BACKGROUND
nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science.
it is particularly popular among plant virologist as it is susceptible to a large number of different plant viruses.
it is also widely used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification.
in addition, its susceptibility to pathogens has been used to transiently express proteins, using either engineered viruses or syringe-infiltration of agrobacterium tumefaciens  <cit> .
the syringe-infiltration method proved to be extremely powerful tool to identify and characterize many viral silencing suppressor proteins  <cit> , pathogens effector proteins  <cit>  and also components of the plant nonsense-mediated mrna decay system  <cit> .
n. benthamiana is an herbaceous plant endemic to australia  <cit> , belonging to the suaveolentes section of the nicotiana genus of solanaceae family.
n. benthamiana is an allotetraploid species with  <dig> chromosome pairs, resulting from the hybridization of two unknown progenitors.
the estimated genome size of n. benthamiana is 3 gb  <cit> .
the draft genome sequence of the n. benthamiana nb- <dig> accession has been published recently  <cit> .
the origins of the n. benthamiana accessions used for research are generally not known.
however it has been suggested that only a single accession of n. benthamiana, or a collection of closely related accessions, are being used by the plant research community today, since they are very similar in both susceptibility to pathogens and also in phenotype  <cit> .
the genomes of economically important close relatives of n. benthamiana, such as potato , tomato  and pepper , genomes have been also sequenced recently .
these species has either smaller or very similar genome size to n. benthamiana.
the potato  genome size is 844 mb  <cit> , the tomato  genome size is approximately 900 mb  <cit>  and both species has twelve chromosomes.
other economically important members of the solanaceae family is the pepper and the eggplant.
the widely cultivated c. annuum accession zunla- <dig> genome size is estimated to be  <dig>  gb  <cit> , while the genome size of the eggplant is approximately  <dig>  mb  <cit> .
gene expression is regulated at several layers to ensure optimal spatial and temporal accumulation of proteins.
rna silencing is an evolutionarily conserved, important gene expression regulator of various cellular processes in eukaryotes, whereby 21-24 nt small rnas  direct silencing of endogenous genes or pathogens.
plant srnas have two major categories based on their biogenesis: small interfering rna  processed from perfectly double stranded rna; and microrna  derived from single stranded rna transcripts that form imperfectly double-stranded stem loop precursor structures  <cit> .
mir genes encode the precursor transcripts which are cleaved into mirnas by dicer-like  <dig> , and subsequently loaded into an rna induced silencing complex , containing an argonaute  protein.
mirna guided riscs are able to inhibit expression of genes with at least partial homology to the mirna by either transcript cleavage or translational arrest  <cit> .
mirnas have important roles in different cellular processes including developmental/ physiological processes and abiotic/biotic stress  <cit> .
mirnas are classified into families, whereby mirnas with the same or very similar sequences are grouped into the same family.
it has been shown that only a small number of mirna families are present across phylogenetically distant species .
the majority of plant mirnas are evolutionarily young and are only present in one to few species and it is likely that mir loci in plant genomes are in a constant dynamic evolutionary state  <cit> .
although the draft genome of n. benthamiana is available and transcriptome-wide expression studies have been reported , the mirna profile of n. benthamiana has not been analysed.
in this study, we used a comprehensive molecular and bioinformatic approach to detect and experimentally validate mirnas and their target mrnas from five different tissues of two n. benthamiana lines .
in our study, we used illumina sequencing to identify small rnas, their cleaved target mrnas followed by northern blot validation of the mirnas.
furthermore, we examined cleaved mirna targets from five different tissues of two aforementioned n. benthamiana accessions.
we identified  <dig> conserved and  <dig> novel micrornas and validated their target mrnas in n. benthamiana with a genomic scale method.
the targets were found to be involved in various biological processes including transcription, rna binding, dna modification, signal transduction, stress response and metabolic processes.
we anticipate that this study will serve as a valuable resource of n. benthamiana mirnas and their validated target mrnas to the whole plant research community.
