Genomics 113 (2021) 2350–2364

Contents lists available at ScienceDirect

Genomics
journal homepage: www.elsevier.com/locate/ygeno

Original Article

Identification and characterization of long non-coding RNA and their

response against citrus bark cracking viroid infection in Humulus lupulus
Vishnu Sukumari Nath a, Ajay Kumar Mishra a, *, Praveen Awasthi a, Ankita Shrestha a,
Jaroslav Matoušek a, Jernej Jakse b, Tomáš Kocábek a, Ahamed Khan a
a
Biology Centre, Czech Academy of Sciences, Institute of Plant Molecular Biology, Branišovská 31, 37005 České Budějovice, Czech Republic
b
Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

A R T I C L E I N F O A B S T R A C T

Keywords: Long non-coding RNAs (lncRNAs) are a highly heterogeneous class of non-protein–encoding transcripts that play
Citrus bark cracking viroid an essential regulatory role in diverse biological processes, including stress responses. The severe stunting disease
Genomics caused by Citrus bark cracking viroid (CBCVd) poses a major threat to the production of Humulus lupulus (hop)
Humulus lupulus
plants. In this study, we systematically investigate the characteristics of the lncRNAs in hop and their role in
Long non-coding RNAs
Plant defense
CBCVd-infection using RNA-sequencing data. Following a stringent filtration criterion, a total of 3598 putative
RNA-sequencing lncRNAs were identified with a high degree of certainty, of which 19% (684) of the lncRNAs were significantly
differentially expressed (DE) in CBCVd-infected hop, which were predicted to be mainly involved in plant-
pathogen interactions, kinase cascades, secondary metabolism and phytohormone signal transduction. Besides,
several lncRNAs and CBCVd-responsive lncRNAs were identified as the precursor of microRNAs and predicted as
endogenous target mimics (eTMs) for hop microRNAs involved in CBCVd-infection.

1. Introduction
Eukaryotic gene expression and regulation are immensely complex
orchestrated events, conditioned by the molecular interactions between
different components such as RNA polymerase II (RNAP II), general
transcription factors (GTF), gene-specific transcription factors (TFs) and
the mediator (Med) complex [1,2]. The intrinsically stochastic nature of
molecular interactions can regulate gene expression at many different
levels with various mechanisms such as epigenetic, transcriptional, post-
transcriptional, translational, and post-translational controls [3]. The
increasing body of research shows that the major portion of the
eukaryotic genome is transcribed into RNAs, only a small portion har­
bors the protein-coding mRNAs, whereas a considerable proportion is
transcribed as non-coding RNAs (ncRNAs), which lack discernible
protein-coding potential [4,5]. Once considered as spurious transcrip­
tional noises, ncRNAs are now increasingly recognized as key regulators
in a number of biological processes via various mechanisms [6,7]. Based
on length, ncRNAs can be broadly classified into two categories: short
ncRNAs containing less than 200 nt [microRNAs (miRNAs), small
interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs)] and long
ncRNAs (lncRNAs), which are generally longer than 200 nt [8]. Among
them, lncRNAs constitute the major proportion, which has gained
widespread attention in recent years due to their mRNA-like structures,
tissue- and cell-specific expression and their diverse gene expression
regulatory potential at the transcriptional, post-transcriptional, trans­
lational and epigenetic level [9,10].
The lncRNAs are defined as a heterogeneous group of ncRNAs, which
are generally more than 200 nt and possess fewer exons than mRNAs
[10,11]. Based on their position relative to neighbouring protein-coding
genes in the genome, lncRNAs are distinguished into long intergenic
non-coding RNAs (lincRNAs), natural antisense transcripts (NATs),
intronic RNAs (incRNAs), and overlapping lncRNAs that partially
overlap with protein-coding genes [12,13]. Recently, based on their
functional mode or type of interaction, lncRNAs have been classified
into those that form ribonucleoprotein complexes, RNA-DNA hybrids,
RNA-RNA duplexes and a subclass that regulates nearby protein-coding
genes via unknown mechanisms [14]. Most lncRNAs are similar in
structure and biogenesis to messenger RNAs (mRNAs) and are generally
transcribed by the RNA polymerase II, which undergoes 5′ -end capping,
polyadenylation and alternative splicing [13]. Nevertheless, plant
lncRNAs are also transcribed by RNA Polymerase IV or RNA Polymerase
V, which results in the absence of the poly-A tail and exhibits extremely

* Corresponding author.
E-mail address: ajaymishra24@umbr.cas.cz (A.K. Mishra).

