viruses
Article
A Novel Self-Cleaving Viroid-Like RNA Identified in RNA
Preparations from a Citrus Tree Is Not Directly Associated with
the Plant
Beatriz Navarro 1 , Shuai Li 1,2, Andreas Gisel 3,4 , Michela Chiumenti 1 , Maria Minutolo 5 ,
Daniela Alioto 5 and Francesco Di Serio 1,*
1 Institute for Sustainable Plant Protection (IPSP), National Research Council (CNR), Via Amendola 122/D,
70126 Bari, Italy
2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
3 Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, 70126 Bari, Italy
4 International Institute of Tropical Agriculture, Ibadan 200001, Nigeria
5 Dipartimento di Agraria, Università degli Studi di Napoli Federico II, 80055 Portici, Italy
* Correspondence: francesco.diserio@ipsp.cnr.it
Abstract: Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs
reported in plants only so far. Some viroids (family Avsunviroidae) and viroid-like satellite RNAs share
self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity
strands. Using a homology-independent method based on the search for conserved structural motifs
of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified
a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like
RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that
circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated
Citation: Navarro, B.; Li, S.; Gisel, A.; hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric
Chiumenti, M.; Minutolo, M.; Alioto, pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the
D.; Di Serio, F. A Novel Self-Cleaving typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the
Viroid-Like RNA Identified in RNA absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it
Preparations from a Citrus Tree Is
was not found again in the original citrus source when repeatedly searched in the following years,
Not Directly Associated with the
suggesting that it was actually not directly associated with the plant. Therefore, the possibility that
Plant. Viruses 2022, 14, 2265.
https://doi.org/10.3390/v14102265 this novel self-cleaving viroid-like RNA is actually associated with another organism (e.g., a fungus),
in turn, transiently associated with citrus plants, is proposed.
Academic Editors: Marc F. Fuchs and
Luisa Rubino Keywords: circular RNAs; infectious non-coding RNAs; satellite RNAs; viroids; hammerhead
Received: 25 September 2022 ribozyme; self-cleavage; high-throughput sequencing
Accepted: 12 October 2022
Published: 15 October 2022
Publisher’s Note: MDPI stays neutral
1. Introduction
with regard to jurisdictional claims in
published maps and institutional affil- Viroids and viroid-like satellite RNAs (Vd-LsatRNAs) are small, infectious, non-
iations. protein-coding and circular RNAs reported in plants only so far [1–4]. They differ from
each other at the biological level, with viroids replicating and infecting their hosts in the
absence of any helper virus, and Vd-LsatRNAs relying on a helper virus for their infectivity.
Other small, circular, non-coding RNAs containing hammerhead ribozymes, differing
Copyright: © 2022 by the authors. from viroids and other infectious viroid-like RNAs, have been reported in plants, such
Licensee MDPI, Basel, Switzerland. as (i) the retroviroid-like RNA known as carnation stunt-associated viroid-like RNA [5]
This article is an open access article that is, however, non-infectious and has a DNA counterpart integrated in the genome of
distributed under the terms and a pararetrovirus [5,6] and of the host plant [7], and (ii) retrozymes, which are ribozyme-
conditions of the Creative Commons containing retrotransposons [8].
Attribution (CC BY) license (https:// According to structural, biological and functional features, viroids are classified in
creativecommons.org/licenses/by/ the families Pospiviroidae and Avsunviroidae. Members of the family Pospiviroidae adopt a
4.0/).
Viruses 2022, 14, 2265. https://doi.org/10.3390/v14102265 https://www.mdpi.com/journal/viruses
Viruses 2022, 14, 2265 2 of 14
rod-like or quasirod-like conformation of minimal free energy, which contains a central
conserved region (CCR) involved in their nuclear replication mediated by an asymmetric
rolling circle mechanism [9]. This replication pathway contemplates the formation of
circular RNAs in only one polarity strand [10]. Viroids of the family Avsunviroidae lack
the CCR but contain in both polarity strands the structural elements needed to form
hammerhead ribozymes (HHRzs) [11]. These ribozymes are inactive in the rod-like or most
stable branched conformations adopted by the circular viroid RNAs, but adopt the active
conformation during transcription, thus, providing the self-cleaving activity needed to
complete the viroid replication through a symmetric rolling circle mechanism. The hallmark
of this replication mechanism is the accumulation in vivo of circular RNAs of both polarity
strands [12,13]. HHRzs have also been reported in Vd-LsatRNAs. However, while viroid
RNAs contain one HHRz in either polarity strand, VL-satRNAs may also contain the HHRz
in only one RNA polarity. The other polarity strand may lack self-cleaving activity or may
contain another ribozyme named hairpin [2]. Both viroid and Vd-LsatRNA populations
infecting a single host are composed of closely related sequence variants, thus, showing
the typical features of quasispecies [14] previously reported for viruses [15]. Sequence
variability observed in the populations of these infectious agents generally preserve the
most stable conformation of the RNA and some critical structural motifs of major relevance
for their infectivity [16].
Whether viroids or Vd-LsatRNAs also exist in organisms other than plants is not
known. However, indirect evidence was provided for two small circular RNAs containing
HHRz in each polarity strand (cscRNA1 and cscRNA2), which have been isolated from
cherry leaves [17,18]. They are not able to replicate autonomously in cherry plants and have
been proposed to be Vd-LsatRNAs of mycoviruses associated with a fungus (Apiognomonia
erythrostomam) infecting cherry leaves [19–24]. Recently, two new Vd-LRNAs containing
aHHRz in each polarity strand, named fig hammerhead viroid-like RNA 1 and 2 (FHVd-LR1
and FHVd-LR2), have been reported from fig trees grown in Hawaii, but their biological
nature remains undetermined [25]. These findings suggest that Vd-LRNAs containing
HHRzs may be more common than currently thought and that their hosts may include
organisms other than plants.
In the last few years, several new viroids of the family Pospiviroidae have been identified
in plants by high-throughput sequencing and the following search for de novo assembled
contigs with sequence similarity to viroids previously annotated in databases [26–30].
However, this approach is unable to identify novel viroids and viroid-like RNAs not
sharing significant identity with those already known and is expected to be less efficient in
the identification of HHRz-containing viroids. In fact, members of the family Avsunviroidae
share little-to-no sequence identity. Instead, relationships between viroids of this family are
based on structural features, such as the quasirod-like or branched secondary structure,
tertiary interactions (i.e., kissing-loops) and the type of hammerhead ribozymes embedded
in both polarity strands of their circular RNAs [11]. Therefore, it is not surprising that the
most recently reported members of the family Avsunviroidae, apple hammerhead viroid,
and a potential new viroid of the same family, grapevine hammerhead viroid-like RNA,
were identified using HTS-based methods that are sequence homology-independent and
identify potential novel viroids and/or viroid-like RNAs by looking for de novo assembled
contigs with terminal direct repeats, a hallmark of potential circular RNAs [31,32].
In an attempt to identify novel viroids of the family Avsunviroidae and other viroid-like
RNAs, we focused our attention on HHRz, a specific hallmark of these infectious agents
to develop a homology-independent method to search for HHRzs in reads or de novo-
generated contigs from HTS libraries. By searching in the RNA preparations from a citrus
tree grown in the field, a novel Vd-LRNA containing one HHRz in either polarity strand was
identified. The molecular and biological features of such a novel circular RNA, tentatively
named citrus transiently-associated hammerhead viroid-like RNA 1 (CtaHVd-LR1) are
presented and discussed here.
Viruses 2022, 14, 2265 3 of 14
2. Materials and Methods
2.1. Plant Material, RNA Isolation and High-Throughput Sequencing
In the frame of a study aimed to identify the potential causal agent of cristacortis, a
virus-like disease of still unknown etiology, a Citrus reticulata plant grown in Southern
Italy (isolate 14A) and showing the typical symptoms of this disease was analyzed. Very
young green sprouts were collected in spring 2018 and stored at −80 ◦C. A sample of the
collected material (5 g) was extracted with buffer-saturated phenol and the preparation was
enriched in double-stranded (ds) RNAs partitioning the nucleic acids by chromatography
on non-ionic cellulose CF-11 (Whatman, Maidstone, UK) with STE (50 mM Tris-HCl,
pH 7.2, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA)) containing 16%
ethanol [33]. Following digestion with TURBO DNase (Ambion, Foster City, CA, USA) and
removing ribosomal RNAs (rRNAs) by the Ribo-Zero Plant leaf kit (Illumina, San Diego,
CA, USA), an RNA-seq cDNA library was generated using the ScriptSeq v2 RNA-Seq
Library Preparation Kit (Epicentre) according to the manufacturer’s protocol in which
an initial denaturation step (95 ◦C for 5 min) was added. The cDNA library was pair-
end sequenced (2 × 150) on an Illumina NextSeq500 analyzer (Illumina, San Diego, CA,
USA). Nucleic acid preparations enriched in highly structured RNAs were obtained with
buffer-saturated phenol followed by CF-11 chromatography, as described above [34], using
STE buffer containing 35% ethanol instead of 16%. DNA extracts were obtained with the
DNeasy Plant mini kit (Qiagen, Hilden, Germany) from 100 mg of tissue following the
manufacturer’s instructions.
