the wasps’ host. For example, Pteromalus puparum negative-strand RNA virus 1, an artovirus found in  numbers .Sitophilus oryzae (Coleoptera: Curculionidae), a serious insect pest of farm-stored grains, is prevalent mainly in the south and the center of China  elegans, which can parasitize the rice weevil larvae in nature, have great potential in biological control of S. oryzae , weevil wasp positive-strand RNA virus 2 (WWPSRV-2), Anisopteromalus calandrae positive-strand RNA virus 1 (AcPSRV-1), Anisopteromalus calandrae negative-strand RNA virus 1 (AcNSRV-1), Anisopteromalus calandrae negative-strand RNA virus 2 (AcNSRV-2), and Lariophagus distinguendus negative-strand RNA virus 1 (LdNSRV-1). Moreover, we analyzed the genome organization and phylogeny of these viruses. Our results revealed that WWPSRV-1, WWPSRV-2, and AcPSRV-1 were related to picornaviruses and AcNSRV-1, AcNSRV-2, and LdNSRV-1 were related to mononegaviruses.The rice weevil, of China . It coulof China . Three h. oryzae , 16. To A. calandrae (8), L. distinguendus (3), and T. elegans (18)  or negative-strand RNA virus (18)  . The majrus (18) . All thrrus (18) .10.1128/mSphere.00331-21.9TABLE S3Anisopteromalus calandrae; LD, Lariophagus distinguendus; and TE, Theocolax elegans. Download Table S3, XLSX file, 0.01 MB.Virus-like contigs identified in three different wasps’ transcriptomes. AC, Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (i) WWPSRV-1. Three large contigs obtained from the wasps’ transcriptomes  (ontig_1)  were higontig_1) . By sequontig_1)  and 2A. ontig_1) . The fouontig_1) . The leaontig_1) . A palinontig_1) . The traontig_1) .10.1128/mSphere.00331-21.1FIG S1FIG S1, TIF file, 0.4 MB.Assemble strategy view of the contigs from the different wasps’ transcriptomes. Download Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00331-21.10TEXT S1Anisopteromalus calandrae; LD, Lariophagus distinguendus; and TE, Theocolax elegans. Download Text S1, TXT file, 0.09 MB.Contigs from these three wasps. AC, Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00331-21.7TABLE S1Table S1, XLSX file, 0.02 MB.Primers used in this study. Download Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (ii) WWPSRV-2. Four contigs  (Text S1) were assembled into a big virus-like contig , which had a high similarity with Diabrotica virgifera virgifera virus 1 (DvvV-1) polyprotein   . Moreovea 2,995) . By sequa 2,995) . The coma 2,995) . The flu2 genome . Of note. oryzae .10.1128/mSphere.00331-21.2FIG S2Sitophilus oryzae adults. In lanes 1 to 7 were the detection results from using specific primers from WWPSRV-1, WWPSRV-2, AcPSRV-1, AcNSRV-1, AcNSRV-2, LdNSRV-1, and actin (weevil), respectively. The primers used to detect viral genome sequence are shown in FIG S2, TIF file, 0.3 MB.PCR detection of the six viruses in the Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (iii) AcPSRV-1. AC_Contig_4 (Text S1) was 7,558 bp in length, including poly-A, and contained regions similar with viral RNA-dependent RNA polymerase (RdRp) of Dicistroviridae sp.  AcNSRV-1. Four contigs related to negative-strand RNA virus were discovered in A. calandrae. The two long contigs  (Text S1), which both had high identities with Linepithema humile rhabdo-like virus 1, were provisionally designated AcNSRV-1 and AcNSRV-2, respectively  AcNSRV-2. The complete genome of AcNSRV-2 failed to be confirmed by PCR. The known length was 12,118 nucleotides and G+C pairs comprised 37.85% of the nucleotides. The genome contained six ORFs in the 3′ to 5′ direction, located at nt positions 29 to 1,330, 1,518 to 2,624, 2,803 to 3,183, 3,453 to 5,075, 5,214 to 5,429, and 5,572 to 11,886, respectively (ectively . The sixectively . The lea(iii) LdNSRV-1. LD_Contig_3  in L. distinguendus, designated LdNSRV-1, showed a certain sequence similarity with a rhabdovirus, Wuhan ant virus. The complete genome of LdNSRV-1 verified by sequencing was 11,633 nucleotides in length with G+C pairs comprising 45.64% of the nucleotides ssRNA viruses identified in the wasps’ transcriptomes. (A) The polyprotein encoded by WWPSRV-1 ORF1. (B) The polyprotein encoded by WWPSRV-2 ORF. (C) The polyprotein encoded by AcPSRV-1 ORF1. Numbers represent the amino acid positions from the N terminus to C terminus. Download Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00331-21.4FIG S4Dicistroviridae , Iflaviridae , and unclassified Nora virus-related viruses  are shown with the corresponding sequences of WWPSRV-1, WWPSRV-2, and AcPSRV-1. (B) The three-dimensional structural models of the helicase domains of these three viruses predicted by the Phyre2 web. The positions of motifs A to C are marked. Motif A, magenta; motif B, red; motif C, blue. Confidence in the models of WWPSRV-1, WWPSRV-2, and AcPSRV-1: 172 residues (95%) modeled at >90% accuracy, 166 residues (97%) modeled at >90% accuracy, and 178 residues (98%) modeled at >90% accuracy, respectively. (C) Multiple sequence alignment of the putative protease domains of these 3 viruses and 12 other picorna-like viruses. Identical residues are shaded in dark blue and similar residues are in light blue. (D) Partial amino acid containing RdRp motifs A to H are shown with their secondary structures . Sequence logos for RdRp motifs A to H of 12 positive-strand RNA viruses are shown with the corresponding sequences of these 3 viruses. (E) The three-dimensional structural models of the RdRp domains of these three viruses predicted by the Phyre2 web. The positions of motifs A to H are marked. Motif A, magenta; motif B, orange; motif C, yellow; motif D, cyan; motif E, wheat; motif F, blue; motif G, red; motif H, white. Confidence in the models of WWPSRV-1, WWPSRV-2, and AcPSRV-1: 374 residues (100%) modeled at >90% accuracy, 442 residues (100%) modeled at >90% accuracy and 388 residues (100%) modeled at >90% accuracy, respectively. Full names and references of these viruses are shown in FIG S4, TIF file, 1.4 MB.Structure of helicase, protease, and RdRp domains of WWPSRV-1, WWPSRV-2, and AcPSRV-1. (A) Partial amino acids containing helicase motifs A to C are shown with their secondary structures . Sequence logos for helicase motifs A to C of 12 positive-strand RNA viruses belonging to the families Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ORF2 protein (244 aa and pI = 4.79) contained three coils, which was similar with tropomyosin, according to InterProScan. VIRALpro suggested that it was capsid protein (distance = 0.4141) and had the tail sequence (distance = 0.1172). ORF3 (526 aa and pI = 9.04) and ORF4 (355 aa and pI = 4.48) proteins were also suggested to encode capsid proteins .To determine the relationship of WWPSRV-1 with other picorna-like viruses, we conducted a maximum likelihood phylogenetic analysis based on the amino acid core sequences of RdRp. Notably, WWPSRV-1 clustered as a distinct lineage in Nora virus-related clade, indicating the need for a novel virus genus or family .10.1128/mSphere.00331-21.8TABLE S2Table S2, XLSX file, 0.01 MB.Viral sequences used for phylogenetic analysis, sequence alignments, and protein prediction in this study. Download Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Only a big ORF was predicted in WWPSRV-2 genome, which encoded a large polyprotein. The putative domains in the polyprotein were arranged successively in the order CP1, CP2, CP3, helicase, protease, and RdRp, corresponding to that in iflaviruses .The polyprotein  was a large transmembrane polyprotein that consisted of four noncytoplasmic domains, three cytoplasmic domains, and six transmembrane regions. All three predicted picornavirus-like capsid protein domains appeared in the first noncytoplasmic domain. A typical helicase domain  was present in the third noncytoplasmic domain. Moreover, a relatively small motif  predicted as a zinc finger domain was also in that noncytoplasmic domain. The putative protease  and RdRp  domains were in the last noncytoplasmic domain B and S4.To determine the phylogenetic relationship of WWPSRV-2 in other iflaviruses, phylogenetic analysis was performed using the highly conserved RdRp region. WWPSRV-2 could form a clade with the known members of iflaviruses, such as sacbrood virus (SBV) and Lygus lineolaris virus 1 (LyLV-1) .Two big ORFs were present in the AcPSRV-1 genome, which encoded nonstructural and structural proteins, corresponding to that in dicistroviruses. The nonstructural protein encoded by ORF1 was a large transmembrane polyprotein  that consisted of two noncytoplasmic domains, one cytoplasmic domain, and two transmembrane regions. The helicase (aa 67 to 248), protease (aa 595 to 831), and RdRp  domains were in the different two noncytoplasmic domains C and S4.Triatovirus , black queen cell virus (BQCV), and Triatoma virus (TrV), which belong to genus atovirus .By comparing the 5′ and 3′ untranslated regions and intergenic regions of the AcNSRV-1, AcNSRV-2, and LdNSRV-1, we pinpointed for each ORF the putative termination signals (TS), intergenic spacer (IS), and transcription initiation (TI). A highly conserved motif was identified in the 5′ and 3′ untranslated regions and intergenic regions of the AcNSRV-1, AcNSRV-2, or LdNSRV-1, respectively.In AcNSRV-1 genome, the conserved transcription initiation motif of 3′-UUUCA(C/U/G)-5′ was identified upstream of each putative ORF. The transcription termination motif of 3′-AUA(A/U)AUCUUUUU-5′ was detected downstream of every ORF. The conserved intergenic spacer 3′-C(.)A-5′ was found in each intergenic region .In AcNSRV-2 genome, the conserved transcription initiation motif of 3′-UUCA(G/C)-5′ was identified upstream of each putative ORF, and the transcription termination motif of 3′-UAAAUCUUUUU(U)-5′ was detected downstream of every ORF. The conserved intergenic spacer 3′-GAG-5′ was found in the intergenic region among N/P, P/M, and M/L .In LdNSRV-1 genome, each ORF was separated by a highly conserved gene junction with the consensus 3′-UAAU(U)CUUUUU(U)(G/A)GUUCC(U/G)U-5′. The conserved transcription initiation motif of 3′-UUCC(U/G)U-5′ was identified upstream of each putative ORF. The transcription termination motif of 3′-UA(A)U(U)(C/A)UUUUU(U)(U)-5′ was detected downstream of every ORF. The conserved intergenic spacer 3′-(G/A)(G)-5′ was found in each intergenic region .The general properties of the tentative proteins encoded by the AcNSRV-1 and AcNSRV-2 genome were shown in Paramyxoviridae , within the InterProScan. The AcNSRV-2 ORF4 protein showed 25.12% identity with the putative glycoprotein of Blattodean arli-related virus OKIAV101. Moreover, only the ORF4 proteins of the two viruses were recommended to have the potential N-linked sites. All of these results indicated that ORF4 encoded the viral glycoprotein (G).Only ORF4 proteins encoded by