https://doi.org/10.1016/j.ygeno.2021.05.029
Received 2 December 2020; Received in revised form 22 April 2021; Accepted 25 May 2021
Available online 27 May 2021
0888-7543/© 2021 Elsevier Inc. All rights reserved.
V.S. Nath et al.
low expression and stability [15,16]. In contrast to the small ncRNAs,
lncRNAs are poorly conserved and regulate gene expression by different
mechanisms. The mechanisms underlying the lncRNA-associated regu­
lation of gene expression are very diverse and it is a daunting task to
categorize the myriad functions of lncRNAs, since a large number of
their target transcripts are associated with a variety of biological pro­
cesses such as plant growth, development, abiotic and biotic stress re­
sponses in different plant species [17–20]. The recent explosion of
knowledge suggests that lncRNAs exert their regulatory potential
through either cis- or trans-acting mode with the association of a wide
range of interacting partners, including RNA-binding proteins, splicing
factors, transcription factors, chromatin-modifying and remodeling
complexes, chromatin, microRNAs, nascent and mature transcripts and
several different components of transcriptional and translation pro­
cesses [21,22].
Recent advances in next-generation sequencing, microarray and
RNA-sequencing technologies, coupled with bioinformatics approaches
have immensely accelerated the systematic identification and classifi­
cation of large numbers of lncRNAs in different plant species such as
Arabidopsis [23], maize [24], wheat [25] and rice [19] and brought
these largely ignored molecular players of various biological processes
to the forefront [10,18]. Undoubtedly, these platforms have been
instrumental in the rapid, robust and efficient cataloging of lncRNA in
plant biotic responses. Several lncRNAs on the genome-scale have been
characterized in direct or indirect biotic responses in rice and A. thaliana
infected with the fungal pathogens, Magnaporthe oryzae and Fusarium
oxysporum, respectively [20,26]. Recently, Zhang et al. [27] reported
that the silencing of two core lncRNAs GhlncNAT-ANX2 and GhlncNAT-
RLP7, which are involved in the regulation of lipoxygenase activity
(LOX1 and LOX2), resulted in enhanced resistance to pathogens Verti­
cillium dahliae and Botrytis cinerea in cotton. Similarly, in A. thaliana, the
lncRNA ELF18INDUCED LONGNONCODING RNA1 (ELENA1) has been
shown to improve resistance to Pseudomonas syringae by interacting with
Mediator subunit 19a, which in turn positively regulates pathogenesis-
related 1 (PR1) gene expression [28]. In tomato, the overexpression of
lncRNA16397 was shown to induce glutaredoxin expression, decrease
reactive oxygen species production, and confer resistance to Phytoph­
thora infestans [29]. Furthermore, a comprehensive set of 529 putative
lncRNAs were identified using RNA-seq method in response to Tomato
yellow leaf curl virus (TYLCV) infection in tomato [30].
Hop (Humulus lupulus L.) is a well-known, economically important
dioecious perennial crop belonging to the Cannabaceae family, widely
cultivated throughout the temperate regions of the world [31]. The fe­
male inflorescences (hop cones or “hops”), which consist of membrane-
like bracts, contain yellow glandular trichomes known as lupulin glands
which biosynthesize the spectrum of bioactive secondary metabolites
such as flavonoids (flavonol glycosides, prenyl flavonoids and proan­
thocyanidins), bitter acids and essential oils [32]. The secondary
metabolite products of hop are important ingredients in the brewing
industry due to their contribution as bitter flavor, herbal aroma, foam
and microbiological stability of beer [33]. In addition to their use as a
flavoring agent in the food industry, hop extract has been traditionally
utilized in folk medicine for several thousand years and has recieved
increasing attention as a model plant for drugs due to its pharmaco­
logically important properties such as antioxidant, antiinflammatory,
anticarcinogenic, antiglycemic, neuroprotective and sedative effects
[34]. Among the various biotic constraints, the disease caused by viroids
can negatively impact the yield and quality of hop cone production
worldwide [35]. Currently, four species of viroid, namely Hop stunt
viroid (HSVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid
(HLVd), and Citrus bark cracking viroid (CBCVd) are known to be
endemic in hop-growing regions around the world [36]. Among them,
CBCVd infection is the most aggressive, causing drastic morphological
and anatomical changes, ranging from severe bine stunting, leaf
epinasty, yellowing, premature flowering, reduction in cone size, dry
root rot, stunted growth and complete death of plant [37].
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Genomics 113 (2021) 2350–2364
Interestingly, viroids are plant-restricted parasites that serve as a
distinct model system to unveil the various aspects of host-pathogen
interaction at the genome level. The genome of a viroid is a single-
stranded, circular naked and non-coding RNA of about 250 to 400 nt
that replicates by rolling circle replication mechanisms and spreads in
plants [38]. The replication cycles of viroids lead to the accumulation of
viroid-specific small RNAs (vsRNA), which can be involved in the
transcriptional silencing of endogenous genes (TGS) via RNA-directed
DNA methylation (RdDM) of cytosine residues of cognate nuclear DNA
[39], direct interaction with plant proteins and/or post-transcriptional
gene silencing (PTGS) [40]. The proposed mechanism sustaining this
view of viroid pathogenesis and symptoms induction has been circum­
stantial and does not exclude alternative mechanisms. Intriguingly, vi­
roids are also envisioned to subvert the endogenous lncRNA-directed
regulatory networks in the host to complete their life cycle, and the
associated developed symptoms are a result of large-scale disruptions in
plant gene expression imprinted by viroid interference in lncRNA-
directed regulatory networks [41,42]. Despite the profound progress
in understanding the regulatory role of lncRNA in various develop­
mental processes and responses to biotic or abiotic stress in plants
[10,43], the role of lncRNA in viroid-mediated reprogramming of the
host’s transcription machinery and in induced diseases in plants remains
to be explored. To date, only one report is available concerning the role
of lncRNAs in viroid infection, in which the authors report on the dif­
ferential regulation of 44 lncRNAs in response to Potato spindle tuber
viroid (PSTVd) infection in tomato [44], thus soliciting the big picture
research initiative for a better understanding of the viroid-induced
regulation of host lncRNAs.
In this study, we have systemically identified the landscape of
lncRNAs and characterized their regulatory role in hop. Furthermore,
we used a comparative analysis of the CBCVd-responsive RNA-seq
datasets to investigate the changes in lncRNAs expression during
CBCVd-infection in the hop. The results presented here will improve our
understanding of the regulatory potential of lncRNAs in plant-viroid
interaction and also serve as a resource material for further studies on
viroid immunity.
2. Materials and methods
2.1. Plant material, CBCVd-infection, and NGS sequencing
The clonally propagated virus and viroid-free three-month-old leaves
of hop plants (cv. Saaz, Osvald’s 72 clone) were biolistically inoculated
with microcarrier gold particles (1 μm) carrying an infectious dimeric
cDNA of CBCVd driven by the CaMV 35S promoter as described previ­
ously [45]. Each hop plant was inoculated in total with 250 ng cDNA of
CBCVd by five shots into leaves and immediately placed in darkness for
24 h after covering with polyethylene bags to prevent drying of the shot-
wound leaf area. Control plants were inoculated with the empty pLV07
plasmid. The CBCVd-inoculated and control plants were grown under
natural conditions and inspected regularly for symptom development.
To confirm CBCVd-infection, the inoculated plants were examined by
reverse transcription PCR (RT-PCR) and RNA gel blot assay after 4, 14
and 28-months post inoculation (mpi) using CBCVd specific primers as
described previously [45].
Total RNA was extracted from approximately 100 mg of four control
and four systematically infected CBCVd hop leaves (14 mpi) using a
Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, MO, USA) following
the manufacturer’s instructions. RNA samples were digested with
TURBO™ DNase (Ambion, Austin, TX, USA) to remove DNA contami­
nation. Total RNA concentration was determined by means of absor­
bance A260/280 using NanoDrop 2000 spectrophotometer (Thermo
Fisher Scientific, Waltham, USA) and RNA integrity analysis (RIN) was
performed by Agilent Bioanalyzer 2100 electrophoresis using RNA 6000
Nano Kit (Agilent Technologies, CO, USA). Each sample (5 μg) used for
sequencing was RNA pooled in the same batch from three individual
V.S. Nath et al.
leaves and used for cDNA synthesis using cDNA Synthesis System
(Roche, Basel, Switzerland) and library construction using Illumina
TruSeq Stranded Total RNA Sample Prep with Ribo-Zero (plant) kit
(Illumina, San Diego, CA). After the evaluation of library quality using
the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara,
CA), the paired-end sequencing (2 × 100 bp) was performed on an
Illumina Hiseq™ 2500 platform. The complete raw RNA-seq datasets for
four biological replicates of CBCVd-infected and control hop plants were
deposited in the NCBI Sequence Read Archive under accession number
SRR7904254 and SRR6206403, respectively.
Fig. 1. The streamlined bioinformatics pipeline used for the identification lncRNAs from transcriptome datasets of CBCVd-infected (CI) and healthy control (CT) leaf
samples of hop.
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Genomics 113 (2021) 2350–2364
2.2. Processing of raw reads and transcriptome assembly
The raw RNA-seq reads were filtered to remove adaptor sequences,
low-quality reads and k-mer contamination using the fastp tool with
additional parameters of overrepresentation analysis and correction
[46], whereas the quality of trimmed reads after processing was assessed
using the FastQC tool [47]. Additionally, ribosomal RNA contamination
was also removed from the remaining data by alignment to the SILVA