2.2. Bioinformatics Analysis
Trimmed and high quality filtered reads from the sequenced RNAseq library were as-
sembled de novo using SPAdes [35], and to identify potential infecting viruses, the resulting
contigs were searched for similar sequences in the National Center for Technology Informa-
tion (NCBI) databases by BlastN and BlastX. The same datasets were used for searching for
RNAs potentially containing hammerhead ribozymes by using PatSearch [36] and applying
a specific pattern syntax developed to find conserved nucleotides and secondary structure
motifs conserved in these ribozymes. HTS reads were mapped to a reference sequence
using Bowtie [37]. Multiple alignments of nucleotide sequences were performed using
Clustal Omega [38]. The secondary structure of minimal free energy of RNAs was predicted
by RNAfold software [39]. Pairwise identities were calculated by Clustal Omega multiple
alignment [38]. Confidently predicted domains, motifs and features were searched for in
SMART [40].
2.3. RT-PCR, Cloning and Sequencing
dsRNA enriched preparations were reverse transcribed using Superscript IV reverse
transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and random
primers following the manufacturer’s instructions. PCR amplification of the cDNA was
performed using 2 µL of the reverse transcription reaction, specific primers (Table S1) at a
final concentration of 0.5 µM, 200 µM of dNTPs, 3% of DMSO, 1× Phusion HF buffer and
0.4 units of Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham,
MA, USA) in a final volume of 20 µL. The cycling conditions were initial denaturation at
98 ◦C for 30 s, followed by 33 cycles at 98 ◦C for 10 s, 63 ◦C for 15 s, 72 ◦C for 15 s and a final
extension step at 72 ◦C for 7 min. Amplified cDNAs of the expected sizes were purified on
agarose-gel and an adenine-residue overhang was added at their 5′ ends by GoTaq DNA
polymerase (Promega, Madison, WI, USA), cloned into a pGEM-T Easy vector (Promega,
Madison, WI, USA) and sequenced by Sanger Sequencing Custom Service (Macrogen,
Amsterdam, the Netherlands).
2.4. Northern Blot Hybridization Assays
Northern blot hybridization assays were performed as reported previously [41]. Briefly,
one µg of nucleic acid preparations enriched in highly structured RNAs were separated on
Viruses 2022, 14, 2265 4 of 14
5% polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (8 M urea and
1× TBE buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3)). RNA fragments
generated by in vitro transcription of a dimeric construct of (-) FHVd-LR [25] and an ss-
RNA ladder (New England Biolabs, Ipswich, MA, USA) were used as molecular markers.
After ethidium bromide staining of the gel, the nucleic acids were electrotransferred to
a nylon membrane (Hybond-N, Amersham, Little Chalfont, UK) in 0.5× TBE buffer and
hybridized in DIG easy Hyb (Roche Applied Science, Germany) at 65 ◦C with DIG-labeled
riboprobes complementary to each polarity strand of CtaHVd-LR1. Hybridization sig-
nals, revealed with an anti-DIG alkaline phosphatase conjugate (AB fragments) and the
chemiluminescence substrate CDP-star (Roche Diagnostics GmbH, Mannheim, Germany)
following the manufacturer’s instructions, were visualized and recorded with a ChemiDoc
Touch Imaging system (Bio-Rad, Hercules, CA, USA). The DIG riboprobes were generated
by in vitro transcription of the pGEM-T easy vector containing the full-length cDNA of
the sequence variant CtaHVd-LR1_3 (accession number OP418121) linearized with the
appropriated restriction enzymes using a Dig-RNA labeling mix and quantified through
direct detection via spot test following the protocol provided by the manufacturer (Roche
Diagnostics GmbH, Mannheim, Germany).
2.5. In Vitro Transcription, Self-Cleavage and 5′ Rapid Amplification of cDNA Ends
(RACE) Analysis
Monomeric transcripts of both polarity strands were obtained by in vitro transcription
of the pGemT-easy plasmid containing the full-length cDNA sequence of CtaHVd-LR1
(variant 3). The recombinant plasmid was linearized by digestion with SpeI or NcoI and
transcribed with T7 (Thermo Fisher Scientific, Wattham, USA) or SP6 RNA polymerase
(New England Biolabs) to obtain HVd-VL1 RNA transcripts of plus and minus polarity,
respectively. After degradation of the DNA template with RQ DNase (Promega, Madison,
Wisconsin, USA), the in vitro transcription reaction was analyzed in a 5% PAGE under de-
naturing conditions (see above). The full-length monomeric RNAs and the 3′ self-cleavage
products generated during the in vitro transcription were eluted from the acrylamide gels
by excising the band and grinding with one volume of water-saturated phenol and one
volume of elution buffer (100 mM Tris-HCl pH 8.9, 1 mM EDTA, 0.5% SDS) [34]. The RNAs
were recovered by ethanol precipitation and used for analysis of self-cleavage and the 5′
RACE, respectively.
For the analysis of RNA self-cleavage, the eluted RNAs were resuspended in 1 mM
EDTA pH 6, boiled for 2 min, snap-cooled on ice and then incubated at 40 ◦C for 1 h in
self-cleavage buffer (50 mM Tris·HCl, pH 8, 5 mM MgCl2, 0.5 mM EDTA). The self-cleavage
reaction was analyzed in 5% PAGE under denaturing conditions as indicated above. The 5′
terminal sequence of the eluted 3′ self-cleavage RNA products was determined by 5′ RACE
as described [25] using the oligonucleotides indicated in Table S1.
3. Results and Discussion
3.1. Identification of an RNA Containing HHRzs by In Silico Search
Viroids of the family Avsunviroidae and most viroid-like RNAs contain one HHRz in
either RNA polarity strand. The active conformation of these catalytic motifs assume a
conserved secondary structure, with typical loops and stems. By comparing all natural
HHRzs known so far, we developed a specific pattern syntax (Figure S1) to look for se-
quences adopting a similar conformation in the reads and/or in the de novo assembled
contigs from RNAseq libraries sequenced by HTS. Since most HHRzs consist of approx-
imately 60 nucleotides, it is expected that some of the 150 nt-long reads generated by
HTS may contain the full-length HHRz sequence of an infecting viroid and or viroid-like
RNA. When the search was performed using the reads sequenced from an RNAseq li-
brary generated in 2018 from young sprouts of the C. reticulata tree isolate 14A, more than
600 redundant reads containing the signatures of HHRzs were found. Most of these reads
corresponded to two non-redundant reads that were mapped to a single contig of 651 nt
Viruses 2022, 14, 2265 5 of 14
(NODE_11827_length_651_cov_57.163636), which was composed of a sequence fragment
of 550 nt followed by a partial direct repeat of 101 nt (Figure S2). As mentioned before,
the presence of direct repeats in a contig is generally considered as a possible indication
that it could correspond to a multimeric or circular RNA in vivo. In this specific case,
the presence of a HHRz in each polarity strand of the same RNA further supported its
possible circularity because such a situation is a typical hallmark of viroids of the family
Avsunviroidae [11] and of most Vd-LsatRNAs and Vd-LRNAs reported so far [1,2]. However,
pairwise identity with these viroids ranged from 37.7 to 55.9% and no significant identity
with sequences in GenBank was found by BlastN searches.
The identified RNA contained one ORF encoding a potential polypeptide of approx-
imately 100 amino acids (aa) in the plus polarity strand that, however, does not show
any significant similarity with proteins in GenBank and does not contain any confidently
predicted domain, motif and feature according to a search in SMART [40]. These findings
supported the working hypothesis that the monomeric fragment could be a novel viroid-
like RNA that, also considering its biological features (see below), has been tentatively
named citrus transiently-associated hammerhead viroid-like RNA 1 (CtaHVd-LR1). Blast
searches also found contigs with sequences almost identical (99%) to the genomic RNAs of
citrus virus A (CiVA), a coguvirus previously reported in citrus [42], and contigs sharing
high sequence identity with several mycoviruses (i.e., partitiviruses, chrysoviruses and
totiviruses) that were likely only indirectly associated with the citrus isolate 14A, being
infectious agents of fungi or other organisms potentially hosted by the field tree.
3.2. Circular Forms of Both Polarity Strands of CtaHVd-LR1 Do Exist
Circular RNAs can be separated from the respective linear forms by polyacrylamide
gel electrophoresis (PAGE) under denaturing conditions that determine a delayed migration
of circular RNAs with respect to the linear ones [43]. To ascertain whether CtaHVd-LR1
exist as a circular RNA, we performed northern blot assays using probes specific for each
polarity strand. Two major bands were detected in the nucleic acid preparations enriched
in highly structured RNAs extracted from citrus sprouts collected in 2018 using specific
probes for either polarity strand of this RNA (Figure 1), with the upper band identified as
the circular form and the faster migrating band as the monomeric CtaHVd-LR1 linear form
with an expected size of 550 nt. By preliminary northern blot assays using in vitro CtaHVd-
LR1 RNA transcripts, we excluded cross-hybridization between CtaHVd-LR1 strands of
the same polarity under the experimental conditions adopted in this study (Figure S3).