RESULTS
high-throughput sequencing of n. benthamiana small rnas
to identify and comprehensively describe the expression of endogenous srnas in n. benthamiana, srnas containing 5′-phosphate and 3′-oh  were cloned from roots, stems, leaves, flowers and small seedlings  of n. benthamiana plants.
the libraries were then sequenced by high-throughput illumina sequencing.
by using biological replicates of five tissues, ten cdna libraries were made from the n. benthamiana line that is used in our laboratory.
to compare the srna profile of our n. benthamiana line and the line, which has been sequenced , two additional srna libraries were prepared and sequenced from the leaves and small seedlings of nb- <dig> accession.
these twelve cdna libraries yielded approximately  <dig> million reads.
sequences flanked by the 5′ and 3′ solexa adaptors, with a minimum and maximum length of  <dig> and 28 nt, respectively, were compared with the draft n. benthamiana genome sequences.
more than  <dig> million non-redundant reads were perfectly matched to at least one locus, and were analysed further .
the size distribution of sequence reads showed that the majority of srnas in our libraries belonged to the 24 nt size class and it was followed by the 21–22 nt size classes .
our sequence data are in agreement with previous observations which show that in plants, this 24 nt size class is the overall most abundant class of srnas  <cit> .table  <dig> high-throughput sequencing statistics of n. benthamiana srnas
fig.
<dig> the size distribution of sequenced small rna reads.
the size class distribution of redundant and non redundant small rna sequences in the twelve tissue samples.
the percentage of the different size classes  and the different tissues, which includes seedling, root, leaf, stem and flower with biological replicates and bti seedling and leaf cdna libraries  represented in n. benthamiana
identification of known and conserved mirnas in n. benthaniana
mirnas are a well-studied class of regulatory srnas.
they are present in all plant species studied so far and have important functions in plant development and stress responses.
conserved families of mirnas were found in many plant species and analysis of data from high-throughput sequencing approaches identified eight deeply conserved mirna families that are present in all embryophytes  <cit> .
first we looked for known mirnas by comparing our libraries to known mirnas from other plant species and we identified  <dig> known or conserved mirna families in the twelve srna libraries generated .
as expected, all of the deeply conserved mirna families  were present in our data sets .
we analysed the number of reads for conserved mirnas and mir <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were represented most frequently in the libraries .
as expected mirna families expressed at very different levels and some mirna families showed clear tissue-specific expression changes .
for example, the expression level of mir <dig> and mir <dig> families were high, while the expression of mir <dig> and mir <dig> families were similarly low in all tissues .
however, for mir <dig> and mir <dig> low expression is not surprising because their expression level is generally low under normal growth conditions as they are induced by sulphur or phosphate deficiency respectively  <cit> .
a recent comprehensive atlas of mirnas from  <dig> representative species across vascular plants identified  <dig> known mirna families that were present across several terrestrial plants.
these families formed eight groups depending on their distribution across lineages and species.
mirnas belonging to group  <dig> were ubiquitous and generally highly expressed across all terrestrial species  while group  <dig> mirna families were represented in all taxonomic lineages but could be absent or present at very low abundance levels in some species   <cit> .
most of the group  <dig> and group  <dig> mirna families were present in our data sets except the mir <dig>  mir <dig> and mir <dig> families .
similar to our result, these three mirna families were absent or their abundance was very low  or in some cases 11– <dig> rpm) in srna libraries prepared from different solanaceae species such as petunia, pepper, potato, tomato and tobacco  <cit> .
furthermore, the remaining six mirna groups were distributed across species with diverse lineage enrichment and one of them  was shown to be predominant in solanaceae species  <cit> .
as expected, in our libraries we identified several mirna families that belong to group  <dig> such as mir <dig>  mir <dig>  mir <dig> and mir <dig>  among the group  <dig> mirna families the mir <dig> abundance level was the highest in our libraries similar to the tobacco libraries reported by chavez montes et al.
<cit> .
some of the group  <dig> mirna families were missing from our libraries , however those mirna families also showed low abundance  in the reported tobacco libraries   <cit> .fig.