the two viruses were predicted to be transmembrane polyproteins and to possess a cleavable signal peptide. Compared to the three transmembrane regions present in the AcNSRV-1 ORF4 protein, only one was in the AcNSRV-2 ORF4 protein . The AcN10.1128/mSphere.00331-21.5FIG S5FIG S5, TIF file, 0.2 MB.Graphical representation of the G proteins encoded by the (−)ssRNA viruses identified in the wasps’ transcriptomes. (A) The polyprotein encoded by AcNSRV-1 ORF4. (B) The polyprotein encoded by AcNSRV-1 ORF4. (C) The polyprotein encoded by LdNSRV-1 ORF4. Numbers represent the amino acid positions from the N terminus to C terminus. Download Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The AcNSRV-1 ORF5 and the AcNSRV-1 ORF6 were supposed to encode the viral large polymerase proteins (L). Both L proteins exhibited more than 40% similarity with the putative RdRp-complex of Linepithema humile rhabdo-like virus 1. Through the InterProScan, the putative RdRp, mRNA-capping enzyme , and methyltransferase (MT) domains were successively arranged in the L protein .10.1128/mSphere.00331-21.6FIG S6Lispiviridae  and Rhabdoviridae  are shown with the corresponding sequences of AcNSRV-1, AcNSRV-2, and LdNSRV-1. (B) The three-dimensional structural models of the RdRp domains of these three viruses predicted by the Phyre2 web. The positions of motifs A to F and GHP are marked. Motif GHP, red; motif A, yellow; motif B, magenta; motif C, cyan; motif D, orange; motif E, white; motif F, blue. Confidence in the models of AcNSRV-1, AcNSRV-2, and LdNSRV-1: 828 residues (100%) modeled at >90% accuracy, 851 residues (99%) modeled at >90% accuracy, and 877 residues (100%) modeled at >90% accuracy, respectively. (C) Partial amino acids containing PRNTase motifs A to E are shown with their secondary structures . Sequence logos for PRNTase motifs A to E of 12 negative-strand RNA viruses are shown with the corresponding sequences of these three viruses. (D) The three-dimensional structural models of PRNTase domains of these three viruses predicted by the Phyre2 web. The positions of motifs A to E are marked with orange. Confidence in the models of AcNSRV-1, AcNSRV-2, and LdNSRV-1: 235 residues (100%) modeled at >90% accuracy, 235 residues (100%) modeled at >90% accuracy, and 237 residues (100%) modeled at >90% accuracy, respectively. (E) Partial amino acid containing methyltransferase motifs (I to IV and VI to X) are shown with their secondary structures . Sequence logos for methyltransferase motifs of 12 negative-strand RNA viruses are shown with the corresponding sequences of these three viruses. (F) The three-dimensional structural models of the methyltransferase domains of these three viruses predicted by the Phyre2 web. KDKE observed in motif X, motif IV, motif VI, and motif VIII, are marked with red, magenta, cyan, and orange, respectively. Motif I is marked with blue. Confidence in the models of AcNSRV-1, AcNSRV-2, and LdNSRV-1: 195 residues (100%) modeled at >90% accuracy, 197 residues (100%) modeled at >90% accuracy, and 194 residues (100%) modeled at >90% accuracy, respectively. Full names and references of these viruses are shown in FIG S6, TIF file, 2.2 MB.Structure of the RdRp, PRNTase, and methyltransferase domains of AcNSRV-1, AcNSRV-2, and LdNSRV-1. (A) Partial amino acid containing RdRp motifs A to F and GHP (Gly-His-Pro) are shown with their secondary structures . Sequence logos for RdRp motifs A to G and GHP of 12 negative-strand RNA viruses belonging to the families Copyright © 2021 Wang et al.2021Wang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Based on the conserved genomic orientation of mononegaviruses (3′-N-P-M-G-L-5′), ORF2 protein of AcNSRV-1 (pI = 5.90) or AcNSRV-2 (pI = 5.32), containing 47 or 52 potential phosphorylation sites, was most likely the phosphoprotein (P). ORF3 protein of AcNSRV-1 (pI = 6.89) or AcNSRV-2 (pI = 7.75), containing 15 or 20 phosphorylation sites, may be a phosphorylated matrix protein (M). In addition, the AcNSRV-2 had an additional small protein P6 (71 aa and pI = 7.76) lying between the G and L proteins. This was also confirmed by the RNA-seq reads’ fluctuating distribution .Lispiviridae  as closest relative, AcPSRV-1 belongs to genus Triatovirus  and Rhabdoviridae  have been identified in the hymenopteran insects  of mononegaviral genomes . The mRN40–In conclusion, we have expanded the diversity of RNA viruses in the parasitoid wasps, and these RNA viruses may play an important role in the biocontrol of rice weevils. For better application of this information, much work should be accomplished. Just like PpNSRV-1 , it alsoS. oryzae) were initially collected from a cereal storehouse in Hangzhou, China in 2017. They were successively reared on wheat seeds in the laboratory at 30 ± 1°C, 80% relative humidity, and 12-h light/12-h dark photoperiod. The mated adult beetles were provided with the fresh wheat seeds to lay eggs for a week (the seeds were replaced weekly). After 10 to 15 days, eggs developed into elder larvae used as hosts for the wasps. The colonies of A. calandrae, L. distinguendus, and T. elegans were initially collected from the parasitized rice weevil hosts of the same sites. The adults of the three wasp species were independently reared on 10% (vol/vol) honey at 25 ± 1°C, 50% relative humidity, 12-h light/12-h dark photoperiod. For parasitism, the mated adults of the three wasp species were placed together with the wheat seeds that contained the host larvae. These parasitized beetle larvae were reared in the same conditions as were the wasp adults. The adults of A. calandrae or L. distinguendus emerged after 15 days. The adults of T. elegans emerged after 20 days. Each wasp colony was independently reared in the laboratory at least three generations before the experiments.Rice weevils . The RNA quality was assessed using NanoDrop 2000  and an Agilent 2100 bioanalyzer . RNA concentration was measured using an Invitrogen Qubit 2.0 Fluorometer . RNA samples (RNA integrity number > 8) were sent to Novogene company  for the subsequent library construction. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep kit for Illumina  following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE cluster kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated. The low-quality, adaptor-polluted, and high content of unknown base (N) reads were removed. The clean reads were performed with de novo assembly using Trinity program . Molecular weight and isoelectric point (pI) of the predicted ORFs were calculated via open-source ProtParam (https://web.expasy.org/protparam/). The viral proteins were queried through InterPro (http://www.ebi.ac.uk/interpro/) and Phyre2 for protein modeling, prediction, and analysis .Nucleotide sequence analysis and assembly were performed using DNAStar Lasergene software version 7.0 . Viral ORFs were predicted using open-source NCBI ORF finder ssRNA viruses (negative-sense single-stranded RNA), noncoding viral genome regions were analyzed using the open-source MEME suite 5.3.0 for the motifs analysis .To identify the viral genomic RNA distribution in these three wasps’ transcriptomes, clean RNA-seq reads from all three wasps were mapped to the complete genome of the six novel RNA viruses using the CLC Genomics Workbench 12.Multiple alignments of amino acid sequences were performed using Clustal Omega webserver  and editSitophilus oryzae is PRJNA719021.The GenBank accession numbers of WWPSRV-1, WWPSRV-2, AcPSRV-1, AcNSRV-1, AcNSRV-2, and LdNSRV-1 are MW864600, MW864601, MW864599, MW864602, MW864603, and MW864604, respectively. BioProject accession for the RNA-seq of three parasitoid wasps of the rice weevil


Second, we tested two types of three-chamber apparatuses, in which choice fish were presented in either a face-to-face or side-by-side location. Test fish (regardless of sex) spent most of the time associating with choice fish in the compartments. However, their sexual preference, calculated as an association ratio, was poorly reproduced when the locations of the choice fish were swapped. Third, the sexual preferences of males quantified using the manual-counting and either of the three-chamber strategies did not correlate (r = 0.147 or 0.297). Hence, we concluded that, even for individuals of a species like medaka, which spawn every day, sexual preference could not be reliably evaluated using the three-chamber strategy. Optimization of the protocol may solve this problem; however, the explanation for the observation that animals that are ready for spawning persist with never-accessible mating partners must be reconsidered.The three-chamber experiment, in which one test animal can choose between two animals placed in physically inaccessible compartments, is a widely adopted strategy for studying sexual preference in animals. Medaka, a small freshwater teleost, is an emerging model for dissecting the neurological/physiological mechanisms underlying mate choice for which intriguing findings have been accumulating. The three-chamber strategy has rarely been adopted in this species; therefore, here we investigated its validity using medaka colour variants that mate assortatively. First, a total of 551 movies, in which a test male and two choice females interacted for 30 min under a free-swimming condition, were manually analysed. The sexual preference of the males, calculated as a courtship ratio, was highly consistent between human observers ( Mate choice in animals has been attracting the attention of zoologists as a research subject. The choice is largely made by the female in many species, which requires the male to exaggerate himself  or defeat his rivals  to be chosen. Nevertheless, there are many cases in which mate choice lies with males . The sexOryzias latipes) is small freshwater fish that is native to the Far East and has been used in research for more than a century  mm), but obtained preliminary results that were not promising (data not shown). Manual counting of male approaches in a three-chamber apparatus could somehow reproduce the sexual preferences of ividuals , but theAt present, the most reliable strategy for quantifying the sexual preference of medaka remains manual observation; i.e., counting the number of approaches, dances or copulation attempts by males –16, and The three-chamber strategy tested here was not valid for medaka mate-choice studies. Although negative, this result demonstrated the difficulty of applying the three-chamber strategy even to a species that spawns every day, which should be widely noted by ethologists interested in mate choice. Medaka might be a mere exception of no importance, or a similar problem may lie dormant among other species.S1 Data(XLSX)Click here for additional data file.


GammaproteobacteriaEscherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling ∼20 to 25% of the entire transcriptome. While the model FirmicutesBacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3′→5′ exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3′ UTR cleavage fragments that contain stabilizing 3′ structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5′ and 3′ ends, efficiently degrading mRNA 3′ UTR fragments. Here, we propose that the lack of 5′→3′ exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3′ UTR ends as the “default” setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3′ UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model  Escherichia coli and Salmonella enterica serovar Typhimurium. Recently, it has become clear that regulatory sRNAs can be generated from a variety of sources in Gammaproteobacteria, including mRNA 5′ untranslated regions (UTRs) (rne) , which litter the transcriptome and have been extensively characterized in the model Gram-negative enteric bacteria s (UTRs) , 2, intes (UTRs) , 3, and s) (rne) –9. TheseGammaproteobacteria like E. coli and Salmonella and the model Gram-positive FirmicutesB. subtilis and S. aureus. Using examples, we propose that 3′ UTR sRNAs represent the latter stages of a stepwise continuum of increasing regulatory independence for mRNA 3′ UTRs. Finally, we propose that 5′→3′ exoribonuclease activity may be a barrier to this stepwise progression of regulatory 3′ UTR evolution in Gram-positive Firmicutes, which has contributed to the depreciation of sRNA chaperone Hfq and ProQ function.Here, we highlight some of the key differences in RNA metabolism between Gammaproteobacteria and the FirmicutesB. subtilis and S. aureus, bulk RNA turnover is carried out by a large multiprotein complex termed the RNA degradosome  that contains the conserved catalytic N-terminal region of the protein and can functionally substitute for rne in E. coli  that is commonly associated with the RNA degradosome complex of Gram-positive Firmicutes. There has recently been evidence of a 5′→3′ exoribonuclease within E. coli , the 3′-terminal RNA fragments (containing the transcription terminator) of more than 50% of mRNAs were increased, highlighting the importance of RNase J1 in degradation of 3′-terminal RNA decay intermediates  and is expected to have 5′→3′ exoribonuclease activity.Chaperones play critical roles in facilitating sRNA regulation by increasing the association rate of sRNA-mRNA interactions to physiologically useful rates. The sRNA chaperone Hfq binds many sRNAs through their poly(U) tail and acts as a matchmaker within Gram-negative bacteria to anneal sRNAs with complementary mRNAs, while ProQ binds to the 3′ UTR of mRNAs to protect the free mRNA 3′ end against 3′→5′ exoribonucleolytic degradation. Some Gram-positive organisms, such as  or ProQ . In factrmicutes , 22. In nditions ; however changes . In S. a mutants , 26. Thehimurium ) and hasifficile , 29, andry sRNAs , and theB. subtilis and S. aureus may be a barrier to evolution of independent regulatory 3′ UTR sRNAs along this continuum.In the section below, we propose that there exists a continuum of regulatory 3′ UTR independence and that the 5′→3′ exoribonuclease activity of RNase J in E. coli largely appear in ancestral genomes before their cognate target mRNA binding sites, suggesting that sRNAs are first produced and then drive evolution of target mRNAs . The simplest arrangement of a regulatory UTR and target mRNA is found in S. aureus where the 3′ UTR of icaR mRNA loops on itself (or between icaR mRNAs) and base pairs to the ribosome-binding site (RBS) of its own 5′ UTR to block translation and promote RNase III-dependent degradation  (E. coli [trans-acting regulatory 3′ UTRs could be a widespread mechanism of regulation in Firmicutes. An advantage is that trans-acting regulatory 3′ UTRs like the hly 3′ UTR are protected from the 5′→3′ exoribonucleolytic processing of RNase J (to the extent that the mRNA is protected).A slightly more complex variation where mRNA 3′ UTRs act in NA prsA2 . This mR protein . The hlydes (nt)  (compare(E. coli ), suggesS. aureus, the regulatory 3′ UTR-derived sRNA RsaC is generated by endoribonucleolytic cleavage of the polycistronic mntABC-rsaC transcript by RNase III (mntABC mRNA (encoding a manganese transporter) and while they share a transcriptional activation signal (repressed in the presence of Mn2+ by MntR), RNase III cleavage separates these transcripts so that they have independent fates within the cell. RsaC retains mRNA targets that are functionally coherent with the mntABC-rsaC operon, repressing the Mn2+-dependent superoxide dismutase (SodA), and other metal-dependent pathways. RNase III cleavage of mntABC-rsaC generates a free 5′ end that should render RsaC highly susceptible to RNase J exonucleolytic attack. However, a notable feature of RsaC is the 25-nt stem-loop that sequesters the 5′ end in a duplex preventing exoribonuclease attack and stabilizes the 3′ UTR-derived sRNA  allowing coordination between these functionally related genes . RNase E cleavage after the oppF stop codon releases OppZ from the parent transcript. In a regulatory circuit that parallels the 5′ UTR-3′ UTR looping autoregulation of icaR in S. aureus, OppZ binds the upstream RBS of oppB to silence expression and control the cellular levels of the precursor oppBCDF transcript through a feedback loop  transporter in Salmonella. The narK mRNA is expressed during anaerobic respiration (2−) transporter nirC, which is located within the nirBDC-cysG operon and controls cytoplasmic nitrate levels during anaerobic growth , and further experiments confirmed that DapZ negatively regulates the synthesis of major ABC transporters through the repression of both dpp and opp mRNAs , suggesting that independently transcribed or processed 3′ UTRs are less abundant in these Gram-positive organisms (Many Gram-negative organisms lack a 5′→3′ exoribonuclease and rely on endoribonucleolytic cleavage (such as RNase E) and 3′→5′ exoribonucleases to degrade RNA within the cell. In the model Gram-negative bacteria y 175 nt , generaty 175 nt . It seemsequence . In contdy state . This isp codons , likely himurium . In S. arganisms  to D.E. coli and Salmonella, it is plausible that this pool of 3′ mRNA fragments provides a source of transiently stable, unconstrained RNA sequence space for the selection and evolution of regulatory RNA features. It follows that RNase J exoribonucleolytic activity in many Gram-positive organisms may pose a significant barrier to evolution of new 3′ UTR regulatory RNAs. Without first acquiring stabilizing 5′ structures, these 3′ RNA fragments are rapidly degraded and would have limited opportunity to gain a foothold on the ladder to positive selection.In The functional importance of Hfq appears to be significantly expanded in many Gram-negative organisms, and we propose that this may be linked to the availability of “stable” 3′ UTR degradation intermediates . Hfq bin62–Firmicutes where the function of Rho terminator may have been depreciated because the pioneering ribosome lags behind the elongating RNA polymerase, exposing the nascent transcript to premature Rho interactions and potentially toxic transcription termination .An analogous scenario has occurred in many Gram-positive mination . In contE. coli and Salmonella allows transient stabilization of cleaved mRNA 3′ UTRs, which has provided abundant raw materials for the evolution of 3′ UTR sRNAs and the expansion of sRNA regulatory networks. This in turn has centralized the function of sRNA chaperones like Hfq in E. coli and Salmonella as the sRNA network has expanded. In Gram-positive Firmicutes that encode a 5′→3′ exoribonuclease (RNase J), cleaved 3′ UTRs are rapidly degraded. In these bacteria, 3′ UTR sRNAs must first acquire 5′ stems or internal transcription start sites before they are stabilized. We propose that RNase J represents a major evolutionary barrier to the expansion of the 3′ UTR sRNA network and has depreciated the function of the sRNA chaperones Hfq and ProQ.All bacteria appear to use regulatory RNA to control gene expression posttranscriptionally; however, it is clear that differences exist in the distribution and importance of RNA chaperones that facilitate sRNA-mRNA interactions. We propose that mRNA 3′ UTRs are a major evolutionary source for regulatory sRNAs and highlight some of the potential intermediate stages of 3′ UTR sRNA evolution. In addition, we propose that the lack of 5′→3′ exoribonucleases in hly mRNA in Listeria) that are not cleaved to protect the 5′ end of the UTR. Evolution of internal transcription start sites should be the preferred mechanism of 3′ UTR release, as this provides a protective 5′ triphosphate.Some testable predictions arise from the above. (i) As the relative importance of Hfq for global gene regulation is uncovered in more bacteria, the size of the Hfq regulon should be negatively correlated with the presence of a functional RNase J. (ii) Endoribonuclease cleavage of 3′ UTR sRNAs should be less prevalent in RNase J-encoding bacteria. These bacteria may make more widespread use of regulatory mRNA 3′ UTRs (like


In plants, the translocation of molecules, such as ions, metabolites, and hormones, between different subcellular compartments or different cells is achieved by transmembrane transporters, which play important roles in growth, development, and adaptation to the environment. To facilitate transport in a specific direction, active transporters that can translocate their substrates against the concentration gradient are needed. Examples of major active transporters in plants include ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion (MATE) transporters, monosaccharide transporters (MSTs), sucrose transporters (SUTs), and amino acid transporters. Transport via ABC transporters is driven by ATP. The electrochemical gradient across the membrane energizes these secondary transporters. The pH in each cell and subcellular compartment is tightly regulated and yet highly dynamic, especially when under stress. Here, the effects of cellular and subcellular pH on the activities of ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters will be discussed to enhance our understanding of their mechanics. The relation of the altered transporter activities to various biological processes of plants will also be addressed. Although most molecular transport research has focused on the substrate, the role of protons, the tiny counterparts of the substrate, should also not be ignored. Transporters play important roles in the transport of nutrients, hormones, and metabolites for the purposes of growth, development, and adaptation to stresses. To deliver the substrates in a specific direction, active transporters, which can transport substrates against their concentration gradients, are required. Transmembrane transporters mediate the transport of substrates across biological membranes. The polypeptides of transmembrane transporters contain transmembrane segments (TMSs), which are integrated in the membrane . The seg+/H+ antiporter (NHX) [+, which is then exported out of the cell