Therefore, since equalized probes and RNA preparations were used, the more intense
hybridization signal generated by one polarity strand also indicated that this is the polarity
accumulating at higher levels in vivo, to which, as previously conducted for viroids and
other viroid-like RNAs [1,2], the (+) polarity was assigned. These findings support the
view that CtaHVd-LR1 very likely replicates through the symmetric pathway of the rolling
circle replication mechanism previously proposed for viroids of family Avsunviroidae [44]
and other viroid-like RNAs containing self-cleaving ribozymes in both polarity strands [2].
Indeed, the detection of circular RNAs of both polarity strands, together with the presence
of self-cleaving ribozymes in both polarity strands, are generally considered the hallmark
of this replication mode [1].
Viruses 2022, 14, 2265 6 of 14
Viruses 2022, 14, x FOR PEER REVIEW 6 of 14 
 
(nt) 1 2 1 2
3000
2000 Circular
1000
812
618
552 Linear
500
358
260
194
(-) polarity (+) polarity
 
FiFgiugurere1 1. . Deetteeccttiion of ciircullarr aanndd lilnineeaar rfofromrms osfo bfobthot phoplaorliatyri stytrasntrdasn odfs CotfaCHtVadH-VLRd1-L bRy1 nboyrthneorrnt hern
blboltoht yhbyrbidriidziaztaiotinona sassasyasy.sL. aLnaen1e , 1R,N RANpAr eppraerpaatrioatniofnro mfrocmit rcuistriusos laistoela1t4eA 1t4eAst etdespteods itpivoesittiovCe ttaoH Vd-
LRC1tabHyVHd-TLSRa1n bdyR HT-TPSC aRn;dla RnTe-2P,CRRN; Alanper e2p, aRrNatAio nprfreopmaraatisoanm fprolemo fa csiatrmusplies oolfa tceit1ru4Os itseosltaetde 1n4eOga tive
tested negative to CtaHVd-LR1 by RT-PCR (negative control); size of RNA markers are indicated 
tooCn ttahHe Vledft-. LIdRe1nbtiycaRl Ta-lPiqCuRot(sn oefg tahteiv seamcoen RtrNolA); psirzeepaorfaRtiNonAs mwearrek elorasdaerde iinnd piacraatleldel oinn tthhee slaemft.eI gdeeln; tical
alaifqtuero ntsuoclfeitch eacsiadm treanRsNfeAr, tphree mpaermatbiroannse wearse culota vdeerdticianllyp,a arnaldl eelaicnh thhaelfs mameme bgrealn; ea fwtears nhuycblreidic- acid
triaznesdf ewr,itthh eqmuaelmizbedra DnIeGw-parsocbuest tvoe drteitceacltl yth, ae n(+d) eaancdh thea l(f−)m peomlabrirtayn setrwanads ohfy CbtraiHdiVzedd-LwR.i tHhyebqruida-lized
DiIzGat-iporno bsiegsntaolsd ferotemct btohteh (m+)emanbdratnhees( w−e)rpe orleacroirtydesdtr sainmduoltfanCetoauHsVlyd. -TLhRe. pHoysibtiroindsiz oaft icoirncusilagrn aanlsdf rom
bolitnheamr efomrbmrsa nofe Cs wtaHerVe dre-LcoRr1d aerde sinimdiucalttaende oonu tshlye. rTighhet.p Tohseit (io+)n psoolfarciitryc uwlaars aasnsdiglnineeda, rbyfo cromnsveonf tCiotnaH, Vd-
to the CtaHVd-LR1 polarity strand that accumulates at a higher level in vivo and generated a 
LsRt1roanrgeerin hdyibcraitdeidzaotinonth seigrniaglh (tl.efTt hpean(e+l)).p olarity was assigned, by convention, to the CtaHVd-LR1
polarity strand that accumulates at a higher level in vivo and generated a stronger hybridization
si3g.n3a. lT(hleef St epqauneenlc).e Variability Detected in CtaHVd-LR1 Populations Preserves a Rod-Like 
3.S3e.cTonhdeaSrye qSuternuccteuVrea roifa MbiilnitiymDale ftreecet eEdneinrgCy taHVd-LR1 Populations Preserves a Rod-Like
SecondTahrye eSxtrisutcetnucree ooff CMtianHimVadl-FLrRe1e iEnn tehreg yoriginal RNA preparations used for generating 
the TRhNeAesxeiqs tleibnrcaeryo fwCatsa iHniVtidal-lLyR c1oninfirtmheedo rbiyg iRnTal-PRCNRA upsirnegp sapraectiiofinc spurismedersfo dregseignneerda ting
thine RthNe Acosnetqigl i(bTraabrlye wS1a, sdiantiat inaollty schoonwfinr)m. Nedewb ytoRtaTl -RPNCRA upsrienpgarsaptieocnisfi, cepxtrriamcteerds udseisnigg ned
different aliquots of the original sample of young sprouts (collected in 2018 and stored at 
in−8t0h e°Cc)o, nwtiegre( Taalsbol eteSst1e,dd bayta RnTo-Pt CshRo uwsinn)g.  tNhreewe dtoiftfaelreRnNt sApepcrifeipc aprraimtioern sp,aeirxst,r aadcjtaecdenuts ing
daifnfder oefn ot pa◦ plioqsuitoet spoolfatrhitey,o trhigati nwaelrsea dmepsilgenoefdy toou anmgpslipfyro fuutlsl-l(ecnoglltehc cteDdNiAns2 o0f1 8thaen cdircsutolarre d at
−C8t0aHCV)d, -wLRer1e (aTlasbolet eSs1te).d WbiythR aTl-lP pCriRmuers ipnagirtsh, raeme pdliifcfoenresn otf stpheec eifixpcepcrteimd esrizpe awiresr,ea dobja-cent
antadinoefdo (pFipgousriet e2)p, ocolanrfiitrym, tihngat thwee irdeednetisfiicganteiodn toof acmircpulliafyr CfutallH-lVendg-LthR1c DRNNAAss wofitthh aenc iarlc-ular
CttearHnaVtidv-eL Rm1et(hToadbl. eCSl1o)n.inWgi tohf athllep armimpelrifpicaaitriso,na mprpoldicuocntss aonfdth seeqexupenecctinedg soifz ae wtoetarel oofb 3ta0i ned
(Fcilgounrees 2(F),igcuornefi Sr4m) isnhgowtheedi dtheenyt icfiocraretisopnonodf ecdir ctou lsaerqCuetanHceV vda-rLiaRn1tsR oNf CAtsaHwVithd-aLnR1a litdeernn-ative
mtieftiehdo din. sCililcoon ainngd ocofntfhiremaemd pthliafit ctahteiiorn sipzer oisd furcotms  a5n50d tose 5q5u1e nntc, iwngitho af  Ga  t+o Cta lcoonfte3n0t colfo nes
(F4i9g.u5%re, Sw4h)ischho iws ae dvathlueey tcyoprirceasl poof nodtheedr tVoLs-eRqNuAensc. eSivnacrei athnets aomfpClitcaoHnVs dw-eLrRe 1geidneenrattiefide d in
siuliscionga nthdreceo ndififfremreendt tphraimt tehr epiraisrisz ieni sinfdreopmen5d5e0ntto re5a5c1tionnt,s,w witeh haadG t+heC ocpopnotretunnt iotyf 4o9f .5%,
wihnivcehstiisgatvinaglu tehet yvpairciabl iolfitoyt ihne trhVe Lre-gRiNonAs st.aSrginectedt hbey aemacphl picroimnserw pearier gbeyn serqautedncuinsgin tgheth ree
daifmfeprleincotnpsr gimeneerrpataeidrs winithin tdhe poethnedre pnatirse aocft piornims,ewrse, fhinaadllyth aessoepspsionrgt uthnei tsyeqouf einvce svtaigria-ting
thaebivliatyri oafb tilhiety coinmtphletere CgtiaoHnsVtda-rLgRe1t eRdNbAy (eFaigchurpe rSi4m).e Trhpea sireqbuyenseceqdu eCntacHinVgdt-hLeRa1m vaprli-cons
gaentesr awterde wbeitwh etehne 9o9t.h1e arnpda 9ir9s.7o%f ipdreimntiecrasl, tfio nealclhy oatshsers saindg ttheeirs mequulteinpcle avliagrniambeilnit y of
thaellocwomedp tlhetee idCetnatHifiVcadt-iLonR o1fR sNeqAue(nFcieg uvareriaSb4i)l.itTyh aet sevqeurealn pceodsitCiotnasH iVn dth-eL CRt1aHvaVrdia-nLtRs1w, ere
bwetiwthe aepnp9ro9x.1imaantdely9 90.7 p%oliydmeonrtpichailc ptooseitaicohnso itdheenrtiafined itnh tehier muultlitpiplel ealaiglingmnemnet n(Ftigaullroew ed
thSe4)i.d Tehnet icfiocnasteinosnuos fsesqeuqeunecnec oef vtahrei ambuillittiyplae tasliegvnemraelntp sohsaitrieodn sthien htihgeheCstt aidHeVntdit-yL Rw1it,hw ith
approximately 90 polymorphic positions identified in the multiple alignment (Figure S4).