<dig> conserved and other known mirnas in n. benthamiana.
normalized read numbers of conserved and other known mirnas across the tissues included in this study.
expression profiles are expressed in reads per million  genome matching reads.
heat map colours represents absolute normalized levels of mirna expression ranging from less than  <dig> rpm  to more than  <dig> rpm  as indicated in the colour key.
we have identified  <dig> known or conserved mirna families in the twelve srna libraries generated.
all of the deeply conserved mirna families  were present in our data sets.
the expression levels of different mirna families were different and we also observed clear tissue-specific expressional changes within some mirna families as it was expected
we also identified several mirna families which were identified previously as tobacco  specific mirnas such as mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig>  <cit> .
these mirna families generally showed low abundance across all tissues in our libraries with the exception of mir <dig>  which was expressed at medium levels in all samples  and mir <dig>  which was relatively abundant in leaf and seedling samples  .
we checked the size distribution and starting nucleotide of the conserved and known mirna families in our libraries.
in  <dig> mirna families, 21 nt long mirnas were the most significant size class .
in several cases they were accompanied by additional size classes, mostly  <dig> nucleotide long sequences or in some cases  <dig> nucleotide sequences were present in significant proportions .
three mirna families exhibited a strong preference for  <dig> nucleotide sequences and in seven mirna families the  <dig> nucleotide long sequences were the most abundant size class .
our observation that the mir <dig> has a predominant  <dig> nucleotide size in our libraries is consistent with previous result and it might suggest a functional consequence since its size and sequence is conserved in terrestrial plant species  <cit> .
it was shown in several cases that size specificity has a functional consequence, like the  <dig> nucleotide preference of phase initiating mirnas associated with the generation of tasirnas  <cit> .
we found seven mirna families with the  <dig> nucleotide sequence dominance and two of them  belongs to the described phase initiating mirnas  <cit> .
it is likely they are also involved in tasirna generation in n. benthamiana.fig.
<dig> size distribution and starting nucleotide of the conserved and other known mirnas.
the relative abundance of different size categories , from  <dig> to  <dig> nucleotides is shown for the conserved and other known mirnas presented in fig.
<dig>  the relative abundance of the 5′-nucleotide  is shown for the conserved and other known mirnas presented in fig.
<dig>  in  <dig> mirna families, 21 nt long mirnas were the most significant size class.
in  <dig> of the  <dig> conserved mirna families, the majority of sequences started with uracyl at their 5′-end, although a portion of these have a different nucleotide composition at position  <dig> to a variable degree.
three mirna families exhibited a preference for an adenine at position 1
sorting of srnas into ago complexes is influenced by their 5′ terminal nucleotide.
hence, we checked the 5′-nucleotide distribution of the conserved and known mirna families in our libraries, since it is an important feature that is correlating to their biogenesis and function  <cit> .
in  <dig> of the  <dig> conserved mirna families, the majority of sequences started with uracyl at their 5′-end, although a portion of these have a different nucleotide composition at position  <dig> to a variable degree .
three mirna families exhibited a preference for an adenine at position  <dig>  although one of them has a different nucleotide composition at position  <dig> to a variable degree .
in three mirna families the most frequently found first nucleotide at their 5′-end was cytosine .
mirnas mostly are incorporated into the ago <dig> effector complex to give sequence specificity to either slice or translationally repress their target mrnas  <cit> .
ago <dig> harbours mirnas that favour a 5′ terminal uridine  <cit> .
in line with this observation, most of the conserved and known mirna families in our libraries started with uracyl at their 5′-end.
we also found several mirnas that start with either adenine or cytosine .
it was reported in many different plant species that mirnas belonging to these mirna families are starting with the same nucleotide at the 5′-position  <cit> .
many plant mirnas show tissue-specific and developmental-stage-specific expression patterns and they have important roles during development and stress adaptation  <cit> .
to validate the tissue-specific expression patterns of selected conserved and known mirnas we carried out northern blot analysis using seedling , root , leaf , stem  and flower  samples isolated from our n. benthamiana line; and leaf  and seedling  samples from n. benthamiana nb- <dig> accession that were obtained from the boyce thompson institute for plant research.