database [48] using the Bowtie2 software package [49] to maximize
reads mapping to transcripts. The clean reads of each RNA-seq dataset
were mapped to a reference hop genome (downloaded from http://h
opbase.cgrb.oregonstate.edu/downloadTeamaker.php) separately with
the TopHat2 program [50] using the parameter max-multihits = 1 to
V.S. Nath et al.
obtain the uniquely mapped proper mate paired hits. The mapped reads
were assembled separately for each dataset into transcripts based on the
reference genome with StringTie (v1.3.1) software using the default
parameters [51]. The transcripts from each library were merged using
the “merge” feature in StringTie software [51] to generate final tran­
scripts. All transcripts with strand information, number of fragments per
kilobase of transcript per million mapped reads (FPKM) greater than 0.5
in multiple exons in at least one sample and length greater than 200 bp
were retained for downstream analysis.
2.3. Identification and characterization of lncRNAs
A series of different software tools (Fig. 1) were used to systemically
identify putative and CBCVd-responsive lncRNAs in the hop. As an in­
tegral step, all transcripts over 200 bp were aligned with the BLASTx (E-
value cut-off of 1e− 10, coverage ≥80% and identity ≥90%) searches
against UniProtKB [52] and National center for biotechnology infor­
mation (NCBI) non-redundant (nr) protein database using locally
installed NCBI-BLAST software (v2.11.0) [53]. Transcripts matched
with known protein-coding genes were excluded from further analysis.
The coding potential of the remaining transcripts was evaluated using
coding potential calculator (CPC2) software (http://cpc.cbi.pku.edu.cn/
) [54], Coding Noncoding Index (CNCI) software (https://github.co
m/www-bioinfo-org/CNCI) [55] and PLEK tool (predictor of long non-
coding RNAs and messenger RNAs based on an improved k-mer
scheme) [56]. All transcripts with CPC2, CNCI or PLEK scores less than
0 were used for further analysis. After these filtering steps, the
remaining transcripts were searched against the Pfam protein family
database (https://pfam.xfam.org/) to examine a set of candidate partial
domains and candidates which showed the absence of domain annota­
tions were set for open reading frames (ORFs) determination using the
getORF utility of the EMBOSS package (http://emboss.bioinformatics.
nl/cgi-bin/emboss/getorf). Transcripts with the potential to code for
less than 100 amino acids were retained and aligned against the Rfam
database (https://rfam.xfam.org/) to discriminate between candidate
lncRNAs and potential small non-coding RNA transcripts (tRNAs,
snRNAs and SnoRNAs). The remaining transcripts were considered as
putative lncRNAs and queried against the CANTATAdb v 2.0 [57]
NONCODE v 5.0 [58] and the GreeNC v 1.12 [59] database for sequence
conservation analysis.
The candidate lncRNAs were classified into different classes (i, j, o, u,
and x) according to their location in the hop genome using the cuff­
compare program in the Cufflinks suite [60]. The class code ‘u’ repre­
sents unknown and intergenic transcript, class code ‘x’ represents long
noncoding natural antisense transcripts (lncNAT) on the opposite
strand, class code ‘j’ potentially novel isoform (fragment) with at least
one splice junction shared with a reference transcript, class code ‘i’
represents the intronic transcripts, and o, generic exonic overlap with a
reference transcript.
2.4. Differential expression analysis of lncRNAs
The quantification of the expression level of protein-coding tran­
scripts and candidate lncRNAs was calculated based on the FPKM values
using StringTie software [51]. The differentially expressed lncRNAs (DE-
lncRNAs) between the CI and CT samples were identified using the R
package DESeq2 based on a negative binomial distribution, and the
resulting data were adjusted via the Benjamini and Hochberg approach
for controlling the false discovery rate [61]. Transcripts with an absolute
log2fold change >1 or < − 1 and a FDR adjusted p-value <0.05 were
considered and identified as DE-lncRNAs [62]. The DE-lncRNAs were
visualized as a volcano plot using ggplot2 (version 3.2.1, https://ggp
lot2.tidyverse.org/) and a K-means clustered heatmap was created
using the clustvis online visualization tool [63].
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Genomics 113 (2021) 2350–2364
2.5. Prediction of cis/trans-targets of candidate lncRNAs and DE-
lncRNAs
The lncRNAs are known to regulate the expression of nearby protein-
coding genes (cis-regulation) or distantly located protein-coding genes
(trans-regulation). To classify lncRNAs/DE-lncRNAs cis-target genes, we
screened for closest coding genes within a 100 kb window upstream and
downstream of lncRNAs/DE-lncRNAs using the BEDTools v 2.25.0
program [64]. To classify the trans-target gene candidates for the
lncRNAs/DE-lncRNAs we determined the mRNA- lncRNAs/DE-lncRNAs
interaction ability using the RIblast algorithm (version 1.0, https://gith
ub.com/fukunagatsu/RIblast) [65] and those interactions showing a
hybridization energy threshold of less than − 30 kcals mol− 1 were
considered as potential trans-targets of lncRNAs/DE-lncRNAs [66]. A
pairwise correlation of expression levels of DE-lncRNAs/lncRNAs and
their cis/trans target mRNAs were computed using the Pearson corre­
lation coefficient (PCC), and highly co-expressed lncRNAs/DE-lncRNAs
-target mRNA pairs (R > 0.60 for positive correlation and R < − 0.60 for
negative correlation) were considered as significant.
2.6. Functional annotation and pathway enrichments
To understand the putative functions of lncRNA/DE-lncRNAs in cis
and trans-acting gene regulation, we performed functional gene
ontology (GO) classifications of the cis and trans target candidate genes
using the Blast2GO software package [67]. The GO enrichment analysis
of lncRNA/DE-lncRNAs was conducted using the clusterProfiler R
package [68]. The BlastKOALA online tool was used to map the path­
ways associated with cis/trans-target genes of lncRNA/DE-lncRNAs by
BLAST search in the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database [69]. A Gene Set Enrichment Analysis (GSEA) was performed
using the online WebGestalt tool [70] for cis/trans mRNAs targets in the
KEGG pathway, assuming a minimum number of genes ≥ 5 in each
pathway. GO terms and KEGG pathways with FDR-adjusted p-values
<0.05 were considered to be significantly enriched. Furthermore, cis
and trans-targets of lncRNAs/DE-lncRNAs were annotated using the
MapMan Mercator tool (http://mapman.gabipd.org/web/guest/mercat
or) and subjected to MapMan analysis (version 3.6) [71] to gain an
unbiased, systematic overview of important biological functions regu­
lated by lncRNAs and coordinated response of cis and trans target genes
of CBCVd-responsive lncRNAs in the hop.
2.7. Identification of lncRNA-miRNA interactions
The interaction between miRNA and lncRNA is considered essential
for gene regulations, as lncRNAs may be targeted by miRNAs, function
as miRNA precursors, or miRNA sponges (competing endogenous RNAs,
ceRNAs) [72]. We, therefore, predicted the lncRNAs targeting the po­
tential miRNA and vice versa by uploading identified lncRNAs and
previously identified miRNAs of hop [73] to psRNATarget online anal­
ysis tool (http://plantgrn.noble.org/psRNATarget/) [74]. The following
previously described criteria [75] were used for the prediction of
lncRNA-miRNA interaction: (1) a perfect complementary nucleotide
pairing at 5’end of miRNA with a bulge of no more than 2–4 nt (2) a total
of three mismatches with no more than two consecutive nt mismatches
were allowed in the seed region of miRNA (3) no protrusions were
allowed in the non-intermediate region of the miRNA (4) a maximum
expectation of 3.0. Moreover, miRNAs targeting the putative cis/trans
target genes of lncRNAs were also predicted using online software
psRNATarget with similar parameters as described above.
The lncRNAs acting as precursors of miRNAs were predicted by
BLAST analysis against the mature miRNAs in the miRbase (htt
p://www.mirbase.org) and those lncRNAs with a percentage coverage
ratio between lncRNA and miRNA sequence greater than 90% and e-
value =1e− 5 were considered as potential miRNA precursors [76]. The
Vienna RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAW
V.S. Nath et al.
ebSuite/RNAfold.cgi) was used to plot the predicted secondary struc­
tures of lncRNA and miRNA precursors.
2.8. Validation of selected DE-lncRNAs and potential target genes by
qRT-PCR
To validate the expression levels of DE-lncRNAs and their predicted
mRNA targets, total RNA was isolated from CI and CT hop plants using
Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) and treated with DNase
I (Invitrogen) to eliminate genomic DNA contamination according to the
manufacturer’s protocol. First-strand cDNA was synthesized from 2 μg of
total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo
Scientific, EU). The first-strand cDNA reaction was diluted 10-fold and 3
μl of the diluted cDNA was used as a template for qRT-PCR. The qRT-
PCR reactions were run on a CFX Connect™ Real-Time PCR Detection
System (Bio-Rad, Hercules, CA, USA) using the TopBio SYBR master mix
(TopBio, Vestec, Czech Republic). Appropriate control reactions were
performed for each primer combination and amplification specificity
was verified by a thermal denaturing step to generate melting curves. Ct
values were determined based on three biological replicates each with
three technical replicates. Relative expression levels (fold-change) of the
target genes or transcripts were calculated using 2-ΔΔCt [77] using DRH1
(DEAD-box ATPase-RNA-helicase) as the reference gene [78]. All
primers used for qRT-PCR analyses were designed using the primer3
software (http://bioinfo.ut.ee/primer3-0.4.0/) and are listed in Sup­
plementary Table S1.
2.9. Construction of lncRNA-mRNA-miRNA interaction networks
To obtain an overview of the interaction potential of the predicted
lncRNAs acting as miRNA targets, mRNAs acting as lncRNA targets or
miRNA targets, we constructed a regulatory network to visualize the
reciprocal interactions between the target pairs of miRNAs–lncRNAs,
miRNAs–mRNAs, and lncRNAs–mRNAs using the Cytoscape v 3.6.0 [79]
and a network analysis was performed using the Cytoscape Network
Analyzer core app. The Cytoscape MCODE plug-in (Version 3.4.0) was