The consensus sequence of the multiple alignment shared the highest identity with the
 
Viruses 2022, 14, x FOR PEER REVIEW 7 of 14 
 
Viruses 2022, 14, 2265 7 of 14
the variant CtaHVd-LR1_3 that was, therefore, considered as the reference CtaHVd-LR1 
variant (accessionva rniauntmCtbaeHrV dO-LPR41_138th1a2t1w)a.s ,Tthheere fsoerec, oconnsdidaerryed sastrthuecrteuferreen coefC tmaHiVndi-mLRu1mvar ifarnet e energy 
(accession number OP418121). The secondary structure of minimum free energy of this
of this variant covrarrieasnpt coornredspeodn dteod tao  arroodd-l-ilkiekceon cfoormnaftoiornminawtihoicnh 7i3n.8 %wohf tihcehre s7id3u.e8s%we roefp atihreed residues 
were paired (Figu(Frigeu 3re).3 ).
Viruses 2022, 14, x FOR PEER REVIEW 7 of 14 
 
the variant CtaHVd-LR1_3 that was, therefore, considered as the reference CtaHVd-LR1 
variant (accession number OP418121). The secondary structure of minimum free energy 
of this variant corresponded to a rod-like conformation in which 73.8% of the residues 
were paired (Figure 3). 
 
Figure 2. AmplificaFtigiourne 2a.sAsamypsli fiucastiionnga sRsaNysAu spinrgeRpNaAraptrieopnarsa tfiornosmfro CmtCatHaHVVd--LLRR1-1po-psitoivseit(isvoela t( eis1o4Alate 14A in 
2018) and -negativien (F21i0g14u8Or)e a2n. dAm-npelgifaictaivtieon(1 a4sOsa)yssa umsipnlge sRNanAd pprreipmarearti
2018) an) ds -anmegaptivlee s(1 4aOn) dsa mpprliems anedr  ppriamierrs p aCirH
poan1isr RsfroCemHv C/1CRtaeHs CH1Rev/CHHvV/Cd-HLR2
2F2orF (oler
F1- op(rol(eslietfifvtt)e).. ( iCCsoHHla3teR3 e1R4vA/C inH 4F
(middle) or CH5Rev/CH6F (right); C, negative control in which ddH O ifnts).t eCaHd3oRfecvD/CNHA4wF (meivd-/CH4F (mid-
dle) or CH5Rev/CH6dFle )( orri CgHh5tR)e;v C/C,H n6Fe (griaghtit)v; Ce,  cneognattivreo clo nintro wl inh wichhich d ddHHO Oins2
as added
2 2  tienads toef acDdN oAf w caDs aNddAed two as added to 
to tthhee aammpplification mix; M, m
the amplification mix; M, mlificoaltieocnu mliax;r M m, maorl
oecleucluarl amr marker is a 100 bp DNA ladder.
ker is aark 1er0 i0s a b 10p0  bDp NDNAA  llaadddedr.e r. 
 
Figure 3. Primary and predicted secondary structures of lowest free energy of CtaHVd-LR1 refer-
Figeunrcee 3v.arPirainmt a(arcycaesnsdiopnr neduimctbeedr sOePco4n18d1a2r1y).s Rtreugcitounrse sinovfollovwede sitnf trheee feonremrgaytioonf Coft a(+H) Vandd-L (R−)1 HreHfeRrze nce
varsitaruncttu(arcecs easnsdio tnhen ruesmpebcetrivOe sPe4lf1-8c1le2a1v)a.gRe seigteios narsei innvdoiclavteedd biny ftlhaegsf oanrmd tahtiicokn aorrfo(w+s), raensdpe(c−tiv) eHlyH, Rz
strwucitthu rbeasrsa nddemthaerkriensgp tehceti nvuecsleloft-icdleas vcaognesesrivteds  ainre minodsti cnaatteudrabl yhaflmagmsearnhdeatdh sictrkuacrturorewss. ,Frilelsepde acntidv ely,
wiothpebna rssymdebmolasr rkeifnerg ttoh (e+)n auncdle (o−t)i pdoelsacriotyn,s reersvpeedctiinvemlyo. sPtonlyamtuorraplhhiac mpomsietirohnesa iddesntrtuifcietudr iens t.hFei lmleudl-and
opteipnles yamligbnomlsenret fwerittho th(+e) oathnedr (c−lo)npedo lvaarritiayn, trse sapree cintidvieclayt.edP boyly cmiroclrepsh ciocnptaoisniitniog nthsei dmeunttaifiteedd niun- the
muclletioptlideea. lNiguncmleeontitdwe cithhanthgeeso pthreesrercvloinnge dthve aprrioapnotsseadr eroidn-dliikcea tseedcobnydcairryc lsetrsuccotunrtea ianrien ign trheed,m wuittha ted
insertions marked by thin arrows. 
nucleotide. Nucleotide changes preserving the proposed rod-like secondary structure are in red, with  
Figure 3. Primary ainnsedrt ipoInrnse tmdhaeir cpktreeoddpbo yssetehdci nrooandrr-dloiwakers .yst rsutcrtuurce,t uthree cse notrfa ll orewgioens tc ofnrteaien se tnhee rHgHyR zosf o Cf tthaeH twVo d-LR1 refer-
ence variant (accessioponla nrituy strands that were located one in front of the other. Interestingly, most polymor-
phiIcn pthosmeitpiboronepsr o dOsiedPd n4roo1td 8m-1loi2kd1eif)sy.t  rtRuhceet gupirroeo,pntohsse eidcn evrnootdrl-avllikereed gs ieiocnno nctdohanerty af iosntrsrumthctaeutrHieo HbneRc zoasufos (ef+ tt)hh eeaytn wdo (−) HHRz 
structures and the rpeoswlapereritecy ctsoitv-rvaean rsdiaestlitfoh-nacstl, ewcaoenvrveaelgrosceiao tsneisdt oeofs nc aeanrinoen firincoandlt iinocftaott hweedoob tbhlyeer  .fblIaanstege-spre aasitrnisn dogr lt ywh, meicroek sl toapcraortleoydmw ionsrt,op r heicspectively, 
with bars demarkinpgols otiothipoesn  (nsFiudgiucdrleen o3o)tt. iImdt ieos dsw icofyrothtnhyse oepfr nrvooeptedo ts hieandt  smreovdoe-rslaitkl  pneoasleytcumoronardpla hhriyca pmsotrsmuitcioteunrrshe mebaaepdc at ous ttshreeut chceetnyutrrwael esr.e Filled and 
open symbols refer toha (m+m) aernhdea d(− r)e gpioonl a(sreiet bye,l orwes).p Aeltcotgievtehelry, .d Patoa loynm thoe rsepqhueicn cpe ovasriitaiboilnitsy  of CtaHVd-
LR1 populations preserving the predicted rod-like secondary structure suggiedste an mtiafijoerd  in the mul-
tiple alignment withr otlhe eo fo ththis estrr uccltounrael dfe avtuarer ifaorn itts  saurvei vianl.d icated by circles containing the mutated nu-
cleotide. Nucleotide changes preserving the proposed rod-like secondary structure are in red, with 
insertions marked by thin arrows. 
 In the proposed rod-like structure, the central region contains the HHRzs of the two 
polarity strands that were located one in front of the other. Interestingly, most polymor-
phic positions did not modify the proposed rod-like secondary structure because they 
were co-variations, conversions of canonical into wobble base-pairs or were located into 
loops (Figure 3). It is worthy of note that several polymorphic positions map to the central 
hammerhead region (see below). Altogether, data on the sequence variability of CtaHVd-
LR1 populations preserving the predicted rod-like secondary structure suggest a major 
role of this structural feature for its survival. 
 
Viruses 2022, 14, 2265 8 of 14
co-variations, conversions of canonical into wobble base-pairs or were located into loops
Viruses 2022, 14, x FOR PEER REVIE(FWig ure 3). It is worthy of note that several polymorphic positions map to the centr8a lof 14 
 hammerhead region (see below). Altogether, data on the sequence variability of CtaHVd-
LR1 populations preserving the predicted rod-like secondary structure suggest a major role
of this structural feature for its survival.