northern analysis usually was in broad agreement with the sequencing data as in the cases of mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> .
in certain cases we observed a discrepancy between the northern blot and sequencing data, as in the cases of mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> .
however, these differences are not unexpected as it has been reported that northern analysis are not always in full agreement with the sequencing data, presumably due to the inherent biases in next-generation sequencing technologies .
theoretically, the oligoprobes used for northern analysis might have detected heterogeneous mirnas.
however, we expect that northern analysis should reveal more accurate in vivo expression patterns.
we also performed rt-qpcr analysis to validate our northern results by checking the expression pattern of mir <dig> and mir <dig> in n. benthamiana tissues.
we found that the results of northern analysis and rt-qpcr were identical .
furthermore, comparing the northern analysis data of our n. benthamiana line to the nb- <dig> accession to detect tissue specific expression pattern of mirnas, we were not able to confirm the differences occasionally observed in the mirna read numbers at certain mirna families  .
our mirna expression data therefore might indicate that these n. benthamiana lines are closely related based on their very similar mirna expression patterns.fig.
<dig> expression analysis of selected conserved and other known mirnas in different n. benthamiana tissues.
total rna was extracted from different tissues including, seedling , root , leaf , stem , flower  from n. benthamiana plants used in our laboratory and from plants from boyce thompson institute .
the rna was separated on page and transferred to nylon membranes for northern blot analysis of the mirnas.
oligonucleotide probes were used to detect specific mirnas, and an u6-specific probe was used to detect u <dig> rna as a loading control for each membrane
targets of conserved and known mirnas
to generate a mirna cleaved target library  from n. benthamiana we applied a high-throughput experimental approach that can identify mrnas targeted by srnas  <cit> .
the poly-a fraction of total rna extracted from seedling , root , leaf , stem , flower  of n. benthamiana plants; and leaf  and seedling  of the nb- <dig> accession were analysed for the identification of target transcripts of conserved, other known, and new mirnas.
we obtained a total number of  <dig> million short sequencing reads representing the 5′ ends of uncapped, poly-adenylated rnas.
after initial processing, equal numbers of 20- and 21-nt sequence reads were obtained, and 48 % of the short sequencing reads could be mapped to the n. bentamiana transcriptome.
in plants, mirna mediated mrna cleavage is highly specific and mirnas have been shown to bind with near perfect complementarity to their mrna targets, which generally leads to the slicing of the mrna between positions  <dig> and  <dig> of the ago <dig> bound mirna.
as a consequence, the cleaved mrna targets should have distinct peaks in the degradome sequence tags at the predicted cleavage site relative to the other regions of the transcript  <cit> .
in our analysis we have applied the paresnip pipeline  <cit>  to identify cleaved targets for both known  and new mirnas in n. benthamiana.
abundance of the sequenced tags was plotted on each transcript and the results are shown on additional file 3: figure s <dig>  the cleaved target transcripts have been categorized into five classes  as it was defined previously in cleaveland   <cit> .
in our target list we kept high-confidence mirna-target gene interactions  and only category  <dig> targets as low–confidence mirna-target pairs.
category  <dig> targets are defined as only one raw read at the expected cleavage position, however it might also be the result of the degradation of the target rna, therefore they were omitted from our target list.
this approach identified a total of  <dig> target mrnas, for conserved and other known mirnas .table  <dig> targets of conserved and other known mirnas in nicotiana benthamiana
a
a the abundance for the degradome fragment  indicating cleavage at that position/total number of fragments x  <dig> ,000
b new target in n. benthamiana for conserved and other known mirnas
altogether we found  <dig> targets for conserved mirnas, from which  <dig> were classed as category  <dig>  six as category  <dig> and fifteen as category  <dig>  for other known mirnas we have found  <dig> targets, from which one was classed as category  <dig>  two as category  <dig> and eight as category  <dig>
we identified targets for  <dig> conserved mirna families out of  <dig>  some of the conserved mirnas without any identified targets  were expressed at a low level in all of the srna libraries , which may explain the lack of detection of cleaved targets.