applied for easy visualization of the highly interconnected networks
(clusters). A node in the networks represented a coding transcript,
lncRNA or miRNA in the modules, and the edge indicated a strong
correlation between them (≥ 0.84).
3. Results
3.1. Overview of transcriptome sequencing
To systematically identify and characterize the lncRNAs and their
response to CBCVd-infection, we performed transcriptome sequencing
of eight cDNA libraries generated from four independent biological
replicates of CI and CT samples of hop [45]. On average, more than
33.75 and 40.02 million raw reads were generated for CI and CT sam­
ples, respectively. Removal of the adapter sequence, low quality reads
and k-mers using fastp software resulted in the retention of more than
90% of reads with an average length of 68.0 bp, 67.0 bp with a Q20 and
Q30 values of 95.15% (CI) and 93.85% (CT), respectively, indicating
high sequencing quality, suitable for subsequent analysis. Approxi­
mately 68.5% of the clean reads in CI and 57.8% of clean reads in CT
samples were uniquely mapped to the reference hop genome by
TopHat2 v 2.0.9 [50]. The successfully mapped reads were assembled
into 42,526 transcripts by the StringTie package, accounting for 93.4%
of all annotated protein-coding transcripts in hop, suggesting a relatively
high coverage and accuracy of the reconstructed transcriptome.
3.2. Global identification and sequence feature analysis of lncRNAs in the
hop
We employed a series of stringent filtering criteria to identify
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Genomics 113 (2021) 2350–2364
putative lncRNAs from the assembled hop transcripts (Fig. 1). We first
screened transcripts with shorter length (< 200 nt) and low abundance
(FPKM <0.5) to minimize background transcriptional noises before
proceeding to the lncRNA identification pipeline. Following a sequential
stepwise criterion, 34,352 transcripts corresponding to known protein-
coding genes, 1434 transcripts with protein-coding potential, 3142
transcripts matched to other known ncRNAs (miRNAs, tRNAs, rRNAs,
snRNAs and SnoRNAs) and conserved protein domains in Pfam database
were eliminated. After applying these stringent criteria, 3598 transcripts
were identified as putative hop lncRNAs.
To understand the genomic characteristics of the putative lncRNAs
and mRNAs, we compared their sequence features including average
transcript length, number of exons, and expression profiles. The lengths
of lncRNAs ranged from 200 to 4600 bp with the majority (85%) having
lengths less than 600 bp (Fig. 2A), while 72.6% of mRNAs were longer
than 500 bp with an average length of 3006 bp. Similar distribution
trends were observed for exon numbers with 27.48% of lncRNAs pos­
sessing a single exon in comparison to 5–25 exons observed in mRNAs
with an average exon count of 5.5 per transcript (Fig. 2B). The expres­
sion levels of lncRNAs and mRNAs in CI and CT samples were estimated
by FPKM values, which revealed that the lncRNAs tend to have a lower
expression (< 10 FPKM) than those of protein-coding mRNAs (Fig. 2C).
Overall, the above results demonstrated that the identified lncRNAs
were shorter in length, possessed fewer exons, and a notably low
expression levels compared to mRNAs which are consistent with previ­
ous reports [10,11].
According to the genomic locations relative to the nearest protein-
coding genes, the lncRNAs identified were categorized into antisense,
intergenic, intronic, and overlapping. The most abundant type of
lncRNAs was derived from intergenic regions, accounting for 37%, fol­
lowed by intron matching lncRNA (35.57%), and intronic lncRNAs
(0.02%). The antisense and overlapping lncRNA gene loci were the least,
accounting for only 0.004% and 0.013% of the entire lncRNA sets
respectively (Fig. 2D).
To investigate the conservation of hop lncRNAs among other plant
species, we performed a BLAST search (e-value <1e− 5) against the
lncRNA databases. Our results showed that 7.3% of hop lncRNAs shared
an average identity of more than 30% (30% query coverage) among the
closest plant species such as Vitis vinifera, Malus domestica and Prunus
persica for which information is available in the lncRNA database
(Supplementary Table S2). Conversely, an identity of below 20% was
observed for distant plant species suggesting less conservation of hop
lncRNAs among other plant species, which was in agreement with
several previously published reports [10].
3.3. Identification of lncRNAs responsive to CBCVd infection in the hop
To investigate the response of lncRNAs to CBCVd infection in hop,
we estimated the expression profiles of each identified candidate
lncRNA based on their FPKM values in CI and CT samples. The FPKM
values for the lncRNAs transcript from the CI and CT samples ranged
from 0.54 to 4814.25 and 0.57 to 10,808.49 respectively, indicating an
extensive modulation of the expression levels of lncRNA transcripts in
response to CBCVd-infection in the hop (Supplementary Table S3).
Following a systematic assessment of the expression profiles of lncRNAs,
we observed that a total of 1417 lncRNAs were expressed in both control
and CBCVd-infected library. Interestingly, 390 lncRNAs were expressed
exclusively in the CI library, while 320 lncRNAs were uniquely
expressed in the CT library (Fig. 3A). A comparative analysis using the
DESeq2 software package showed that a total of 684 lncRNAs (19.01%),
including 484 intergenic lncRNAs (70.76%), 4 antisense lncRNAs, and 9
intronic lncRNAs were differentially expressed in the CI sample (FDR
adjusted p-value <0.05 and log2fold-change >1 or < − 1) (Supplemen­
tary Table S4). Among the DE-lncRNAs, 371 (54.24%) lncRNAs showed
significant upregulation, whereas 313 (45.76%) lncRNAs exhibited
significant downregulation (Supplementary Fig. S1 and Supplementary
V.S. Nath et al.
Fig. 2. Characteristic features of identified hop lncRNAs. (A) The proportion of different classes of lncRNAs identified based on nearby protein-coding genes in hop.
(B) Comparison of length distribution of identified lncRNAs and protein-coding mRNAs in hop. (C) Distribution of exon numbers between hop lncRNAs and protein-
coding mRNAs. (D) FPKM distribution of lncRNAs and protein-coding mRNAs in hop.
Table S4). The above results including the expression profiles of the DE-
lncRNAs implied their plausible role in the regulation of CBCVd-induced
disease development or innate immunity in hop. The clustered heatmap
created using the clustvis online visualization tool portrayed the
expression profiles and inter-relationships of the DE-lncRNAs (Fig. 3B).
3.4. Prediction of lncRNA targets and their functional analysis
To explore the biological regulatory potential of lncRNAs/DE-
lncRNAs on the protein-coding genes, we predicted their mRNA tar­
gets based on the cis and trans mode of interaction and correlated co-
expression profile (R > 0.60 for positive correlation and R < − 0.60
for negative correlation). For predicting the cis-targets, we screened
proximal protein-coding genes located within a genomic window of
100 kb upstream and downstream of the 3598 lncRNAs and 684 DE-
lncRNAs of the hop, which resulted in the identification of 4996 and
771 potential cis-targets for the lncRNAs and DE-lncRNAs, respectively.
A PCC analysis showed that a total of 127 (16.47%) DE-lncRNAs and 331
(6.62%) lncRNAs were co-expressed with their potential target genes
(Supplementary Tables S5 and S6). A total of 9145 and 2399 trans target
genes were identified for lncRNAs and DE-lncRNAs, respectively using
the RIblast program. Among them, 230 (2.51%) and 107 (4.46%) trans
target protein-coding genes were found to be co-expressed with the
lncRNAs and DE-lncRNAs, respectively. The expression correlation
analysis of DE-lncRNAs and their mRNA targets resulted in the identi­
fication of 224 differentially expressed cis and trans target genes (113
down-regulated, 111 up-regulated) between CI and CT samples. Inter­
estingly, our results suggest that some lncRNAs/DE-lncRNAs may
interact with multiple neighbouring target genes, which may imply
functional regulatory relationships between lncRNAs and their target
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Genomics 113 (2021) 2350–2364
protein-coding genes. Remarkably, we found a high correlation in
expression levels (PCC > 0.95) of intergenic DE-lncRNAs and their
neighbouring protein-coding genes compared to those located at the
distant origin (Supplementary Tables S7 and S8).
To gain insights into the functional significance of the identified cis
and trans targets of lncRNA/DE-lncRNAs, we performed their functional
GO and KEGG analysis. The identified 3598 hop lncRNAs that were
transcribed close to 4996 protein-coding genes and 9145 trans target
genes were subject to functional GO analysis. A total of 3469 (69.43%)
cis and 5444 (59.50%) trans target genes of hop lncRNAs are annotated
in the GO database, which revealed that the highest proportion of target
genes of both cis- and trans-acting lncRNAs was involved in “Cellular
process” (GO:0009987) in the biological process category, “Catalytic
activity” (GO: 0003824) in the molecular function category and
“Cellular anatomical entity” (GO:0110165) in the cellular component
(Fig. 4A). In addition, 1332 (26.7%) and 1235 (21.5%) target transcripts
of cis- and trans-acting lncRNAs were annotated in 334 and 317 path­
ways of the KEGG database under different subcategories (Table 1).
Among them, the highest proportion of target genes of both cis- and
trans-acting lncRNAs was associated with “Carbohydrate metabolism”,
“Translation”, “Transport and catabolism”, and “Signal transduction”,
suggesting the vital and diverse regulatory role of hop lncRNAs.
Furthermore, GO enrichment analysis revealed that the predicted target
genes participated in a wide array of biological processes and pathways
(Supplementary Fig. S2). The result shows that a total of 135 GO terms
were populated for the identified cis-targets of lncRNAs and among
them, 81 GO terms (biological process), 20 GO terms (cellular compo­
nent) and 25 GO terms (molecular function) were significantly enriched
(P < 0.05). Among the enriched GO categories steroid biosynthetic
process, carotenoid biosynthetic process, cellular response to nutrient
V.S. Nath et al. Genomics 113 (2021) 2350–2364