3.4. HHRzs Embedded in CtaHVd-LR1 Are Active during Transcription and in the Absence of 
3.P4r. oHteHinRsz s Embedded in CtaHVd-LR1 Are Active during Transcription and in the Absence
of ProteTinhse CtaHVd-LR1 hammerhead ribozymes have a central catalytic core surrounded 
by tThhreeeC htaaHirpVidn-sL, Rtw1oh aomf wmheirchhe a(hdariribpoinzy Im anesd hIIa)v aerae ccelonsteradl bcyat samlyatilcl lcooorepss u(Frrigouurned 4e)d. The 
bysetlhf-rceleeahvaairgpei nssi,tetw iso porfewcehdicehd (bhya iarptyinpiIcaanl dGUII)Aa raencdlo AseUdAb ytrsimnuacllleloootipdses(F iing uthree 4()+.)T ahned (−) 
seplfo-lcaleraitvya gstersaintediss, prreescpeedcetdivbeylya.t ySpuicchal aG UcoAmabnidnaAtUioAn torifn turcinleuoctildeoestiidnetsh ep(r+e)ceadndin(g− t)hpeo -self-
lacrlietyavstargaen dssit,er ewspaesc tpivreelvyi.oSuusclhy aocnolmy brienpaotirotnedo fitnr inthuec le(+o)t idanesdp (r−e)c eHdiHngRzthse osfe lcf-scclReaNvAag1e and 
site was previously only reported in the (+) and (−) HHRzs of cscRNA1 and cscRNA2,
twcsocRviNroAid2-,l itkweoR vNirAoisdr-elipkoer tReNd Atosm reopsot rlitkeedl ytob me soastte llilkiteelyR NbeA ssaoteflalitme yRcNovAirsu osf [a2 4m].yTchoevirus 
G[U24A].t rTinhuec GleUotAid etrwinauscallesootpidrees wenatsa tatlhsoe sparmeseepnot saitti othnse osfatmhee (p+o) sHitHioRnzs ooffF tHhVe d(+-L) RH[H25R] z of 
anFdHVindt-hLeR ([−25)]H anHdR izn othf et h(−e) VHdH-LRsza toRf NthAe sVodf-LvsealvtRetNtAobsa ocfc voemlvoettt tloebvaicrcuos manodttlleu vceirrunse and 
trlauncseirennet tsrtarenaskievnitr ustsre[4a5k, 4v6i]r.uTs h[4e5A,4U6]A. Twhaes ApUreAv iwouassl yprreevpioorutesdlyi rnetphoert(e+d)- sintr athned (R+)N-sAtrand 
ofRtNheAs oatfe tlhliete soafteblalirtlee yofy belalrolweyd yweallrofwv idruwsa[r4f7 ]viarnuds t[w47o] naonvde ltwvior onido-vleikl evRirNoiAd-slilkikee RlyNAs 
asliskoecliyat eadsswociitahtecadr rwotitrhe dcalreraoftv rireuds l[e4a8f] .vTirhues s[e4q8u].e nTchee vsaerqiaubeinlictye ovbasreiarbvieldityin otbhseeHrvHedR zisn the 
alHwHayRszps raelsweravyesd ptrheeseprvredi cttheed parcetdiviceterdib oazctyimvee rsibtrouzcytumre sbtercuacutuserem boesctauchsae nmgeosstw cehraenges 
mwaperpee dmatptphedh atm thmee hrhaemadmleorohpesa,dp rloesoeprsv,i npgretsheervpiontge nthtiea lptoertetinatriyali ntetertriaacrtyio instebreatcwtieoenns be-
sotwmeenu scolemoeti dnuesc,leoortdididesn, otr mdiodd nifoyt bmaosedipfyai briansge opfatihriensgt eomf tsh(eF sigteumres 4()F.igInurfeo u4r). oInf  tfhoeur of 
setqhuee snecqeudevnacreidan vtas,rioannetsc,h oaneg echwaansgfeo uwnads ifnouthnedc ianta tlhyeti cactoarleytoifc tchoere(− o)f HthHe R(−z) wHhHerRezt hwehere 
Uthate pUo saitti opnos3i7ti7own a3s7r7e pwlacse rdepblyaaceCd, bthyu as, Cm, otdhiufys,i nmgothdeifcyoingse trhve dcoCnUseGrAvemd oCtiUf iGnAto ma otif 
CiCnGtoA a mCoCtiGf A(F imguorteif4 ()F. iTghueres a4m). eTmheu tsaatmione hmaustbaetieonnp hreavs iboeuesnly prerepvoirotuedslyin roenpeorvtaerdia innt one 
ofvpareiaacnht olaft peneat cmho lsaateicntv mirooisdai(cP vLiMroVidd )(,PaLnMdVitdh),a asnbde eitn hsahso bwenent hshatowit nd itdhanto itt adfifde cntotth aeffect 
setlhf-ec sleealfv-icnlegacvaipnagb cilaitpyaobfiltihtye omf uthtaet emduHtaHteRdz H[4H9]R. z [49]. 
U U
A
G C U G C U
C G G C G
A
C U U
G G . U
C A A G . C
G G . C C G U G . C
C U G . C C
C U C . G
A A C . G A . U
U G . C
G . C G . U . A
G U C . G
G . C C . G G . C U C . G
U . A U . A
C . G C . G
G A A C G A A C
A U A U C
A G A G
A . U G U A A . U G U A
C . G U . A
G A . U C . G C
C . G C . G
C . G A . U
49 U . G 544 U 348 C . G 405
3’ 5’ 3’ 5’
(+) (-)
 
FiFgiugruere4 .4P. rPimrimarayrya nadndse csoencodnadryarsyt rustcrtuucrteuoref hoaf mhmamermheeardheraidb orziybmozeysm(HesH (RHzHs)Rozfs)(+ o)fa n(+d) (a−n)d (−) 
CCtatHaHVdV-dL-RL1R. 1T.h Tehneu cnlueoctliedoetsidoefst hoef ctahtea lcyatitcalcyotriec ccoonrsee crvoendseinrvmedo sitnn matuorsat lnhaatmurmale rhhaemadmsetruhcetaudre struc-
arteurinesb alure .inT hbeluclee. aTvhaeg eclseiatevaogf e ascihter oibf oezaycmh eriibsoiznydmicaet eisd ibnydiacnataerdr obwy. aNnu acrlreowtid. eNsuincltehoetildoeosp isn the 
oflobopths oHfH bRotzhs HpoHteRnztsia plloyteinvtioallvlye dinivnotlevretida riyn itnetretirarcyti oinntseraarcetmionarsk aerde bmyabrklueedl ibnye sb.luNeu lcinleeost.i dNeuscleo-
intitdhees( +in)  athnde ((+−) )apnodla (r−it)y paorlearniutym aerrea tnedumcoenrsaitdeedr icnogntshideierrpinogs itthioenirs pinotshiteiovnasr iiann tthCet avHaVridan-Lt RC1t_a3H. Vd-
MLuRta1t_io3n. Ms oubtsaetrivoends ionbosethrveredv airni aontthsearr veainridainctast eadrei nincdiricclaetse,dw iinth ctihrcelecos,m wpiethns tahteo rcyommupteantisoantosrayn md uta-
thtoiosensn aont dm othdoifsyei nngott hmeopdriofpyoinsged thseec pornodpaorsyesdt rsuecctounredaorfyth setrHucHtuRrzes oref pthoert eHdHinRzresd r.eported in red. 
The preservation of the hammerhead active structure, regardless of the sequence var-
iability, is indirect evidence that these motifs play a key biological role. We provided ex-
perimental evidence that the HHRzs of CtaHVd-LR1 of both polarity strands are active 
during transcription, generating RNA fragments with sizes consistent with those expected 
considering the activity of HHRzs in the nascent RNA transcript (Figure 5). Moreover, 
when monomeric RNA transcripts were eluted from the gel, denatured and slowly cooled 
 
Viruses 2022, 14, 2265 9 of 14
The preservation of the hammerhead active structure, regardless of the sequence
variability, is indirect evidence that these motifs play a key biological role. We provided
experimental evidence that the HHRzs of CtaHVd-LR1 of both polarity strands are active
Viruses 2022, 14, x FOR PEER REVIEW
 du ring transcription, generating RNA fragments with sizes consistent with those expect9e dof 14 
considering the activity of HHRzs in the nascent RNA transcript (Figure 5). Moreover, when
monomeric RNA transcripts were eluted from the gel, denatured and slowly cooled in a
sienl fa- csleelafv-calgeeavbuagffee rbcuofnfetar icnoinngta5inminMg M5 mgCMl2 ,MmgoCstl2o, fmthoesttr oafn sthcrei ptrtasnelsfc-rcilpeat vseedlf,-gcleenaevreadtin, ggen-
tehreateinxpge tchteed eRxpNeActefrda gRmNeAn tfsr(aFgimguernets5 )(.FTighuerree f5o)r.e ,TthheereHfoHrRe,z tshoef HboHthRzpso loafr ibtyotsht rpanodlasrity 
osftrCantadHs Vodf -CLtRaH1 aVrde-aLlsRo1 aacrteiv aelsino athcetivabe sienn tcheeo afbasneyncper ootfe ainn,yc opnrofitreminin, gcotnhfeirrmibionzgy tmhaet ricibo-
nzaytmuraetiocf nthateuserec aotfa tlhyetiscem caottaiflsy.tic motifs. 