by PM-ATPase [+ results in the proton gradient and electrical potential gradient across the membrane [+ out of the cell or the intrusion of Na+ inside the vacuole by NHX [2+/H+, Na+/H+ and Zn2+/H+ exchangers and phosphate transporter, at the vacuolar membrane [+.In plants, cellular pH has been reported to be mainly regulated by proton pumps including the plasma membrane ATPase (PM-ATPase), the vacuolar-type ATPase (V-ATPase), the vacuolar pyrophosphatase (V-PPase) ,7 and Naer (NHX) . PM-ATPaM-ATPase . Such trmembrane ,10. Undee by NHX . V-ATPase by NHX ,13. In amembrane . The pH Phytohormone transporters, alkaloid transporters, ion and ion chelator transporters, sugar transporters, and amino acid transporters are common active transporters in plants. Examples of phytohormone transporters, alkaloid transporters, and ion and ion chelator transporters include ATP-binding cassette (ABC) transporters and Multidrug Additionally, Toxic compound Extrusion (MATE) transporters, while examples of sugar transporters include MonoSaccharide Transporters (MSTs) and SUcrose Transporters (SUTs). These transporters have been reported to play important roles in various biological processes including cellular detoxification, nutrient transport, and stress adaptation. Previous reports have largely focused on the transport of substrates such as metabolites, sugars, and amino acids by these transporters. However, considering the mechanics of the transport activities, the protons required in exchange of these substrates also deserve the same attention. The awareness of the role of protons, and therefore pH, in transport activities will bring forth a more comprehensive understanding of these transporters.ABC transporters and MATE transporters are the two major types of multidrug transporters that transport various substrates including phytohormones, alkaloids, ion, and ion chelators .The ATP-binding cassette (ABC) transporters belong to one of the largest superfamilies of transporters and are present in many organisms ranging from prokaryotes to human . Driven In plants, ABC transporters were first identified as being involved in cellular detoxification by mediating the storage of xenobiotics inside the vacuole . They haAuxin molecules can migrate by mass flow in the vascular system or being transported from cell to cell . In the ABCBs are mainly expressed in the leaf and root meristem and are related to the transportation of auxin in the apoplast [abcb1abcb19 showed reduced apical dominance, dwarfism, short hypocotyl, and poor fertility [Oryza sativa, under salt stress, it was found that the OsABCB genes in leaf and root had differential expression responses towards the stress [It was reported that ABCB transporters are associated with the active transport of auxin driven by ATP hydrolysis . ABCBs aapoplast . AtABCB1apoplast . The Araertility . It was ertility . Althougertility . Howevere stress . Under se stress . From thatabcg25 knockout mutants exhibited enhanced ABA-sensitive phenotypes at the early growth stage compared to the wild-type [AtABCG40 [atabcg40 mutants had a slower uptake of ABA into the protoplast and a slower rate of stomatal closure under ABA treatment [Lr34res allele from a rice blast-resistant wheat cultivar improved the accumulation of ABA and enhanced the tolerance of rice to rice blast [ABA is involved in the signaling of both abiotic and biotic stresses . One of ild-type . On the AtABCG40 . Comparereatment . Furtherce blast . These e+) or proton (H+) [+ or H+ across the membrane results in an electrochemical gradient that drives the transport of the substrates, which could be macromolecules or ions, in the opposite direction [+ while prokaryotic MATE transporters can use either Na+ or H+ for this purpose [Multidrug and toxic compound extrusion (MATE) transporters are antiporters that transport various molecules across a membrane in exchange for sodium ion (Naton (H+) . The movirection . Most eu purpose . In plan purpose .AtDTX50 is mainly expressed in vascular tissues and guard cells where ABA is synthesized [+ from the medium. It is possible that the pH itself, besides the pH gradient across the membrane, has effects on the protein function. Since MATE is not the sole transporter of ABA, it is possible that other transporters could complement the ABA transport when one transporter is not working at its optimal capacity.AtDTX50 (detoxification efflux carrier) was reported to be a plasma membrane-localized MATE-type protein for ABA efflux . AtDTX50thesized . Under dthesized . Among pthesized . It thereds5 mutant was formerly known as the sid (SA induction deficient) mutant [EDS5 was induced by Pseudomonas syringae [EDS5 and the enhanced exporting activity of the protein promote the resistance to the pathogen.Besides ABA, MATE transporter has also been reported to transport salicylic acid (SA). ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) was reported to be a chloroplast envelope-localized MATE-type protein that transports SA from chloroplast to cytoplasm . The eds) mutant ,50. It w) mutant . Therefo) mutant . The expsyringae . Upon paCoptis japonica, as an antimicrobial compound [Coptis japonica multidrug resistance 1) mediates the transport of berberine. CjMDR1 is expressed in the xylem tissue and encodes a multidrug-resistance protein (MDR)-type ABC transporter to facilitate the transport of berberine from the root to the rhizome [Besides phytohormones, ABC transporter is also able to transport alkaloids. ABC transporters play an important role in vacuolar sequestration, which is an important process for minimizing the effects of toxic compounds and heavy metals ,53. Varicompound . It was  rhizome .Nicotiana tabacum, Nt-JAT1  was reported to be a MATE-type antiporter that mediates the influx of nicotine into the vacuole in exchange of H+ [+ from the vacuole. In this case, the nicotine influx into the vacuole resulted in the increase in cytosolic H+.Besides exporting substrates from the cell, MATE transporters have also been shown to mediate substrate influx into organelles such as vacuoles. In ge of H+ . The uptge of H+ . The lowabcc1abcc2 was hypersensitive to arsenic (As) and had reduced vacuolar uptake of As(III)–PC2 and apoPC2 [AtABCC1 alone could not promote the tolerance to As [AtPCS1 (PhytoChelatin (PC) Synthase) [AtABCC1 or AtABCC2 mediated the microsomal uptake of PC2-As more efficiently than that of apo-PC2 [AtABCC1 and AtABCC2 were shown to mediate the tolerance to Cd and Hg [abcc1abcc2 was also hypersensitive to Cd(II) and Hg(II), and was demonstrated to be impaired in the vacuolar sequestration of Cd [ABC transporters have also been reported to be involved in the detoxification of various toxic heavy metals such as cadmium (Cd), mercury (Hg), and aluminum (Al). In Arabidopsis, compared to the wild-type, the double knock-out mutant d apoPC2 . Howeverce to As , unless ynthase) . Using y apo-PC2 . It was  apo-PC2 . In anotd and Hg . In Arabon of Cd . In addion of Cd . In riceon of Cd . ABC traon of Cd .Xenopus oocytes as the model, it was shown that a decrease in the pH of the medium favored the SbMATE (Sorghum bicolor MATE)-mediated efflux of citrate from the oocytes [+ antiporters. H+-ATPase was suggested as the provider of H+ for MATE transporters in exchange for their substrates [+-ATPase and a MATE transporter mediated the efflux of citrate and played a role in maintaining a stable cytosolic pH [+ is dissociated from citric acid from the TCA cycle [+ is then transported out of the cell by H+-ATPase [+ back into the cytosol [+ level. It was speculated that this drop in cytosolic pH may be related to the pH regulation in the cytosol and the balancing of charges for secondary ion transports [Using  oocytes . Similar oocytes , FRD3 [6 oocytes , and AtD oocytes , were albstrates . When unsolic pH . The Al-CA cycle . The H+ +-ATPase . The rem cytosol . The ext cytosol . This pransports .− influx into the vacuole and regulate the vacuolar turgor [dtx33/dtx35 double mutant is impaired in stomatal opening and is more tolerant to drought. Although DTX33 and DTX35 were demonstrated to function as ion channels with the measured reversal potentials in agreement with the theoretical Nernst potentials at different pH and dependent on the Cl− concentration, it was also demonstrated that their activities were dependent on vacuolar pH [The MATE transporters DTX33 and DTX35 in Arabidopsis were reported to be tonoplast-localized MATE-type proteins that mediate Clr turgor . The dtxuolar pH . Among vuolar pH . However+ concentration [+ concentrations in different cellular compartments, which result in the pH gradient across the biological membrane where the MATE transporters are localized, regulate the activities of the transporters. Under stress, the fluctuation of cellular pH possibly plays a role to regulate the activities of MATE transporters.The above examples show that the export of substrates out of the cell and the import of substrate into the vacuole by MATE transporters could mediate the upregulation of the cytosolic Hntration ,65. On t+-dependent, will be discussed.In plants, MonoSaccharide Transporters (MSTs), SUcrose Transporters , and Sugars Will Eventually be Exported Transporters (SWEETs) are the three major types of sugar transporters ,69. SUTs+/sucrose symporters involved in loading sucrose into the phloem against the concentration gradient and are driven by the proton motive force across the plasma membrane of the sieve element–companion cell complex (SE-CCC) [In photosynthetic cells, sucrose is derived from the photosynthetically fixed carbon and is the major form of sugar transported via the phloem to other tissues ,76. SUTs(SE-CCC) .