Table 1
KEGG categories mapped for co-expressed cis/trans targets of DElncRNAs ac­
cording to blastKOALA analysis.
KEGG categories DElncRNA lncRNA

Cis- Trans- Cis- Trans-

target target target target

Metabolism
Carbohydrate Metabolism 37 26 94 89
Energy metabolism 12 8 48 32
Lipid metabolism 15 21 65 48
Nucleotide metabolism 3 5 16 17
Amino acid metabolism 26 16 102 79
Metabolism of other amino acids 8 8 21 17
Glycan biosynthesis and 6 5 25 14
metabolism
Metabolism of cofactors and 8 10 48 30
vitamins
Metabolism of terpenoids and 7 5 21 17
polyketides
Biosynthesis of other secondary 6 15 31 38
metabolites
Xenobiotics biodegradation and 9 4 24 13
metabolism

Genetic information processing

Transcription 6 17 37 37
Translation 12 24 105 83
Folding, sorting and degradation 9 9 77 47
Replication and repair 4 4 30 23

Cellular Process
Transport and catabolism 17 19 97 55
Cell growth and death 9 5 56 44
Cellular community - eukaryotes 1 1 7 9
Cellular community - 3 1 8 5
prokaryotes
Cell motility 1 1 5 5

Environmental information processing

Membrane transport 1 1 4 1
Signal transduction 25 31 129 91
Others 64 70 135 119

level, response to water was activated while RNA methylation, ribonu­

cleoprotein complex, ribosome, small ribosomal subunit, nuclear lumen
were suppressed (Supplementary Fig. S2A). Likewise, in the lncRNA
trans-targets, the enriched GO terms corresponding to DNA binding
transcription factor activity, transcription regulator activity, regulation
of biosynthetic process were activated, while ribosome, RNA methyl­
ation, large ribosomal subunit were suppressed (Supplementary
Fig. S2B). The KEGG pathway analysis results showed that the identified
lncRNA cis- and trans-target genes were highly enriched for pathways
involved in the biosynthesis of secondary metabolites, phenylpropanoid
biosynthetic pathways, primary metabolic pathways, ribosome biosyn­
thesis and protein processing, etc. (Supplementary Fig. S3), suggesting
the complex regulatory potential of hop lncRNAs in diverse biological
and metabolic pathways. The MapMan analysis revealed that the target
genes of hop lncRNAs are mainly involved in various aspects of plant
physiological process including gene expression regulation, metabolism,
signaling, growth and development (Fig. 5A and B).
Furthermore, to unveil the potential functions of the differentially
CBCVd-responsive lncRNAs, we analyzed GO terms of cis- and trans-
acting target genes. A total of 684 CBCVd-responsive lncRNAs that were
transcribed close to 771 protein-coding genes and 2399 trans target
genes were subject to GO analysis. The result indicated that 598
(77.56%) cis and 1778 (74.11%) trans-acting target genes are annotated
in the GO database and the majority of target genes of both cis- and trans-
acting lncRNAs were involved in “Cellular process” (GO:0009987) and
“Metabolic process” (GO:0008152) in the biological process category,
“Catalytic activity” (GO: 0003824) and “Binding activity”
(GO:0005488) in the molecular function category and “Cellular
anatomical entity” (GO:0110165) in the cellular component (Fig. 4B).
Fig. 3. Expression profiles and distribution of DE-lncRNAs in hop. (A) Venn
diagram showing the unique distribution of DE-lncRNAs in CBCVd-infected (CI)
and healthy control (CT) samples of hop. (B) Cluster heatmap showing
expression of lncRNAs identified in four libraries of CBCVd-infected (CI) and
healthy control (CT) leaf samples of hop.
Based on KEGG pathway analysis, 217 (28.1%) and 357 (28.3%) cis- and
trans-target transcripts of DE-lncRNAs were annotated in 194 and 197
pathways of the KEGG database under different subcategories (Table 1),
which revealed that CBCVd-responsive lncRNAs may regulate genes
involved in many biological processes, including signal transduction,
energy synthesis, metabolism, transcription and translation, in response
to CBCVd-infection.
Furthermore, GO and KEGG enrichment analysis of target genes of
CBCVd-responsive lncRNAs provided a measure of functional signifi­
cance and the specificity of targeted pathways. Interestingly, among the
399 identified GO terms for the DE-lncRNA cis-target genes, 54 GO terms
(biological process), and 8 GO terms (molecular function) were signifi­
cantly enriched (P < 0.05). In the highly enriched GO category, nucleic
acid metabolic process, DNA binding, RNA metabolic process, regula­
tion of transcription, protein binding was activated while peptide