 
FFiiggurree5 5.. ((A))S Scchheemmaatitcicr erepprerseesnetnattaiotinono fotfh tehpe lpaslamsmidsidasn adntdh ethReN RANpAr opdruodctuscgtesn geernaeterdatbeyd ibnyv iintr voitro 
ttrraannssccrriippttiioonn.. PPllaassmmiiddssc coonntataininininggt htheem monoonmomereicriccD cNDANAse qseuqeunecnecoef oCft aCHtaVHdV-LdR-1L_R31i_n3 oipnp oopspitoesite 
oorriieennttaatitoionnssw weerereli lnineaerairziezdedan adndtr atrnasncrsicbreibdetdo tpor opdruodceumceo mnoomnoermiceRriNc ARNs (AMs) (oMf ()+ o)f a(n+d) a(n−d) (p−o)l paroiltayrity 
ssttrraannddss aannddt htheer erespspecetcitvivee5 ′5(′ 5(′5F′)F)a nadnd3 ′3(′3 (′3F′)Ff)r fargamgmenetnst(sl e(flteaftn adnrdig rhigt,hrte,s rpeescpteivcetilvye) ldye)r diveerdivferdo mfrom 
tthhee HHHHRRzzs seelflf--ccleleaavviningga actcitvivitiyt.yP. lPalsamsmididse sqeuqeunecnecseasr eardee dpeicptiecdteidn ignr egerne,ewn,h wilehiilne yinel lyoewlloawnd abnldu eblue 
are the polymerase promoter and CtaHVd-LR1 sequences, respectively. The scissors and arrowhead 
amrearthke thpeo lpymoseirtiaosne porfo tmheo tseerlfa-ncdleaCvtaaHgeV sdi-tLesR. 1Tsheeq ueexnpceecst,erde sspizeect iovfe elya.cThh ferasgcimsseonrst aisn dreaprorortwedh eoand the 
mrigarhkt. t(hBe) pAonsiatliyosniso bf yth 5e%se PlfA-cGleEa vuangdeesri tdees.nTathuerienxgp eccotneddistiiozneso of fe athche ifnra vgimtreon ttraisnrsecprioprttieodn o(nleftth)e and 
rsieglhf-tc. l(eBa)vAagnea lryesaicstbioyn5 i%n tPhAeG aEbsuenndcee rodf eannayt uprriontgeicno (nrdigithiot)n osfo tfhteh peliansmviitdros tcroanntsacirnipintigo n(+()l oefrt )(−a)n mdon-
soemlf-ecrlieca CvatagHe Vreda-cLtiRo1n_i3n ctDheNaAb.s Len, cReNoAf  alnadydperro tweiinth( rsiigzhest) ionfdtihcaetepdla osmn itdhse cloefntt; asienein pga(n+e)l oAr f(o−r) the 
mabobnroemvieartiiconCst aMH,V 3d′F-L aRn1d_ 35c′FD. N(CA) .DLe, tReNrmAinlaadtidoenr owfi tshelsfi-zcelesaivnadgicea steitde obny t5h′e RleAftC; Ese eofp 3a′nFe lfrAagfmorent. 
tSheeqaubebnrceivnigat eiolencstMro,p3h′eFraongdra5m′Fs. (oCf )5D′ eRtAerCmEin partoiodnuocftss eolff -tchl ea (v+a)g aensdit e(−b)y 35′F′  RfrAaCgmE eonf t3s′ Farfrea sghmoewnnt. on 
Stheqe ulefntc aingd erliegchtrt,o rpehsepreocgtrivamelys,o wf 5it′hR tAheC E5′ pterromduincatsl onfutchleo(t+i)daen (dG)( −in)d3i′cFaftreadg bmye tnhtes areroswho. wThneo enxtra 
tTh eolbesfet ravneddr iagth tth, ere tsepremctiinvaell ye,nwdi tohft hthee5 (′+t)e r3m′Fi nfaral gnmucelenot tcidDeN(GA) (ilnedfti)c actoerdrebsyptohneda rtroo wa .nTohne-texmtrpalate 
Tnuocblseeortvidede tahtatth heatse rlmikienlya lbeenedn oafdtdheed( +b)y 3t′hFe frreavgemrseen ttrcaDnNscArip(lteafste) dcourrriensgp othned ctDoNaAno sny-ntethmepsilsa t[e50]. 
nucleotide that has likely been added by the reverse transcriptase during the cDNA synthesis [50].
5′ RACE experiments followed by cloning and sequencing of the amplification prod-
ucts 5s′hRoAwCeEd etxhpaet rtihmee n3t′ sffroalglomweendt breysculoltniningg faronmd s ethqeu eHncHinRgz oafctthiveiatym pinli fithcaet i(o−n) prooldar-ity 
ustcrtasnsdh ohwaded thteh aetxptheect3e′df rnaugcmleeonttidree sautl ttihneg 5f′r toemrmtihneuHs. HInR tzhaec (t+iv) iptyolianritthye s(t−ra)npdo, laanr ietyxtra 
snturacnledohtiadde twhease xopbescetrevdedn uactl ethoeti d5′e taetrmthien5u′st eorfm thine u3s′ .fIrnagthmee(n+t) gpeonlaerriattyedst rbayn dth,ea n(+e) xHtrHa Rz 
n(Fuicglueoreti d
′ ′
5e). wHaoswoebvseerrv, ethdisa tisth ae n5onte-rtmeminpulsatoef nthuecl3eoftriadgem aedndtegde ntoe rtahtee d3′b eyntdh eo(f+ t)hHe HsyRnzthe-
sized cDNA by the well-documented terminal nucleotidyl transferase activity of reverse 
transcriptases [50]. Altogether these data confirmed the predicted self-cleavage sites for 
the HHRz of both polarity strands of CtaHVd-LR1, thus, supporting their major role in 
the replication of this RNA. 
  
 
Viruses 2022, 14, 2265 10 of 14
(Figure 5). However, this is a non-template nucleotide added to the 3′ end of the synthe-
sized cDNA by the well-documented terminal nucleotidyl transferase activity of reverse
transcriptases [50]. Altogether these data confirmed the predicted self-cleavage sites for
the HHRz of both polarity strands of CtaHVd-LR1, thus, supporting their major role in the
replication of this RNA.
3.5. CtaHVd-LR1 Is Not Associated with a DNA Counterpart
Since HHRzs have also been reported in circular RNAs that have a DNA counterpart
integrated in the host genome (i.e., retrozymes or retroviroids) [5–8], we investigated
whether this is the case for CtaHVd-LR1. DNA and dsRNAs were extracted from the
original young sprouts of isolate 14A (collected in 2018 and stored at −80 ◦C) and tested by
PCR and RT-PCR, respectively, using the primer pair CH2 For/CH5 Rev (Table S1). The
expected amplicons were detected only by RT-PCR. In contrast, no amplicon was generated
by PCR using the DNA preparation, thus, excluding the presence of a CtaHVd-LR1 DNA
counterpart (Figure S5).
3.6. Assessment of the Biological Nature of CtaHVd-LR1
While RT-PCR assays performed using RNA preparations from the original sample
of sprouts collected in 2018 from the isolate 14A were always positive, regardless of the
primer pair (Table S1) used, all the attempts to detect CtaHVd-LR1 with the same primers
in samples collected in 2019, 2020 and 2021 (early spring) from the same isolate always
failed. The RT-PCR assays repeated in all seasons during 2020, including several times in
early spring in 2021, always tested negative to CtaHVd-LR1. To exclude that the viroid-like
RNA was not detected due to its low concentration, a new RNA library was prepared from
sprouts of the isolate 14A collected in 2021 and sequenced by HTS. Contigs of CiVA were
easily identified by de novo assembling of the sequenced reads followed by BlastN and
BlastX searches. Moreover, when the sequenced reads were mapped on the CiVA reference
genomic RNAs by Bowtie, a full coverage of the virus genome (average coverage depth 52
and 121 for RNA1 and RNA2, respectively) was observed, showing that the HTS was deep.
Although 2 contigs related to partitiviruses were identified, no contig sharing significant
sequence identity with chrysoviruses and totiviruses were identified in this second RNAseq
library, suggesting that the mycoviruses detected in 2018 were not associated with the
isolate 14A in 2021, likely because their primary host(s) was not infecting the plant when the
samples were collected. Importantly, no contig sharing sequence identity with CtaHVd-LR1
was found, and no read was retrieved when the mapping by Bowtie was performed using
CtaHVd-LR1 as a reference sequence, confirming the absence of this viroid-like RNA in the
isolate 14A in the sample collected in 2021 and tested by HTS.
In line with the results reported above, the attempts of transmitting CtaHVd-LR1 to
two grapefruit and two sour orange seedlings by grafting bark tissues from stems collected
from the isolate 14A in early spring 2018 also failed, as confirmed by the negative results
obtained when samples from the inoculated seedlings were tested for the next four years by
RT-PCR. In contrast, in all the inoculated seedlings, CiVA was detected by RT-PCR already
six months post-inoculation and during the following years, confirming that this virus
was effectively graft-transmitted to the indicator plants and that the infection was stably
maintained over time.
Altogether, these data support the view that CtaHVd-LR1 was only transiently asso-
ciated with the citrus isolate 14A in 2018 and, possibly, that this circular RNA is not able
to infect this host. Instead, considering the results of this study, the alternative possibility
that CtaHVd-LR1 was infecting another organism transiently associated to the isolate 14A
appears more feasible.
4. Conclusions
The identification of CtaHVd-LR1, by looking for conserved motifs of HHRzs, showed
that homology-independent methods based on the identification of structural elements
Viruses 2022, 14, 2265 11 of 14
may be an effective method to find new viroid-like RNAs. With respect to the previously
reported homology-independent methods, the one used in this study does not rely on
the effective de novo assembly of the full genome plus terminal repeats of the viroid or
viroid-like RNA for its identification. Indeed, when a HHRz is identified in a read that is
not assembled in a contig with terminal repeats, adjacent primers of opposite polarities
can be designed in the read of interest to check, by RT-PCR, whether the HHRz is part of a
potential circular RNA that can be further characterized by sequencing full-length cDNAs
and by northern blot hybridization assays.