+/sucrose antiporter or symporter depending on the pH difference between vacuole lumen and the medium outside [+ from the vacuole; when the medium outside the vacuole was more acidic, sucrose was transported together with proton into the vacuole [+ symporter activity of ZmSUT1 was demonstrated to be pH-dependent [The pH dependence of SUT activities has been reported in various species. For example, in Arabidopsis, AtSUC4 was reported to be a proton/sucrose symporter localized in the vacuole membrane . The pro outside . When th vacuole . Such sw vacuole . The sucependent . It was ependent .Atsuc4 mutants were found to be more sensitive to stresses including salt, osmotic, cold, and ABA treatments compared to the wild-type [Atsuc4 mutants [Ipomoea batatas), similar to AtSUC4, IbSUT4 was found to regulate the accumulation of sucrose in the root and ABA signaling under stress [IbSUT4 in Arabidopsis reduced the sucrose level in the leaf but improved the sucrose level in the root under salt stress [ild-type . Probablild-type . The ABA mutants . Althoug mutants . The accr stress . The ovet stress . This ist stress , which rPhaseolus vulgaris SUT1.1, when expressed in transgenic yeast, was found to have a higher transport activity when the pH of the medium was more acidic [PvSUT1.1 was found to be repressed in the leaf upon heat stress [P. vulgaris was therefore hypothesized to be associated with the repression of PvSUT1.1 under high temperature [+ can leak through membranes [The e acidic . The tone acidic . In the t stress . Since tperature . Howeverembranes . AlthougSUT expressions by factors such as photosynthetic rate, light level, and CO2 concentration are limited [Amaranthus caudatus and Vicia faba as the models for C4 and C3 plants, respectively, despite different kinetics and extents, the C4 and C3 plants both exhibited light-dependent cytosolic alkalization and vacuolar acidification [2 [SUTs under different environmental conditions could not sufficiently explain the regulation of the SUT function.It has been widely accepted that SUTs are responsible for phloem loading. However, the analyses of expression data, including microarray, RNAseq, and quantitative PCR, from 167 experiments on various plant species showed that the effects on  limited . It was  limited . Other r limited . Mechanification . Such alation [2 . Under sBased on the protein sequences, MSTs can be further classified into several clades, including the STP, HXT, PLT-VGT, SBG-GLT-GLUT1, ERD6-like, and TMT clades . MonosacA. thaliana that has recently been resolved [Based on the crystal structure of STP10 from resolved ,90, it wresolved . At the resolved . The subresolved ,90.A. thaliana, AtPLT5 (polyol transporter 5) was reported to be a plasma membrane-localized MST-like protein that mediates the transport of sorbitol, glucose, galactose, ribose, xylose, mannitol, glycerol, and inositol [Xenopus oocytes as the model and glucose and glycerol as substrate examples, the transport activity of AtPLT5 was demonstrated to be pH-dependent [Malus domestica) [Datisca glomerata has the optimal pH for transport activity at pH 4.5 [In inositol . Using tependent . Maximalependent . At pH 6ependent . When thependent . AtPLT5 ependent . Therefoependent . The pH-mestica) . MdSTP13mestica) . Using tmestica) . An incrmestica) . Differet pH 4.5 . In plant pH 4.5 . Since dA. thaliana, AtSTP1 was found to have a consistently high expression level in both the root and leaf among all 14 of the STPs identified, under normal conditions [AtSTP13 was highly inducible by salinity and ABA treatment [ATSTP1 in the root was also induced by salinity, the fold change is much less than that of AtSTP13 [stp1 and stp13 mutants had reduced abilities to uptake glucose and fructose, while stp1 also had reduced galactose uptake [stp13 mutant was enhanced [STPs in O. sativa. In this study, the expressions of STPs were found to be responsive to stresses including cold, high temperature, and submergence [STPs, which also had different expression patterns in different tissues [In nditions . Howeverreatment . Althoug AtSTP13 . Both ste uptake . After senhanced . Based oenhanced . Such dimergence . However tissues .Plants can absorb inorganic and organic nitrogen from the environment via the root system . In soil+-Symporters (PHSs). Members of the ATF family and the APC family usually share similar transport activities and protein structures. These transporters usually function by one of these mechanisms: solute-cation symport, solute-solute antiport, or facilitated diffusion at the plasma membrane [Amino acid transporters are found in diverse plant species. They are categorized into three major families: the Amino acid Transporter Family (ATF)  family), the Amino acid-Polyamine-organoCation (APC) family and the Usually Multiple Acids Move In and out Transporter family (UMAMIT) ,101,102.membrane , and themembrane ,104. UMAmembrane .+-pumping complex featuring a proton-pumping ATPase at the membrane [AAP3, AAP4, or AAP5, the proline uptake rate was increased when the external pH was made to decrease. In other words, the higher external H+ concentration leads to a stronger transportation driving force for the uptake of the amino acid [Amino acid transporters act as secondary active transporters, with the specific amino acid being transported coupled to the proton motive force generated by the primary active Hmembrane ,107,108.membrane ,107,108.membrane . In tranino acid ,110,111.Hordeum vulgare (barley) proline transporter, HvProT, was suggested to be crucial in transporting proline to the root tip region upon salt stress [Triticum aestivum (wheat) proline transporter 3, TaProT3, was upregulated in the root under salt stress whereas TaProT1 and TaProT2 were downregulated [cis-regulatory elements were found within the promoter regions of the amino acid transporter genes [cis-regulatory element analyses were conducted between Brassica napus (rapeseed) and Arabidopsis, and the results were consistent with previous research [Changes in the expressions of amino acid transporters were reported under abiotic stresses such as salt and water stress in Arabidopsis, rice, wheat, and barley ,115,116.er genes . Similarresearch , where mresearch . Based oresearch ,118.+ and Cl−, and the reduced levels of other essential ions such as K+ and Ca2+ [Olea europaea [Cucumis sativus [O. europaea, it was shown that proline treatment reduced the level of Na+ but enhanced the level of Ca2+ in the leaf under salt treatment [C. sativus, it was shown that proline treatment reduced the level of Cl− in the leaf under salt treatment [The accumulation of proline is widely known to protect plants from water-related stresses such as salinity, drought, and freezing . The higand Ca2+ . In diffeuropaea  and Cucu sativus , it was reatment . In C. sreatment . Exogenoreatment . These oPsuedomonas syringae genome [Lysine Histidine Transporter 1 (LHT1), expressed in the rhizodermis and mesophyll in Arabidopsis [lht1 had reduced susceptibility to the hemibiotrophic pathogen, P. syringae [lht1 plants. By studying the accumulation patterns of H2O2 and NO in the mesophyll and the spatial expression patterns of the pathogen-induced LHT1, it was suggested that LHT1 helped to maintain a low reactive oxygen species (ROS) level by keeping the glutamine level high. As a result, the low ROS level hindered the activation of SA-mediated defense responses [Plants carry a readily utilizable form of nitrogen. It is conceivable that the amino acid composition in plants was one of the determinants of plant–pathogen interactions. The importance of amino acid transporters in sustaining pathogen growth is not surprising. For example, there are a large number of transporter protein-encoding genes in the e genome . The cele genome ,126. It e genome ,128. A bbidopsis  was relasyringae . It was esponses .AtCAT1 (Cation Amino acid Transporter 1) incorporated lysine at a higher rate and was more resistant to P. syringae compared to the wild-type and the cat1 mutant [AtCAT1 overexpressor, the resistance to the bacterial pathogen P. syringae was enhanced with an increased SA level in the leaves [In another study, Arabidopsis overexpressing 1 mutant . In the e leaves . Therefoe leaves ,126.+ production during nitrogen assimilation [TaALMT1 was positively correlated with the growth performance of the wheat plant in the alkaline environment. The expression pattern of the amino acid transporter SlCAT9 in Solanum lycopersicum was linked to the exchange of GABA for glutamate and aspartate during fruit ripening, and eventually led to changes in the amino acid composition of the developing fruit [Although amino acids usually carry charges and their transportation across membranes is often coupled with protons, there was no significant direct correlation among amino acid concentrations, transport, and cellular pH ,129. Nevmilation . Moreovemilation . The expng fruit . The resng fruit . The corng fruit . It was ng fruit .− into the vacuole for turgor regulation [+. For example, AtSUC4 was demonstrated to have reversible direction of H+ transport when the H+ gradient between the membrane was made to reverse, although the structural basis of such reverse transport direction by the transporter has remained unclear [phH7 has the substrate binding site close to both sides of the transporter to allow alternating access of glutamate from either side of the transporter [The activities of active transporters have been reported to be pH-dependent. Such property implies the regulation of the transporter activities at biological membranes between various cellular compartments with different pH. An obvious example is the increase in proline uptake by amino acid transporters when the extracellular pH was made to decrease . In addigulation , while tgulation . The pH  unclear . Reverse unclear . For exa unclear . The exi unclear . In anotnsporter . HoweverThe pH of different cellular compartments is highly regulated and yet highly dynamic, especially when under stress. The alteration in pH of different cellular compartments under stress implies the regulation of transporter activities. For example, under drought stress, the pH in xylem sap was found to be increased from 6.1 to 6.7 , which iEDS5 is coupled with possible pathogen-induced cytosolic acidification [2 concentration had limited effects on the expressions of SUTs [Considering the mechanics of the active transporters under stress, it could be deduced that the altered expression of the transporters under stresses could not fully explain the regulation on the transport capacities. In some cases, the altered transporter activity under stress could be coupled with the altered expression of the gene encoding the transporter to improve the transport efficiency. For example, the pathogen-induced expression of fication ,51, whic of SUTs . HoweverThe modulation of the expression of active transporters in plants has been considered a common approach to modulate the accumulation of desired metabolites . Since p+ across the biological membranes results in electrochemical proton gradient, which means the proton gradient as well as the electrical potential gradient across the membrane, to energize the substrate transport. In the above discussion, several proteins, such as AtEDS5 [The movement of Hs AtEDS5 ,51, AtSUs AtEDS5 ,79,81, as AtEDS5 ,125, wers AtEDS5 , AtDTX33s AtEDS5 , AtDTX35s AtEDS5 , PvSUT1.s AtEDS5 , AtPLT5 s AtEDS5 , MdSTP13s AtEDS5 , and HvPs AtEDS5 , the traExamples of the activities, pH dependence, and the biological significance of ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters are summarized in ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters are involved in the transport of ions, toxic compounds, sugars, hormones, and amino acids. The transport of these molecules is important in the growth, development, and stress adaptations of plants. Since these transporters are driven by proton motive force, the activities of these transporters are highly regulated by cellular pH, which is usually influenced by both abiotic and biotic stresses. Besides directly participating in the transport activity by providing the proton motive force, pH also influences the charges of the substrates to be transported. In addition, the pH itself could possibly affect the transporter function. These factors together affect the transport efficiency. The role of the proton in molecular transports deserves more attention than it has received thus far. In-depth structural and mechanical studies will be needed to delineate the effects of pH, proton gradient, and electrical potential gradient on the transporter functions.