however, both mir <dig> and mir <dig> were expressed at a considerable level in at least some of the tissues  without mrna targets identified in the degradome libraries.
as an alternative explanation these mirnas might be engaged in translational repression, however in different plant species cleaved target mrnas were identified as their target which makes this explanation less plausible  <cit> .
we have confirmed  <dig> conserved targets of conserved mirnas already identified in different plants  <cit> .
many of the identified conserved targets are members of different families of transcription factors, such as squamosal promoter-binding , myb, arf, nac, hd-zipiii, ccaat-binding, ap <dig> and tcp.
in addition to these targets we also identified  <dig> new targets in n. benthamiana for conserved mirnas .
we found one target of the non-conserved mir <dig> which was already identified in tomato  <cit> .
we also identified nine new targets of seven non-conserved mirnas .
most of the non-conserved mirna families for which no target had been identified were expressed at a very low level in all tissues , except mir <dig>  mir <dig>  mir <dig> and mir <dig>  .
n. benthamiana specific mirnas
we used strict criteria to identify new mirnas.
briefly, the srna reads were mapped to the n. benthamiana genome and secondary structures were predicted for each locus.
based on the hairpin prediction, the presence of mirna* strands in the srna libraries and validated target mrna in the degradome libraries we found  <dig> new mirna candidates .
nb_mirc1_3p was the most abundant nicotiana specific mirna in our srna libraries and it expressed in all of the tissues.
the sequence of nb_mirc1_3p was also reported previously from a large scale srna dataset as endogenous small rna of nicotiana attenuata  <cit> , however it was not further investigated which small rna class it belongs to.
the second most abundant mirna was the nb_mirc2_3p and it was also present in all of the small rna libraries.
for the remaining n. benthamiana specific mirnas the relative number of reads in all tissues was low .
however, this result is in line with the observation shown previously, that mirna abundance decreases as the conservation of the sequence decreases  <cit> .fig.
<dig> novel mirnas in n. benthamiana.
normalized read numbers of novel mirnas across the tissues included in this study.
expression profiles are expressed in reads per million genome matching reads.
heat map colours represents absolute normalized levels of mirna expression ranging from less than  <dig> rpm  to more than  <dig> rpm  as indicated in the colour key
as a next step, we checked the size distribution and starting nucleotide of the new n. benthamiana specific mirnas in our libraries.
the 21 nt long mirnas were the most significant size class and half of the new mirnas belong to this group .
in several cases they were accompanied by additional size class,  <dig> nucleotide long sequences were present in significant proportions .
three mirnas exhibited a strong preference for  <dig> nucleotide sequences  and in five mirna families the  <dig> nucleotide long sequences were the most abundant size class .
we found that two mirna families showed the coexistence of 20– <dig> nucleotide variants with the  <dig> nucleotides being the most abundant variants  .fig.
<dig> size distribution and starting nucleotide of the novel n. benthamiana specific mirnas.
the relative abundance of different size categories , from  <dig> to  <dig> nucleotides is shown for the novel mirnas presented in fig.
<dig>  the relative abundance of the 5′-nucleotide  is shown for the novel mirnas presented in fig.
5
next we checked the 5′-nucleotide distribution of the new mirna families in our libraries.
seven started with uracil at their 5′-end  .
eight mirna families exhibited a preference for an adenine at position  <dig>  although six of these have a different nucleotide composition at position  <dig> to a variable degree .
in three mirna families the most frequently found first nucleotide at their 5′-end was cytosine .
one mirna family exhibited a preference for a guanine at position  <dig>  although a significant fraction has uracil at the 5′-position  .
although the majority of plant mirnas have a uracil at position  <dig>  it is not unusual that some mirna families contain adenine, cytosine and even guanine in significant proportions at the 5′-position  <cit> .
young mirnas are often weakly expressed, processed imprecisely or lack targets  <cit> .
our data fits well with these previous observations since most of the n. benthamiana specific mirnas showed low relative abundance in our libraries  and in several cases they were accompanied by additional size classes for various degrees.
furthermore, some of the new mirnas identified in our srna libraries are produced from long hairpin precursors that may also indicate the young evolutionary origin  .