By showing that CtaHVd-LR1 is a circular RNA endowed of an active HHRz in either
polarity strand, we provided solid evidence that it is indeed a novel viroid-like RNA,
confirming the reliability of the searching method. The detection of circular RNA forms
of both polarity strands strongly support that this RNA replicates through the symmetric
pathway of a rolling circle mechanism previously reported for several viroids and viroid-
like RNAs [1,2]. The biological nature of CtaHVd-LR1 remains unknown. In this respect,
CtaHVd-LR1 resembles cscRNAs and FHVd-LRs reported previously from cherry and
fig trees, respectively [17,25]. As in the case of CtaHVd-LR1, these RNAs were also not
graft-transmissible and were associated with mycoviruses [24,25]. In the case of cscRNAs,
the mycoviruses were characterized as new species in the genera Chrysovirus, Partitivirus
and Totivirus [19–23]. Interestingly, CtaHVd-LR1 was also identified in the isolate 14A
together with contigs with the highest sequence identity to viruses of the genera Chrysovirus,
Partitivirus and Totivirus. The fact that CtaHVd-LR1 was not found in the same isolate in the
following years strongly supports the conclusion that it was only transiently, and possibly
indirectly, associated with citrus plants. In this respect, the possibility that it could be an
infectious agent of an organism other than citrus is quite likely. In conclusion, our study
highlights the high sensitivity of HTS approaches associated with homology-independent
bioinformatics tools to identify novel viroid-like RNAs. At the same time, we showed that
the finding of a novel viroid-like RNA in a plant RNAseq library must not be considered as
evidence of a direct association of the former with the latter. As also previously reported
for plant viruses [51,52], the possibility that the viroid-like RNAs are only transiently
associated with the plant, because actually infecting another organism must be considered,
thus, highlights the need for implementing bioinformatics with biological data to clarify
the nature of novel viroid-like RNAs.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/v14102265/s1, Table S1: Primers used in this study; Figure S1:
Pattern syntax used to search for hammerhead ribozyme structure in citrus RNA seq library; Figure S2:
Contig containing the sequences conserved in most natural hammerhead ribozymes in each polarity
strand and a terminal direct repeat; Figure S3: CtaHVd-LR1 (+) or (−) transcript RNAs detected by
northern-blot hybridization; Figure S4: Multiple-sequence alignment of full-length cDNAs of citrus
transiently associated hammerhead viroid-like RNA 1 variants; Figure S5: PCR and RT-PCR assays
using DNA preparations from isolate 14A.
Author Contributions: Conceptualization, B.N., A.G. and F.D.S.; data curation, B.N., A.G. and M.C.;
formal analysis, B.N. and F.D.S.; funding acquisition, F.D.S.; investigation, B.N., S.L., M.C., A.G., M.M.,
D.A. and F.D.S.; methodology, B.N., A.G. and F.D.S.; project administration, F.D.S.; Resources, M.M.
and D.A.; software, A.G. and M.C.; supervision, B.N., D.A. and F.D.S.; validation, B.N.; visualization,
B.N. and S.L.; writing—original draft, B.N. and F.D.S.; writing—review and editing, B.N., S.L.,
A.G., M.C., M.M., D.A. and F.D.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was partially funded by the European Union’s Horizon 2020 research and in-
novation program under the Marie Skłodowska-Curie grant agreement No. 734736 (project ‘VirFree’).
This paper reflects only the authors’ views, and the agencies are not responsible for any use that may
be made of the information it contains.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Viruses 2022, 14, 2265 12 of 14
Data Availability Statement: Full-length sequence variants of hammerhead viroid-like RNA 1 are
available in GenBank (accession numbers OP418120-OP418149).
Acknowledgments: This work is dedicated to the memory of Giovanni Paolo Martelli, an example
of a true visionary scientist, and for some of us, irreplaceable mentor and guide. We had the privilege
of meeting him almost daily and sharing ideas and projects with him. We also had the opportunity to
appreciate his human qualities and his innate ability to see beyond, guiding everyone in the right
direction to accommodate their scientific curiosities.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Navarro, B.; Flores, R.; Di Serio, F. Advances in Viroid-Host Interactions. Annu. Rev. Virol. 2021, 8, 305–325. [CrossRef] [PubMed]
2. Navarro, B.; Rubino, L.; Di Serio, F. Small circular satellite RNAs. In Viroids and Satellites; Hadidi, A., Flores, R., Randles, J.W.,
Palukaitis, P., Eds.; Academic Press: Cambridge, UK, 2017; pp. 659–669.
3. Gago-Zachert, S. Viroids, infectious long non-coding RNAs with autonomous replication. Virus Res. 2016, 212, 12–24. [CrossRef]
[PubMed]
4. Katsarou, K.; Rao, A.L.; Tsagris, M.; Kalantidis, K. Infectious long non-coding RNAs. Biochimie 2015, 117, 37–47. [CrossRef]
[PubMed]
5. Daròs, J.A.; Flores, R. Identification of a retroviroid-like element from plants. Proc. Natl. Acad. Sci. USA 1995, 92, 6856–6860.
[CrossRef] [PubMed]
6. Vera, A.; Daròs, J.A.; Flores, R.; Hernández, C. The DNA of a plant retroviroid-like element is fused to different sites in the
genome of a plant pararetrovirus and shows multiple forms with sequence deletions. J. Virol. 2000, 74, 10390–10400. [CrossRef]
[PubMed]
7. Hegedus, K.; Dallmann, G.; Balazs, E. The DNA form of a retroviroidlike element is involved in recombination events with itself
and with the plant genome. Virology 2004, 325, 277–286. [CrossRef] [PubMed]
8. Cervera, A.; Urbina, D.; de la Peña, M. Retrozymes are a unique family of non-autonomous retrotransposons with hammerhead
ribozymes that propagate in plants through circular RNAs. Genome Biol. 2016, 17, 135. [CrossRef]
9. Di Serio, F.; Owens, R.A.; Li, S.F.; Matoušek, J.; Pallás, V.; Randles, J.W.; Sano, T.; Verhoeven, J.; Vidalakis, G.; Flores, R.; et al. ICTV
Report Consortium. ICTV Virus Taxonomy Profile: Pospiviroidae. J. Gen. Virol. 2021, 102, 001543. [CrossRef] [PubMed]
10. Branch, A.D.; Robertson, H.D.; Dickson, E. Longer-than-unit-length viroid minus strands are present in RNA from infected plants.
Proc. Natl. Acad. Sci. USA 1981, 78, 6381–6385. [CrossRef]
11. Di Serio, F.; Li, S.F.; Matoušek, J.; Owens, R.A.; Pallás, V.; Randles, J.W.; Sano, T.; Verhoeven, J.; Vidalakis, G.; Flores, R. ICTV
Report Consortium. ICTV Virus Taxonomy Profile: Avsunviroidae. J. Gen. Virol. 2018, 99, 611–612. [CrossRef] [PubMed]
12. Branch, A.D.; Robertson, H.D.A. replication cycle for viroids and other small infectious RNAs. Science 1984, 223, 450–455.
[CrossRef] [PubMed]
13. Flores, R.; Gago-Zachert, S.; Serra, P.; Sanjuán, R.; Elena, S.F. Viroids: Survivors from the RNA world? Annu. Rev. Microbiol. 2014,
68, 395–414. [CrossRef] [PubMed]
14. Codoñer, F.M.; Darós, J.A.; Solé, R.V.; Elena, S.F. The fittest versus the flattest: Experimental confirmation of the quasispecies
effect with subviral pathogens. PLoS Pathog. 2006, 2, e136. [CrossRef] [PubMed]
15. Andino, R.; Domingo, E. Viral Quasispecies. Virology 2015, 479–480, 46–51. [CrossRef] [PubMed]
16. Steger, G.; Perreault, J.P. Structure and Associated Biological Functions of Viroids. Adv. Virus Res. 2016, 94, 141–172. [CrossRef]
17. Di Serio, F.; Darós, J.A.; Ragozzino, A.; Flores, R. A 451-nt circular RNA from cherry with hammerhead ribozymes in its strands
of both polarities. J. Virol. 1997, 71, 6603–6610. [CrossRef] [PubMed]
18. Di Serio, F.; Darós, J.A.; Ragozzino, A.; Flores, R. Close structural relationship between two hammerhead viroid-like RNAs
associated with cherry chlorotic rusty spot disease. Arch. Virol. 2006, 151, 1539–1549. [CrossRef]
19. Coutts, R.H.A.; Covelli, L.; Di Serio, F.; Citir, A.; Açıkgöz, S.; Hernández, C.; Ragozzino, A.; Flores, R. Cherry chlorotic rusty spot
and Amasya cherry diseases are associated with a complex pattern of mycoviral-like double stranded RNAs. II. Characterization
of a new species in the genus Partitivirus. J. Gen. Virol. 2004, 85, 3399–3403. [CrossRef] [PubMed]
20. Covelli, L.; Coutts, R.H.A.; Di Serio, F.; Citir, A.; Açıkgöz, S.; Hernández, C.; Ragozzino, A.; Flores, R. Cherry chlorotic rustyspot
and Amasya cherry diseases are associated with a complex pattern of mycoviral-like double-stranded RNAs. I. Characterization
of a new species in the genus Chrysovirus. J. Gen. Virol. 2004, 85, 3389–3397. [CrossRef] [PubMed]
21. Kozlakidis, Z.; Covelli, L.; Di Serio, F.; Citir, A.; Açıkgöz, S.; Hernández, C.; Ragozzino, A.; Flores, R.; Coutts, R.H.A. Molecular
characterization of the largest mycoviral-like double-stranded RNAs associated with Amasya cherry disease, a disease of
presumed fungal aetiology. J. Gen. Virol. 2006, 87, 3113–3137. [CrossRef]
22. Covelli, L.; Kozlakidis, Z.; Di Serio, F.; Citir, A.; Açikgöz, S.; Hernández, C.; Ragozzino, A.; Coutts, R.H.; Flores, R. Sequences of
the smallest double-stranded RNAs associated with cherry chlorotic rusty spot and Amasya cherry diseases. Arch. Virol. 2008,
153, 759–762. [CrossRef] [PubMed]
Viruses 2022, 14, 2265 13 of 14
23. Carrieri, R.; Barone, M.; Di Serio, F.; Abagnale, A.; Covelli, L.; García-Becedas, M.T.; Ragozzino, A.; Alioto, D. Cherry chlorotic
rusty spot and cherry leaf scorch: Two similar diseases associated with mycoviruses and double stranded RNAs. J. Plant Pathol.