RNA-based therapeutics have entered the mainstream with seemingly limitless possibilities to treat all categories of neurological disease. Here, common RNA-based drug modalities such as antisense oligonucleotides, small interfering RNAs, RNA aptamers, RNA-based vaccines and mRNA drugs are reviewed highlighting their current and potential applications. Rapid progress has been made across rare genetic diseases and neurodegenerative disorders, but safe and effective delivery to the brain remains a significant challenge for many applications. The advent of individualized RNA-based therapies for ultra-rare diseases is discussed against the backdrop of the emergence of this field into more common conditions such as Alzheimer’s disease and ischaemic stroke. There remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system; coupled with the accelerated development expected over the next decade, the true potential of RNA-based therapeutics to transform the therapeutic landscape in neurology remains to be uncovered. The messenger RNA (mRNA)-based COVID-19 vaccines have thrust RNA-based therapeutics into the mainstream, but RNA drugs are far from new. Over 40 years of research is now culminating in the rapid expansion of these new classes of drugs, and approvals for diseases involving the nervous system are leading the way with ten approvals to date . RNA-bastrans splicing (SMaRT) . The. The8]. nscripts ,67, howen signal ,69. Morefor FSHD . An addiynamin 2 .The example of using ssAONs to remove a polyglutamine-expanded exon in SCA3 was discussed above. The trinucleotide repeat disorders are also good candidates for AON targeting designed to downregulate toxic protein expression and reduce aggregation. In the case of SCA1, SCA2, SCA3 and SCA7 this has been investigated in mouse models where AONs delivered via intracerebroventricular injection reduced disease protein levels and improved phenotypes highlighting significant promise for AON therapy in this group of diseases . For HunSNCA gene. It is suggested that decreasing SNCA expression could delay disease onset or modify progression; this hypothesis has been tested using AONs. An amido-bridged nucleic acid (AmNA)-modified AON efficiently reduced SNCA mRNA and protein levels in-vitro and was efficiently delivered to the mouse brain via intracerebroventricular injection resulting in an amelioration of neurological defects in a PD mouse model expressing human wild-type SNCA . ,93. traned decay . In the ystrophy . For SMA protein . In the survival . Exon 10ng SMaRT . SMaRT cFTDP-17) . SMaRT cneurones  and can auopathy . Thus, tDMD exon 2 leading to either mRNA containing a single copy of exon 2 or no copies at all, the latter resulting in a functional but N-terminally truncated protein isoform [Another more nuanced alterative to standard AONs takes advantage of the U-rich small nuclear ribonucleoproteins (snRNPs) which mediate pre-mRNA splicing. The U7 snRNP however does not play a role in splicing but rather processes the 3ʹend of replication-dependent histone mRNAs. The U7 snRNP histone binding domain can be custom-modified to target the splicing of a gene of interest and delivered using traditional gene therapy vectors such as adeno-associated viral (AAV) vectors  [3]. Thi isoform . Planned isoform . A potenIn principle many neurological and neurodegenerative disorders could be treated or greatly diminished via gene silencing, however delivery roadblocks have slowed progress in comparison to siRNA delivery elsewhere such as the liver . The oneet al. observed minimal evidence for immune stimulation demonstrating its high tolerability as well as minimal off-target effects. Alternative conjugated-based delivery systems are being pioneered by Alnylam Pharmaceuticals who have used the sugar molecule, N-acetylgalactosamine  to effectively target the liver, the company have presented on the development of a similarly novel conjugate system to target the central nervous system with data in non-human primates on the silencing of β-catenin, a component of the Wnt signalling pathway and have an siRNA targeting APP in the pipeline [Until recent advances, when directly injected into the brain, siRNA affects only nearby cells for a short duration, leading to the undesirable requirement for frequent administration. However, potent and sustained huntingtin gene silencing throughout the central nervous system in small and non-human primate animal models has now been achieved using a divalent chemical scaffold (di-siRNA) delivered via a single injection into the cerebrospinal fluid . Di-siRNpipeline .BACE1 in an Alzheimer’s disease mouse model showing improved cognitive capacity [3 platform developed by Bioasis works via peptide conjugation  and receptor-mediated transcytosis. Proof-of-concept has been obtained for the silencing of NOX4 which plays a role in ischaemic stroke [An siRNA that efficiently crosses the BBB, Gal-NP@siRNA, has shown to effectively target capacity . Gal-NP@capacity . Anotherc stroke . After ic stroke .Many studies have explored the potential for siRNA to treat glioblastoma, the most aggressive form of glioma. These include for example the use of synthetic protein nanoparticles to target signal transducer and activator of transcription 3 (STAT3)  and siRNsiRNA also holds promise for the many genetic diseases discussed above for AON therapy. For example, there are numerous studies on the development of siRNA for the SCAs using various experimental systems, of which siRNA for SCA3/Machado-Joseph disease (MJD) has seen the most extensive effort towards allele-specific gene silencing . SimilarRNA aptamers are RNA molecules with a stable three-dimensional structure that are selected to bind a target with high affinity and specificity; targets include proteins, ions, whole cells and viruses . SelectiThe specificity of RNA aptamers offers a functional advantage over small-molecule therapy owing to reduced toxicity and off-target effects and their immunogenicity is limited due to chemical modification . Currentet al. show that aptamer treatment in the context of intracranial haemorrhage can be reversed using a specific antidote, thus presenting a safer approach to the treatment of stroke. Similarly, an antidote-controlled RNA aptamer targeting Von Willebrand Factor has also been shown to significantly reduce stroke volumes in a dog model [MS is characterized by pro-inflammatory leukocyte infiltration. Two therapeutic RNA aptamers targeting interleukin-17 (IL-17) and midkine (a heparin-binding growth factor) have both shown reduced inflammation in the experimental autoimmune encephalitis mouse model which resembles MS . For iscog model . For epiog model . Targetsog model . For neuog model . For exaog model . Furtherog model ,131.ex-vivo and redelivered. A first in human phase I/II study (NCT04573140) of an RNA-loaded nanoparticle vaccine is also underway for glioblastoma [Administering mRNA as a therapeutic to treat neurological disorders has several advantages such as being able to produce proteins in their native form in mature non-dividing cells, without having to enter the nucleus. However, like all RNA-based therapeutics, delivery poses a challenge since mRNA is not particularly stable and is strongly immunogenic. There are currently no approved mRNA drugs to treat neurological diseases and clinical research is limited; but spurred by the success of COVID-19 vaccines, renewed efforts are underway for example to develop an mRNA vaccine for glioblastoma immunotherapy ,133. Sucblastoma .Figure et al. who, using a novel polyplex nanomicelle carrier, delivered brain-derived neurotrophic factor (BDNF) expressing mRNA via daily intranasal administration to a mouse model of olfactory dysfunction. They showed increased neurological recovery of olfactory function as well as the repair of olfactory epithelium to a nearly normal architecture [More in line with the other RNA-based therapeutics approaches discussed thus far, the feasibility of using mRNA to treat a sensory nerve disorder has been demonstrated by Baba itecture . An mRNAitecture .Circulating endogenous non-coding RNAs such as miRNAs, circular RNAs (circRNAs) and long non-coding RNAs are increasingly recognized for their potential use as accessible biomarkers for the diagnosis and prognosis of CNS diseases. Underlying disease mechanisms often begin pre-symptomatically and earlier diagnosis could be achieved (without direct access to brain tissue) through profiling the expression of miRNAs and other RNA species which are secreted into circulation. For example, many individual studies show that Alzheimer’s disease and/or mild cognitive impairment can be differentiated from cognitively normal controls through peripheral miRNA biomarkers , though circDLGAP4 (a known mir-143 sponge) is downregulated in the brains of a rodent stroke model as well as in the plasma of human acute ischaemic stroke patients [circDLGAP4 delivered using a lentivirus to the lateral brain ventricle ameliorated stroke outcomes and improved neurological function in a mouse model of stroke [Improvements in sequencing technologies and bioinformatic pipelines have also revealed the potential for circRNAs as biomarkers in, for example, Alzheimer’s and Parkinson’s diseases where their association with pathological processes is well established . Howeverpatients . The ovef stroke .There is now a long-standing recognition of the value of miRNAs as biomarkers, but as with circRNAs they are also viable drug targets and therapeutic agents themselves. The expression of target genes can be increased or decreased using miRNA inhibitors  and synthetic miRNA mimics respectively. miRNA mimics and inhibitors have been identified for application in CNS injuries such as stroke, traumatic brain injury and spinal cord injuries . SimilarThe rapid development of new chemistries and delivery systems for RNA-based therapeutics warrants careful screening for unwanted on- and off-target side effects. There is limited information on the toxicological properties of RNA aptamers but hybridization-dependent and hybridization-independent effects are well documented for AONs . HybridiHybridization-independent effects are more common than the above on- and off-target effects caused by Watson–Crick base pairing. AON toxicities induced by RNA-protein interactions are common and depend on the delivery system and chemistry used . For exaRegardless of the chemistry used, after intravenous administration a very high concentration of AON is found in the liver and kidneys (high exposure organs) where it accumulates as basophilic granules. This accumulation is generally considered non-adverse since the effect is reversed when treatment is terminated. However, some gapmers do induce a more severe and sequence-specific acute liver toxicity in mice after only a single treatment ,152. ThuThe possibilities to develop RNA-based drug modalities to treat neurological diseases appear limitless. Whilst progress is rapid across basic, translational and clinical research for most disease categories, there remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system. Efficient, and safe, delivery remains an important hurdle but there is precedent to overcome such challenges from for example the discovery of GalNAc conjugation for liver targeting. With the advent of n-of-1 therapies, we are likely to witness changes in the regulatory processes for individualized medicine to best accommodate the requirement for the rapid treatment of rare progressive brain diseases, as well as the need to collectively measure the success of this approach across many different disease types. In parallel to the excitement surrounding n-of-1 therapies, the field is fast emerging out of the rare genetic disease research communities and witnessing the testing of RNA-based drugs in more common and chronic conditions. This is an essential development since AON drug approvals to date have been based on testing only small numbers of patients. Thus, their true potential to transform the neurological therapeutic landscape is yet to be uncovered.