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V.S. Nath et al.
Fig. 4. Blast2GO functional gene ontology (GO) annotation of the differentially expressed cis/trans- target genes of lncRNAs identified in hop. (A) The GO terms of
the cis/trans- target genes of differentially expressed hop lncRNAs. (B) The GO terms populated for the cis/trans- target genes of hop DE-lncRNAs identified in
this study.
metabolic process, peptide biosynthetic process, translation, nucleotide
biosynthetic process were suppressed (Supplementary Fig. S4A). In the
case of CBCVd-responsive lncRNA trans-target, 11 GO terms in the bio­
logical process category were significantly enriched (P < 0.05) (Sup­
plementary Fig. S4B). Notably, among the highly enriched GO terms,
RNA splicing was found to be activated while organelle organization,
chromosome organization, secondary and pyruvate metabolism were
suppressed suggesting the intricate transcriptional reprogramming
exerted by CBCVd-infection in the hop. KEGG pathway enrichment
analysis revealed that the majority of the cis and trans-regulated target
genes of DE-lncRNAs were enriched in primary and secondary metabolic
biosynthetic pathway (Supplementary Fig. S5).
We mapped cis and trans-regulated target genes of DE-lncRNAs to
different functional categories in the MapMan tool, which revealed their
role in transcriptional regulation of genes involved in various metabolic
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Genomics 113 (2021) 2350–2364
processes, hormone signaling, immune response in the CBCVd-infection.
Notably, target genes of DE-lncRNAs were represented by an array of
transcription factors, reinforcing their important role in the regulation
of gene expression in the CBCVd-hop interaction (Fig. 5C and D).
3.5. Prediction of DE-lncRNAs acting as miRNA targets and precursors
LncRNAs influence the functions of miRNAs at multiple levels. They
can act as miRNA decoys/sponges by sequestering miRNAs away from
target mRNAs or by competing with miRNAs for specific binding sites in
mRNAs, which leads to the regulation of gene expression. We have
therefore predicted the potential lncRNAs/DE-lncRNAs acting as miR­
NAs targets using the online tool psRNATarget by uploading our pre­
viously identified hop miRNAs [73]. Our analysis showed that among
3598 lncRNAs, 44 were predicted to interact with 35 miRNAs, while
V.S. Nath et al. Genomics 113 (2021) 2350–2364

Fig. 5. MapMan visualization of significantly differentially expressed transcripts (log2 fold change) related to the hormone signaling, transcription factors, ROS
signaling process and secondary metabolites in CBCVd-infected (CI) compared to the healthy control (CT) samples of hop. (A) predicted lncRNA trans-targets (B)
predicted lncRNA cis-targets (C) predicted DE-lncRNA cis-targets and (D) predicted DE-lncRNA trans-targets. Red and green signals represented in the scale bar
denotes an upregulation and downregulation of transcripts, respectively, in CI relative to the CT samples of hop. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

among the 684 DE-lncRNAs, eight were predicted to interact with 12
miRNAs in the hop (Supplementary Tables S9 and S10). A detailed
assessment of the results revealed three types of interaction between the
DE-lncRNAs and the miRNAs: a single lncRNA interacting with single
miRNAs, or a single lncRNA interacting with multiple miRNAs, or a
single miRNA interacting with more than one lncRNA. Among the
lncRNAs acting as miRNA targets, five lncRNAs were targeted by mul­
tiple miRNAs, 10 lncRNAs targeted by more than two miRNAs and 30
lncRNAs targeted by only one miRNA. Similarly, for the DE-lncRNAs, we
identified one lncRNA targeting three miRNAs, two lncRNAs with two
target miRNAs and six lncRNAs with only one miRNA target. Thus, our
results illustrate that most of the identified lncRNAs were regulated by
multiple miRNAs, which gives an insight into the intertwined and
complex regulatory network of lncRNAs and miRNAs in hops.
Besides their function as targets for miRNAs, lncRNAs can also serve
as precursors for miRNAs. Accordingly, we predicted lncRNAs might
function as miRNA precursors by aligning sequences of hop lncRNAs
against miRNA precursors downloaded from the miRbase using the
BLAST algorithm. A total of 17 lncRNAs were identified as potential
precursors of 11 miRNAs, suggesting that some lncRNAs can encode
miRNAs in hop (Supplementary Table S11). Subsequently, we visualized
the secondary structures of the lncRNA transcripts acting as miRNA
precursors using the RNAfold web server, which indicated that only a
few of the lncRNAs had a stable hairpin/stem-loop structure, which is
required for the formation of miRNA precursors, further confirming our
prediction (Supplementary Fig. S6).
3.6. Validation of selected DE-lncRNAs and target gene expression
profiles
To ascertain the reliability of lncRNA expression profiles generated
from transcriptome sequencing data, we examined the expression pat­
terns of selected eight lncRNAs involved in cis/trans-regulation or acting
as miRNA targets and five target genes that are co-expressed with the
lncRNAs by qRT-PCR. Most of the expression profiles obtained from
qRT-PCR were consistent and closely correlated with the RNA-seq data