2011, 93, 485–489.
24. Minoia, S.; Navarro, B.; Covelli, L.; Barone, M.; García-Becedas, M.T.; Ragozzino, A.; Alioto, D.; Flores, R.; Di Serio, F. Viroid-like
RNAs from cherry trees affected by leaf scorch disease: Further data supporting their association with mycoviral double-stranded
RNAs. Arch. Virol. 2014, 159, 589–593. [CrossRef] [PubMed]
25. Olmedo-Velarde, A.; Navarro, B.; Hu, J.S.; Melzer, M.J.; Di Serio, F. Novel fig-associated viroid-like RNAs containing hammerhead
ribozymes in both polarity strands identified by high-throughput sequencing. Front. Microbiol. 2020, 11, 1903. [CrossRef]
26. Jiang, J.; Zhang, Z.; Hu, B.; Hu, G.; Wang, H.; Faure, C.; Marais, A.; Candresse, T.; Li, S. Identification of a viroid-like RNA in a
Lychee transcriptome shotgun assembly. Virus Res. 2017, 240, 1–7. [CrossRef]
27. Leichtfried, T.; Dobrovolny, S.; Reisenzein, H.; Steinkellner, S.; Gottsberger, R.A. Apple chlorotic fruit spot viroid: A putative new
pathogenic viroid on apple characterized by next-generation sequencing. Arch. Virol. 2019, 164, 3137–3140. [CrossRef] [PubMed]
28. Bester, R.; Malan, S.S.; Maree, H.J. A plum marbling conundrum: Identification of a new viroid associated with marbling and
corky fesh in Japanese plums. Phytopathology 2020, 110, 1476–1482. [CrossRef] [PubMed]
29. Yang, Y.; Xing, F.; Li, S.; Che, H.Y.; Wu, Z.G.; Candresse, T.; Li, S.F. Dendrobium viroid, a new monocot-infecting apscaviroid.
Virus Res. 2020, 282, 197958. [CrossRef]
30. Chiaki, Y.; Ito, T. Characterization of a distinct variant of hop stunt viroid and a new apscaviroid detected in grapevines. Virus
Genes 2020, 56, 260–265. [CrossRef] [PubMed]
31. Wu, Q.; Wang, Y.; Cao, M.; Pantaleo, V.; Burgyan, J.; Li, W.X.; Ding, S.W. Homology-independent discovery of replicating
pathogenic circular RNAs by deep sequencing and a new computational algorithm. Proc. Natl. Acad. Sci. USA 2012, 109,
3938–3943. [CrossRef]
32. Zhang, Z.; Qi, S.; Tang, N.; Zhang, X.; Chen, S.; Zhu, P.; Ma, L.; Cheng, J.; Xu, Y.; Lu, M.; et al. Discovery of replicating circular
RNAs by RNA-seq and computational algorithms. PLoS Pathog. 2014, 10, e1004553. [CrossRef] [PubMed]
33. Navarro, B.; Di Serio, F. Double-Stranded RNA-Enriched Preparations to Identify Viroids by Next-Generation Sequencing. In
Viral Metagenomics: Methods and Protocols. Methods in Molecular Biology; Pantaleo, V., Chiumenti, M., Eds.; Humana Press: New
York, NY, USA, 2018; Volume 1746, pp. 37–43. [CrossRef]
34. Pallás, V.; Navarro, A.; Flores, R. Isolation of a Viroid-like RNA from Hop Different from Hop Stunt Viroid. J. Gen. Virol. 1987, 68,
3201–3205. [CrossRef]
35. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvor-kin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski,
A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19,
455–477. [CrossRef] [PubMed]
36. Grillo, G.; Licciulli, F.; Liuni, S.; Sbisà, E.; Pesole, G. PatSearch: A program for the detection of patterns and structural motifs in
nucleotide sequences. Nucleic Acids Res. 2003, 31, 3608–3612. [CrossRef] [PubMed]
37. Langmead, B.; Trapnell, C.; Pop, M.; Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the
human genome. Genome Biol. 2009, 10, R25. [CrossRef] [PubMed]
38. Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Söding, J.; et al. Fast,
scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011, 7, 539.
[CrossRef] [PubMed]
39. Lorenz, R.; Bernhart, S.H.; Höner Zu Siederdissen, C.; Tafer, H.; Flamm, C.; Stadler, P.F.; Hofacker, I.L. Vienna RNA Package 2.0.
Algorithms Mol. Biol. 2011, 6, 26. [CrossRef] [PubMed]
40. Letunic, I.; Khedkar, S.; Bork, P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Res. 2020, 49,
D458–D460. [CrossRef] [PubMed]
41. Hajizadeh, M.; Navarro, B.; Bashir, N.S.; Torchetti, E.M.; Di Serio, F. Development and validation of a multiplex RT-PCR method
for the simultaneous detection of five grapevine viroids. J. Virol. Met. 2012, 179, 62–69. [CrossRef]
42. Navarro, B.; Zicca, S.; Minutolo, M.; Saponari, M.; Alioto, D.; Di Serio, F. A negative-stranded RNA virus infecting citrus trees:
The second member of a new genus within the order Bunyavirales. Front. Microbiol. 2018, 9, 2340. [CrossRef] [PubMed]
43. Hanold, D.; Vadalamai, G. Gel Electrophoresis. In Viroids and Satellites; Hadidi, A., Flores, R., Randles, J.W., Palukaitis, P., Eds.;
Academic Press: Cambridge, UK, 2017; pp. 357–367.
44. Flores, R.; Daròs, J.A.; Hernández, C. Avsunviroidae family: Viroids containing hammerhead ribozymes. Adv. Virus Res. 2000, 55,
271–323. [CrossRef] [PubMed]
45. Haseloff, J.; Symons, R.H. Comparative sequence and structure of viroid-like RNAs of two plant viruses. Nucleic Acids Res. 1982,
10, 3681–3691. [CrossRef] [PubMed]
46. Keese, P.; Bruening, G.; Symons, R.H. Comparative sequence and structure of circular RNAs from two isolates of lucerne transient
streak virus. FEBS Lett. 1983, 159, 185–190. [CrossRef]
47. Miller, W.A.; Hercus, T.; Waterhouse, P.M.; Gerlach, W.L. A satellite RNA of barley yellow dwarf virus contains a novel
hammerhead structure in the self-cleavage domain. Virology 1991, 18, 711–720. [CrossRef]
48. Babalola, B.M.; Schönegger, D.; Faure, C.; Marais, A.; Fraile, A.; Garcia-Arenal, F.; Candresse, T. Identification of two novel
putative satellite RNAs with hammerhead structures in the virome of French and Spanish carrot samples. Arch. Virol. 2022.
[CrossRef] [PubMed]
Viruses 2022, 14, 2265 14 of 14
49. Ambrós, S.; Flores, R. In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket.
Nucleic Acids Res. 1998, 26, 1877–1883. [CrossRef]
50. Chen, D.; Patton, J.T. Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5′-RACE and primer extension.
Biotechniques 2001, 30, 574–582. [CrossRef] [PubMed]
51. Hily, J.M.; Candresse, T.; Garcia, S.; Vigne, E.; Tannière, M.; Komar, V.; Barnabé, G.; Alliaume, A.; Gilg, S.; Hommay, G.; et al.
High-Throughput Sequencing and the Viromic Study of Grapevine Leaves: From the Detection of Grapevine-Infecting Viruses to
the Description of a New Environmental Tymovirales Member. Front. Microbiol. 2018, 9, 1782. [CrossRef] [PubMed]
52. Ma, Y.; Fort, T.; Marais, A.; Lefebvre, M.; Theil, S.; Vacher, C.; Candresse, T. Leaf-associated fungal and viral communities of wild
plant populations differ between cultivated and natural ecosystems. Plant-Environ. Interact. 2021, 2, 87–99. [CrossRef]