We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous The online version contains supplementary material available at 10.1007/s12104-021-10047-2. The 5'-untranslated regions (5'-UTR) of Betacoronavirus RNA genomes contain several highly conserved, structured RNA elements that play essential roles in viral RNA synthesis. SL1, the first of these RNA stem-loops, has been structurally characterized by NMR spectroscopy in Mouse hepatitis virus (MHV), Bovine coronavirus (BCoV), and the human coronavirus HCoV-OC43  according to the manufacturer’s instructions and linearized with HindIII prior to in vitro transcription with T7 RNA polymerase . After 1 h incubation time, yeast inorganic phosphatase [9.6 µg/mL /4.8 µg/mL (uniformly 15N labelled RNA) final concentration] was added. Transcription reactions (6 h at 37 °C and 70 rpm) were terminated by addition of EDTA  and NaOAc . After transcription, RNAs were precipitated by adding 1 volume equivalent of ice-cold 2-propanol and incubation for 1 h at − 20 °C. For purification, RNA fragments were separated on 12 % denaturing polyacrylamide (PAA) gels and visualized by UV shadowing at 254 nm. SL1 RNAs were excised from the gel and incubated at − 80 °C for 30 min, followed by 15 min at 65 °C in 0.3 M NaOAc. Elution was achieved overnight by passive diffusion into 30 mL 0.3 M NaOAc solution. RNAs were precipitated by addition of 4 volume equivalents of ethanol at − 20 °C overnight. If the absorption ratio 220/260 nm of the RNA after dissolving in water was higher than 1.5, RNA was desalted via PD10 columns  for the following HPLC. Residual PAA was removed by reversed-phase HPLC using a Kromasil RP-18 column and a gradient of 0–40 % 0.1 M acetonitrile/triethylammonium acetate. After freeze-drying of RNA-containing fractions and cation exchange by LiClO4 precipitation [2 % (w/v) in acetone], the RNA was folded in water by heating to 80 °C followed by rapid cooling on ice. Buffer exchange to NMR buffer  was performed using Vivaspin centrifugal concentrators . Purity of SL1 was verified by denaturing PAA gel electrophoresis and homogenous folding was monitored by native PAA gel electrophoresis, loading the same RNA concentration as used in NMR experiments .RNAs were synthesized by 15N labelled RNA sample and a 1.2 mM uniformly 15N,13C-labelled RNA sample, each in NMR buffer with 5 % (v/v) D2O for a 5 mm Shigemi tube and 7 % (v/v) D2O for a 1.7 mm NMR tube, a 1.33 mM uniformly 15N,13C-labelled RNA in 99.95 % (v/v) D2O and an 0.87 mM selectively 15N,13C(A/C)-labelled RNA in NMR buffer (5 % (v/v) D2O).Using this protocol, four NMR samples of 5_SL1 were prepared and used for the assignment presented herein: A 0.64 mM uniformly Based on our previously reported assignment of the base-paired imino groups, the amino groups of base-paired cytidines and the adenosine H2 protons for 5_SL1  NOEs. Within the H1′–H6/H8 region of the NOESY, also the pyrimidine  H5–H6 and adenosine H1′i–H2(i+1) intra−strand, (i+1) cross−strand) NOE signals are typically observed. The 2D NOESY experiment, in combination with a 2D 1H,1H-TOCSY experiment showing only the pyrimidine H5–H6 cross peaks, thus allowed the unambiguous assignment of all pyrimidine H5 and adenosine H2 protons. All protonated nucleobase carbons as well as the C1’ carbons were assigned in 1H,13C-HSQCs optimized for the respective CH-transfer CCH-COSY experiment CNC experiments -labelled sample.Starting from these available assignments and following the classical NOE-based strategy, we first assigned all anomeric H1′ protons and all aromatic H6 (pyrimidine)/H8 (purine) protons via one single “sequential walk” in a 2D NOESY spectrum acquired in D2JNN coupling for U13N3-A26N1 was 4.5 Hz as derived from the intensity ratio of cross peak to diagonal peak according to Icross/Idia = – tan2(πJNNτ) Below is the link to the electronic supplementary material.


Viroids are tiny, circular, and noncoding RNAs that are able to replicate and systemically infect plants. The smallest known pathogens, viroids have been proposed to represent survivors from the RNA world that likely preceded the cellular world currently dominating life on the earth. Although the small, circular, and compact nature of viroid genomes, some of which are also endowed with catalytic activity mediated by hammerhead ribozymes, support this proposal, the lack of feasible evolutionary routes and the identification of hammerhead ribozymes in a large number of DNA genomes of organisms along the tree of life have led some to question such a proposal. Here, we reassess the origin and subsequent evolution of viroids by complementing phylogenetic reconstructions with molecular data, including the primary and higher-order structure of the genomic RNAs, their replication, and recombination mechanisms and selected biological information. Features of some viroid-like RNAs found in plants, animals, and possibly fungi are also considered. The resulting evolutionary scenario supports the emergence of protoviroids in the RNA world, mainly as replicative modules, followed by a further increase in genome complexity based on module/domain shuffling and combination and mutation. Such a modular evolutionary scenario would have facilitated the inclusion in the protoviroid genomes of complex RNA structures , likely needed for their adaptation from the RNA world to a life based on cells, thus generating the ancestors of current infectious viroids and viroid-like RNAs. Other noninfectious viroid-like RNAs, such as retroviroid-like RNA elements and retrozymes, could also be derived from protoviroids if their reverse transcription and integration into viral or eukaryotic DNA, respectively, are considered as a possible key step in their evolution. Comparison of evidence supporting a general and modular evolutionary model for viroids and viroid-like RNAs with that favoring alternative scenarios provides reasonable reasons to keep alive the hypothesis that these small RNA pathogens may be relics of a precellular world. Containing only ∼250–430 nucleotides (nts), they are able to replicate autonomously (i.e. in the absence of a coinfecting helper virus) in their hosts (restricted so far to higher plants) and to invade them systemically and, in some instances, cause disease. In addition to their small size, all viroids share: (1) a circular structure, (2) a high self-complementarity, leading to compact secondary structures, (3) replication through a rolling circle mechanism with only RNA intermediates, and, most importantly, (4) a lack of protein-coding ability .The lack of coding capacity, in particular, distinguishes viroids from RNA viruses, which in all cases (excepting some plant satellite viruses) encode at least the RNA-dependent RNA polymerase (RdRp), the only protein common to all RNA viruses and essential for their replication . MoreoveSeveral scenarios have been proposed to explain the origin of viroids  ribozymes, the key signature of a ‘primordial world’ and, more specifically, of an RNA world . The modIn developing our scheme for the origin and subsequent evolution of viroids, we have complemented phylogenetic reconstructions with both relevant molecular data  and available biological information . In doing so, we have followed an approach previously applied to RNA viruses . Where a2.Pospiviroidae  do. Of note, members of the Avsunviroidae infect a very narrow range of hosts (dicots), restricted to those in which each viroid was initially reported and to some closely related plant species. Below, relevant structural and biological features of these chloroplast replicating RNAs are considered in the frame of possible scenarios for their emergence and evolution.Members of this family adopt rod-like, quasi-rod-like, or multibranched secondary structures and replicate in plastids, mostly chloroplasts, through a symmetric double rolling circle mechanism in which the oligomeric RNA (+) and (−) intermediates self-cleave by hammerhead ribozymes (HHRs)  embedded2.1Avsunviroidae and of the genus Avsunviroid  (in silico (in vitro (in vivo (cis-acting HHRs (Avocado sunblotch viroid (ASBVd), type member of the family nviroid) , is one nviroid) . ASBVd Rn silico , in vitrin vitro , and in Avsunviroing HHRs , thus enAvsunviroidae . Its discovery (Avsunviroidae) for grouping those viroids not included in the first one (Pospiviroidae). In silico  is the type member of the genus iscovery  establisn silico , in vitrin vitro  and in vPelamovir of ∼100 nt) emerging in the RNA world as the result of template switching by a ‘jumping’ RNA polymerase. Having transcribed the lower strand of this domain, the polymerase would have turned back and resumed transcription using the recently synthesized RNA as a template, thereby producing both strands connected by a small loop. This mechanism would explain why the catalytic domain is essentially self-complementary. Moreover, because each strand is itself self-complementary, the catalytic domain can also adopt an alternative cruciform structure , inset . Such a Altogether the size of the RNA to be synthesized nonenzymatically in the primordial scenario would be reduced to less than half the size of the ∼100-nt catalytic domain. This domain (or protoviroid) would then have eventually fused to the multibranched domain, assuming some evolutive advantage for the resulting recombinant; for example, intracellular or intercellular movement. Thus, the multibranched ∼230-nt domain, stabilized by a kissing-loop interaction, might have emerged at a later stage in the cellular world. There is experimental evidence, apart from that obtained for ASBVd, that NEP also catalyzes the polymerization of PLMVd strands in plastids . The higin silico  shows clear similarities with PLMVd, including a catalytic domain of about 130 nt, with stable HHRs in either polarity strand. The secondary structure predicted initially for CChMVd n silico  should bitutions . The catitutions , possiblitutions , similaritutions , was latf CChMVd . The mutf CChMVd  and remif CChMVd .bona fide viroid , initially