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(R = 0.79), indicating the reliable expression profile of the lncRNAs in
the RNA sequencing data sets (Fig. 7). Nevertheless, despite the overall
similar trend in expression profiles, we also found some differences in
the expression levels of some lncRNAs and their predicted target genes,
which could be attributed to their complex and reciprocal cis/trans-
regulatory role, where a trans-acting lncRNA might also regulate the
expression of neighbouring genes and vice versa [80]. Together, our
results suggest that the lncRNAs, mRNAs and miRNAs identified in this
study and their predicted reciprocal interactions are likely to play a role
in response to CBCVd infection in the hop.
3.7. lncRNA-miRNA-mRNA interaction network during CBCVd infection
in the hop
To obtain a comprehensive overview of the concerted and inter­
connected regulatory relationships between lncRNAs, miRNAs and
mRNAs in our study, we constructed a co-expression network
comprising 309 lncRNA cis/trans-target genes, 34 miRNAs and 43
lncRNAs that were either co-expressed with their target genes and
differentially regulated in CT and CI samples or predicted as potential
targets for miRNAs. The reconstituted network projected the complex
regulatory and reciprocal interaction potential between lncRNAs,
mRNAs and miRNAs in in the regulation of various biological processes
triggered as a consequence of CBCVd infection in hop (Fig. 6). Notably, a
larger number of edges was shared by hop-miR2673a, hop-miR39, hop-
miR473a, and hop-miR28 (Fig. 6 and Supplementary Fig. S7), implying
that these miRNAs may play a crucial role in the regulation of multiple
mRNAs by interacting with multiple lncRNA targets in hop. Overall, our
network illustrates the complexity of RNA regulatory interactions and
projects the integral regulatory role that lncRNAs play in the miRNA-
mediated interactions with mRNAs.
4. Discussion
In plants, the recent advances in next-generation sequencing data
and related research paradigms have placed lncRNAs in the forefront as
a potentially new and key player in the regulation of gene expression at
the transcriptional, translational and post-translational levels that
impact a wide range of cellular functions and biological processes,
including biotic stress response [11]. In this study, we systematically
identified lncRNAs of hop and preliminarily analyzed their possible in­
teractions with mRNAs or miRNAs, which led us to reveal their plausible
involvement in several biological processes in hop. Recently, an
increasing number of reports suggested that lncRNAs are important
regulators of the immune response in plants. However, there is still a
paucity of information on the role of lncRNAs in viroid infections and
plant responses. In this study, high-throughput transcriptome
sequencing (RNA-Seq), followed by the sequential application of various
custom computational tools/algorithms and the adoption of a stringent
criterion for filtration of transcripts led to the reliable cataloging of 3598
high-confidence putative lncRNAs with a length of 200 to 2243 bp in
hop. Similarly, the adapted modified pipeline resulted in the identifi­
cation of 684 CBCVd-responsive lncRNAs associated with the tran­
scriptional modulation of several genes associated with plant-viroid
interactions.
4.1. Hop lncRNAs share common lncRNA features and involved in
various biological processes
Although similar to mRNAs, the majority of lncRNAs are transcribed
by RNA polymerase II, possess a 5′ 7-methylguanosine cap and a 3′ poly
(A) tail, diverse features such as shorter length, a fewer number of exons,
low and tissue-specific expression patterns clearly distinguish lncRNAs
from protein-coding mRNA genes [10,11]. LncRNAs are classified with
respect to their genomic location and the direction of transcription into
intergenic, intronic or exonic regions in the sense and antisense
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Genomics 113 (2021) 2350–2364
direction [10]. In this study, a large percentage (approximately 37%) of
the lncRNAs were classified as intergenic lncRNAs, which is not sur­
prising considering that intergenic lncRNA is increasingly recognized as
a major subclass of lncRNAs described in plants so far [81]. Similar
trends were observed for the lncRNA classification in previously pub­
lished reports of Brassica napus [62], banana [82], grapevine roots [83],
and cotton [27]. Additionally, hop lncRNAs identified in this study were
relatively shorter in length, non-conserved in sequence, had fewer exons
with low expression profiles compared to the protein-coding mRNAs and
were amenable for splicing, which was consistent with previous reports
on lncRNAs from other plant species including cucumber, chickpea, and
soybean [84–86]. Intriguingly, hop lncRNAs exhibited less sequence
conservation compared to other closely related species available in the
lncRNA database indicative of the high potential evolution of plant
lncRNAs repertoire [87].
In contrast to mRNAs, the low expression of lncRNAs has raised its
dubious regulatory role during the initial phase of their discovery in
plants [10]. However, mounting research evidence has now uncovered
the diverse regulatory potential of feebly expressed lncRNAs in
numerous biological processes ranging from normal growth and devel­
opment to biotic and abiotic stress responses in several plant species
[43]. To explore the diverse regulatory potential of candidate lncRNAs
in various biological and cellular processes in hop, we predicted their
cis-target genes by searching a 100 kb window upstream and down­
stream of their location and trans targets through RIblast analysis, which
was further accompanied by their functional annotation and pathway
assignments. The predicted target genes were highly enriched in
numerous biological processes including secondary metabolite biosyn­
thetic pathways, starch and sucrose metabolism, mRNA surveillance
pathway, ribosome biogenesis, protein processing and export indicating
the versatile and dynamic functional role of lncRNAs in the hop. The
glandular trichomes (known as lupulin glands) present in hop cones
biosynthesize several valuable secondary metabolites, including the
prenylflavonoid, bitter acids, essential oils and flavanols, which are of
commercial importance due to their diverse application in the brewing
and pharmaceutical industries worldwide [88]. Over the long term,
there is renowned interest in modulating the density and productivity of
such secreting structures including improved production of important
specialized metabolites using metabolic engineering approaches [89].
Our current understanding of the involvement of lncRNAs in the
biosynthesis of secondary metabolites, such as flavonoids, carotenoids
and alkaloids in plants [90] is improving, but the involvement of
lncRNAs in trichome development is still quite fragmentary. In this
study, we predicted several lncRNAs targeting the trichome develop­
mental factors such as the calmodulin-binding transcription activator
and TCP4 transcription factor. In this context, it will be intriguing to
decipher the functional role of these lncRNAs in the glandular trichome
development in hop. In addition, it is worth mentioning that many
lncRNA target genes were predicted to be involved in the biosynthesis of
secondary metabolite. Among the coding target genes in the phenyl­
propanoid biosynthesis pathway, we found a number of transcription
factors such as WRKY1, bZIP and bHLH2 and structural genes such as 4-
coumarate-CoA ligase 2 (4CL2), phenyl ammonia-lyase (PAL) as puta­
tive targets of candidate lncRNAs in hop. From a metabolic point of
view, it is tempting to perform the functional characterization of these
candidate lncRNAs by using gain and loss-of-function lines to establish
their regulatory role in secondary metabolite biosynthesis and glandular
trichome developments in hop. Notably, we have also predicted
lncRNAs to target mitogen-activated kinase, serine kinases, ABC trans­
porters and flowering locus implying the important and diverse regu­
latory potential of lncRNAs exerted in a wide array of biological and
cellular process in hop.
Several studies have suggested that lncRNAs can serve as molecular
sponges, acting as potential miRNA targets by competing with the
mRNA for binding to miRNA resulting in regulation of miRNA target
genes [91]. In addition, they can also act as precursors of miRNA,
V.S. Nath et al. Genomics 113 (2021) 2350–2364

Fig. 6. Interaction network of lncRNAs, their predicted miRNA and mRNAs targets. Blue hexagons represent lncRNAs, green diamonds represent lncRNA cis-targets,
yellow ellipse represents lncRNA trans-targets and red rectangles represent miRNAs. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

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V.S. Nath et al. Genomics 113 (2021) 2350–2364

Fig. 7. Expression levels of selected lncRNAs and their predicted cis and trans-target genes validated by qRT-PCR. 1: DE-lncRNA targeting splicing factor-like protein
1, AGO-T: argonaute 2 (predicted trans-target of DE-lncRNA MSTRG.4305.1), DNA-T: increased DNA methylation 1 (predicted cis-target of DE-lncRNA
MSTRG.3816.2), 2: DE-lncRNA targeting salicylic acid-binding protein 2, RNM-T: ribosomal lysine N-methyltransferase 3 (Predicted cis-target of DE-lncRNA HL.
SW.v1.0.G028223.1), 3: DE-lncRNA targeting disease resistance protein RPP2B, 4: DE-lncRNA targeting mitogen-activated protein kinase, 5: DE-lncRNA targeting
ribosomal lysine N-methyltransferase 3, 6: DE-lncRNA targeting apoptotic chromatin condensation inducer, HL.SW.v1.0.G040433.1: pre-mRNA-splicing factor ATP-
dependent RNA helicase (predicted cis-target of DE-lncRNA MSTRG.3411.1), HL.SW.v1.0.G023511.1: apoptotic chromatin condensation inducer (predicted trans-
target for DE-lncRNA MSTRG.4359.1), 7: DE-lncRNA targeting serine/threonine-protein kinase, 8: DE-lncRNA targeting pre-mRNA-splicing factor ATP-dependent
RNA helicase. The bars indicate ± standard error.

whereby lncRNAs are spliced to shorter miRNAs [91]. Previous studies
have shown a considerable regulatory potential for miRNA and lncRNA
interactions in several biological processes such as vernalization, cell
differentiation, flowering and fruiting, resistance to cold and drought,
and other biotic and abiotic stresses [10]. In the present study we pre­
dicted 43 hop miRNAs as potential targets for lncRNA/DElncRNAs
(Supplementary Tables S9 and S10). Remarkably, only a few of these
miRNAs were predicted to interact with serine/threonine-protein ki­
nases, which are the major class of protein kinases in plants and are
involved in various cellular functions, including metabolism, gene
expression, and cell growth and division and cell death mediating
signaling pathways [92]. In addition, we found 17 lncRNAs that were
predicted to serve as precursors of miRNAs, suggesting that hop lncRNAs
could be targeted by splicing factors to produce miRNAs, which in turn
could regulate the protein-coding genes. In this study, the co-expression
network analysis confirmed the complex reciprocal interactions be­
tween lncRNAs, miRNAs and mRNAs in hop.
4.2. Role of lncRNAs in response to CBCVd infection and symptom
development in the hop
Although lncRNAs have been shown to play an important role in
biotic stress in plants, their involvement in viroid pathogenicity in plants
is still in its infancy. To enrich our knowledge of how the viroid utilizes
lncRNAs to interplay with their host factors and other intriguing roles of
the lncRNA in plant-viroid interaction, we have used CBCVd as a model
system in this study. The differential expression of lncRNAs is an
important feature that allows to reveal their function in the orchestra­
tion of plant defense in a more flexible way. In congruence, our analyses
revealed a large number of hop lncRNAs showed sustained upward or
downward expression trends in expression during CBCVd-infection,
suggesting their putative role in viroid pathogenicity.
Plants have evolved sophisticated layering of the immune system to
defend against invading pathogens. Accumulating data suggest that the
stimulation of the plant defense response during viroid infection is often
associated with the activation of genes of downstream signaling path­
ways such as mitogen-activated protein kinases (MAPKs), calcium-
dependent protein kinases (CDPKs), AMP-activated protein kinase
(AMPK) including transcriptional reprogramming, induction of PR
proteins, alteration in hormone signaling pathways, generation of
reactive oxygen species (ROS) and cell wall dynamics, etc. [44,45].
MapMan pathway analysis of CBCVd-responsive lncRNAs-targeted
genes clearly demonstrated the involvement of lncRNAs in the modu­
lation of several protein kinases genes such as CDPKs, AMPK, serine
protein kinase, wall-associated kinase, etc., implying their plausible
involvement in the stimulation of the signaling cascades.
Emerging evidence of plant-pathogen interactions has shed light on
the role of plant lncRNAs as key players in regulating the phytohormone
levels, phytohormone response and hormone signal transduction [10]. It
is worth mentioning that the DE-lncRNA cis-target genes exhibited a
higher enrichment degree in the ethylene (ET), brassinosteroid (BA),
and abscisic acid (ABA) signal transduction pathways, which was
consistent with previous reports suggesting that the viroid intervenes
with phytohormone-regulated defenses during infection and disease
progression [44,45]. Interestingly, the JA responsive targets were
modulated exclusively in the trans- target subsets indicating their po­
tential involvement and effects on the distal transcriptional apparatus,
rather than direct interaction with the promoter elements or any co-
expressed genes. Overall, our results indicated that the altered
protein-coding targets for CBCVd-responsive lncRNAs are mainly
involved in the biosynthesis and signaling of ET, ABA and BA, but mostly
independent of the salicylic acid (SA) associated pathways.
Transcriptional reprogramming under the subtle control of tran­
scriptional factors (TFs) is one of the critical components during path­
ogen infection that favors defense responses over normal cellular
functions [93]. The mounting research indicated that several TFs fam­
ilies play an important role in the regulation of gene expression during
plant-viroid interactions [44,94]. Interestingly, the results of the target
gene GO-enrichment analysis of DE-lncRNAs indicated their involve­
ment in the dynamic reprogramming of the hop transcriptome in

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V.S. Nath et al.
CBCVd-infection. The increasing evidence suggests that the multiprotein
mediator (MED) complex is an important player in the fine-tuning of the
gene expression and pathway-specific transcriptional reprogramming in
response to biotic stress in plants [95–97]. Interestingly, the lncRNAs
were shown to involve in transcription activation through direct
recruitment and interaction with MED subunits and further facilitate
their binding to the promoter complex [98]. In this study we found that
CBCVd-responsive lncRNA was associated with MED subunits genes
(MED2, MED4 and MED6), whose regulatory role is still enigmatic and
requires further research to shed new light on the understanding of the
interdependent interactions of viroid, mediator and lncRNAs. Compiling
evidence supports the diverse involvement of primary and secondary
metabolites in plant-pathogen interactions, including surveillance
against pathogen attack, enzyme regulation, signal transduction and
antimicrobial activity [99]. In this context and considering the function
of the predicted target genes of DE-lncRNA, it appears that primary and
secondary metabolic pathways are regulated by lncRNAs and play
important roles upon CBCVd-infection in the hop. In addition, it is worth
noting that several CVBCVd-responsive lncRNA target genes were pre­
dicted to be involved in translational reprogramming, epigenetic
silencing, alternative splicing and phosphorylation/dephosphorylation
of proteins, which are hallmarks of viroid pathogenicity, thus reinforc­
ing lncRNAs as an emerging player in gene regulation and disease
pathogenesis. Further studies and research efforts are particularly
needed to establish the credentials of predicted candidate CBCVd-
responsive lncRNAs in epigenetic regulation of gene expression during
viroid pathogenicity. Intriguingly, we found 12 CBCVd-responsive
lncRNAs, which could be involved in competing endogenous RNAs
(ceRNAs) network via target mimicry and modulate other RNA tran­
scripts. The role of lncRNAs as ceRNAs has not yet been studied in viroid
pathogenicity and thus deserves the enticing priority that should be
considered in the coming future.
In conclusion, we have identified and characterized the lncRNAs in
hop on a genome-wide level, the foregoing outcome provides the novel
insight into CBCVd-responsive candidate lncRNAs and their interaction
with the regulatory component involved in viroid pathogenicity.
Moreover, we believe that the knowledge gained from this research can
be employed in functional studies to understand the regulatory roles of
CBCVd-responsive lncRNAs and the development of markers in a
breeding program for viroid resistance.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ygeno.2021.05.029.
Funding sources
This research was supported by MEMOBIC (EU Operational Pro­
gramme Research, Development and Education No. No. CZ.02.2.69/
0.0/0.0/16_027/0008357), and Czech Science Foundation GACR 19-
19629S and 18-10515 J with institutional support RVO:60077344.
Authors statement
We declare that this manuscript is original, has not been published
before and is not currently being considered for publication elsewhere.
We confirm that the manuscript has been read and approved by all
named authors and that there are no other persons who satisfied the
criteria for authorship but are not listed. We further confirm that the
order of authors listed in the manuscript has been approved by all of us.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
We apologize to the many colleagues whose work could not be cited
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Genomics 113 (2021) 2350–2364
in this manuscript due to space limitation. The authors thank Helena
Matoušková and Kavita Mishra from the Biology Centre (CAS) for their
excellent technical assistance.
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