identified as a viroid-like RNA by RNA-seq and a computational algorithm for discovering circular RNAs , was late viroid . AHVd she viroid . CaptureGrapevine hammerhead viroid-like RNA , adopts a quasi-rod-like conformation in silico (in vitro (in vivo (Pospiviroidae) , the type member of the genus n silico , in vitrin vitro , and in Elaviroid(in vivo . A possiiroidae) . Howeveriroidae) .3.Avsunviroidae, these viroids have a CCR that plays a major role in replication (Pospiviroidae are consistent with a modular evolution.Members of this family adopt a rod-like or quasi-rod-like secondary structure and replicate in the nucleus through an asymmetric single rolling circle mechanism in which only the oligomeric (+) RNA intermediates are processed into their monomeric circular form  Ishikaw. Rather lication . As disc3.1Pospiviroidae and of the genus Pospiviroid , was the first viroid discovered (in silico (in vitro (in vivo (Pospiviroidae), variable, terminal left, and terminal right (in planta as a free RNA (PSTVd (359 nt), the type member of the family scovered , 1972 anscovered . PSTVd Rn silico , in vitrin vitro , and in  can be regarded as a protoviroid because it contains structural elements that direct two of the three replication steps, as well as the corresponding cleavage/ligation site. There is also evidence for mechanisms resulting in the enlargement of pospiviroid genomes, such as a simple duplication in the left terminal domain of PSTVd resulting from discontinuous transcription by a jumping RNA polymerase , as wellin vitro-generated noninfectious PSTVd mutant containing a 9-nt deletion has been observed to evolve into an infectious RNA in vivo (in tobacco) via the appearance of a complementary 9-nt deletion that restores the rod-shape secondary structure, thus highlighting its functional relevance , most likely as a result of intracellular RNA recombination between HSVd and one or more pospiviroids known to coinfect common host plants , the type member of the genus nut palm  and predn silico  with fivn silico . Membersn silico . As shown silico . Infecten silico . These en silico . CCCVd in silico , a closen silico  and citrn silico .in vitro self-cleavage of members of the family Avsunviroidae and some viroid-like satellite RNAs (see below). Sequences around the CCCVd self-cleavage site cannot form the HHR or HP ribozyme structures characteristic of other small self-cleaving plant RNAs, however , the type member of the genus d in hop . Its natd in hop  and to sd in hop . In silisubclass . Insteadsubclass  . As in the case of CLVd described above, dahlia latent viroid (342 nt)   contains3.4Apscaviroid, folds into a rod-shaped conformation in silico containing five domains, a CCR distinct from that of PSTVd (Apple scar skin viroid (330 nt), the type member of the genus of PSTVd  and the of PSTVd  , the type member of the genus entified  and sequentified . CbVd-1 entified . CbVd-1 d genera . Its terSome coleus plants are naturally coinfected by CbVd-1 and by two other members of its genus, coleus blumei viroid 2 and 3 . CbVd-2 is composed of two blocks of sequences, one identical to the right-hand portion of the rod-shaped structure of CbVd-1 and the other identical to the left-hand portion of the rod-shaped structure of CbV-3, with sharp demarcation boundaries between the two blocks of sequences. Therefore, CbVd-2 most likely emerged by recombination between CbVd-1 and CbVd-3 coinfecting the same plant . Other c4.The presence of conserved CCR and TCR or TCH regions/motifs located in similar positions within the rod-shaped secondary structure makes it feasible to align the corresponding sequences and to infer a phylogenetic reconstruction, supported in most branches by high bootstrap values, in which members of each genus group together  Di Seri. In this5.Avsunviroidae, some viroid-like RNAs display catalytic activity mediated in most instances by HHRs but also by another two classes of ribozymes. These viroid-like RNAs, some of which have a DNA counterpart, are biologically different from viroids and each other. Present not only in plants but also in animals and possibly in fungi, they offer the opportunity to compare their possible origin and evolution with that of viroids.Resembling members of the family 5.1Pospiviroidae) has also been found occasionally trans-encapsidated by a luteovirus coat protein, with the resulting particles being transmitted plant-to-plant by an aphid , circularity, compact secondary structure, rolling circle replication, and generally lack of protein-coding ability . Unlike an aphid , such prAvsunviroidae), a common origin in the RNA world has been proposed  and its derivative cscRNA2 (∼375 nt), isolated from trees expressing cherry chlorotic rusty spot (CCRS) disease. The sequence variability detected does not affect the HHRs embedded in either polarity strand, arguing that they mediate cscRNA replication. However, cscRNA1 seems more closely related to viroid-like satellite RNAs than to viroids, suggesting that one of the dsRNA mycoviruses also associated with CCRS disease may serve as a helper virus , 2006. Fin vitro head-to-tail dimeric transcripts of the viroid-like RNA have so far been unsuccessful. Therefore, rather than being directly associated with the plant, the possibility that this viroid-like RNA may depend upon a virus infecting a fungus or another organism transiently associated with fig has been proposed as a possible alternative  and containing HHRs in both polarity strands, has been detected in fig trees growing in Hawaii . The exaernative .In both the cases just described, RNA molecules of different sizes coexist in the same host and are likely derived from recombination events , thus su5.3Several carnation accessions have been shown to contain a unique species of small RNA with close similarities to plant viroid and viroid-like satellites RNAs. In addition to its small size (275 nt), circular structure and ability to adopt HHRs in both polarity strands , this cain vivo as abundant noncoding circular RNAs of the size  expected for HHR-mediated self-cleavage and circularization  and (−) strands are detected in planta, suggesting that, like viroids and viroid-like satellite RNAs, they might undergo RNA-RNA replication through a rolling circle mechanism  in humans infected by hepatitis B virus (HBV)  was the viroidae . The repthe HHRs . The resthe HHRs  other ththe HHRs , while tthe HHRs .The modular structure of its antigenomic RNA provides a hint about the evolutionary origin of HDV. Like that of PLMVd and other pelamoviroids, HDV genomic RNA is composed of a viroid-like domain (∼300 nt) containing the ribozymes located in a terminal portion of the rod-shaped conformation. In contrast to viroids, however, this terminal domain is fused to a protein-coding domain. In one plausible scenario, the antigenomic HDV RNA may have evolved from a primordial self-replicating viroid (emerged in the RNA world) by the capture (in the protein/DNA world) of an mRNA encoding a ∂Ag-like protein, thereby enhancing the survival of the fusion product . The widin vivo dissemination of HDV (New metatranscriptomic data provided yet another unexpected twist: HDV-like agents (deltaviruses) have been reported in multiple organisms, including birds , snakes n of HDV  and HDV-n of HDV . These rn of HDV , therebyn of HDV  are founOf particular interest in this regard are HDV-like circRNAs identified in amphibians and termites , which c6.The hypothesis that viroids are host-derived, in the past considered unlikely mainly due to the lack of any traceable sequence identity between the genomes of viroids and their hosts, has been recently revived . Based oin vitro through partial instruction from DNA seeds, thus suggesting that a similar process could also be regarded as a possible original step of the viroid genesis  than to those present in complex cellular habitats, which are divided into membranous organelles crowded with multiple proteins that outperform RNA in their chemical versatility. Moreover, the replicative catalytic module of protoviroids seems a particularly good candidate to have emerged in the RNA world, considering that the three catalytic activities mediating rolling circle replication  can be provided by ribozymes, thus making this replication mechanism plausible in a protein-free world , one might reasonably expect to find some vestiges of such RNAs in cyanobacteria, from which chloroplasts evolved by endosymbiosis   . In the ear gene . PerhapsAvsunviroidae is their narrow host range, restricted to those where they were initially reported , with high-throughput sequencing of other plant species having failed so far to retrieve sequences of these but not of other viroids, including a novel apscaviroid unexpectedly infecting a monocot (an orchid)  . Such obrequency  would suAvsunviroidae and Pospiviroidae families share a common origin? Phylogenetic analyses do not answer this question, but certain hints are consistent with a link: the right terminal domain of HSVd contains a hammerhead-like structure, and the lower strand of the CCCVd CCR may contain a self-cleavage site. Moreover, PSTVd and ASBVd use during replication Pol II and NEP, respectively, with HDV RNA behaving like PSTVd. It is remarkable that viroids and this viroid-like RNA have found the same solution: to divert DNA-dependent RNA polymerases to transcribe RNA templates. The crystal structure of Pol II bound to a scaffold  vast expanses of genetically identical plants (monocultures) close to wild ecosystems that may be reservoirs of viroids, usually latent in the latter but potentially pathogenic for the former, (2) international trade of propagation material, and (3) use of diverse sources of germplasm in plant breeding, which if infected with a vertically transmissible viroid may spread it into new areas . Additio7.Based on the totality of current evidence, we believe that there are still good reasons to keep alive the hypothesis that viroids may be relics of precellular evolution . An evol