this situation is well observable on the nb_mirc <dig> precursor where two mirna duplexes were both identified in the srna libraries and detected by northern blots .
however, we did not identify mirnas without target mrnas in our data set, since we used the presence of cleaved target mrna as a criterion to select new mirnas.
we used northern blot analysis to check the expression level of n. benthamiana specific mirnas in five tissues .
the mirnas that gave signal on the northern blot, in most cases were detectable in all of the tissues tested .
some of the new mirnas were abundant in specific tissues , and many of them showed differential expression in the tissues analysed  .
the relative number of reads in all tissues was very low for most of the mirnas  and this was also reflected in the northern blot analysis, since we did not obtain signal for six mirnas .
the expression profiles obtained by northern blot analysis were different from the sequencing data for nb_mirc3_5p, nb_mirc6_3p, nb_mirc8_3pa, nb_mirc11_5p and nb_mirc17_5p.
however, for the other new mirnas , sequencing data and expression profiles obtained by northern blot analysis showed good correlation .
although the relative read numbers for most of the new mirnas were low, we were able to confirm the presence of 13 n. benthamiana specific mirnas by northern blot.fig.
<dig> expression patterns of novel mirnas found in n. benthamiana.
total rna was extracted from different tissues including, seedling , root , leaf , stem , flower  from n. benthamiana plants used in our laboratory and from plants from boyce thompson institute .
the rna was separated on page and transferred to nylon membranes for northern blot analysis of the novel mirnas.
oligonucleotide probes were used to detect specific mirnas, and an u6-specific probe was used to detect u <dig> rna as a loading control for each membrane
targets of n. benthamiana specific mirnas
we identified  <dig> targets for new mirnas using our degradome libraries.
interestingly, in the case of nb_mirc <dig> we found target for both the mature and the star strand of the new mirna .
based on the degradome data, among the identified target mrnas of the specific, new n. benthamiana mirnas, three were classed as category  <dig>  two as category  <dig>  twenty-two as category  <dig> and five as category  <dig>  comparing the target list of the known  and new mirnas the most obvious difference between them is that the known mirna targets mostly belong to category  <dig> and  <dig> .
based on their molecular function both groups regulate various genes that are included in transcriptional regulation or enzymatic activity .table  <dig> targets of novel mirnas found in nicotiana benthamiana
a the abundance for the degradome fragment  indicating cleavage at that position/total number of fragments x  <dig> ,000
the new mirnas target different genes with a wide variety of predicted functions.
one of the three identified category  <dig> targets of the new mirnas is the mrna of the heterotrimeric gtp binding protein  alpha subunit .
g proteins are highly conserved in eukaryotes.
plant g proteins play an important regulatory role in multiple physiological processes.
they are involved in signal transduction from hormone receptors, including the plant hormones gibberellin and abscisic acid that regulate gene expression; secretion, defence, stomata movements, channel regulation, sugar sensing and cell death  <cit> .
the heterotrimeric g protein complex is minimally composed of three subunits: alpha, beta and gamma.
plants have only a small number of genes encoding the g proteins subunits.
arabidopsis has only one canonical g protein alpha subunit, one beta subunit and three gamma subunit  <cit> .
plant alpha subunits are highly conserved between species, it binds and hydrolyse gtp and relay signals by interacting with downstream effector proteins  <cit> .
interestingly, during convergent evolution human g protein alpha- <dig>  expression is also regulated by mirna  in breast cancer cells  <cit> .
another category  <dig> target is the mrna of peroxisome biogenesis factor  <dig> .
it is cleaved by nb_mirc5_3p and has been demonstrated to be a component of the peroxisomal matrix protein import machinery.
peroxisomes are eukaryotic organelles and they are involved in a wide range of processes.
plant peroxisomes are important in primary and secondary metabolism, development, and responses to abiotic and biotic stresses.
previous studies identified  <dig> pex genes in the arabidopsis genome and demonstrated that they play a pivotal role in the import of peroxisomal matrix proteins  <cit> .
pex  <dig> perform multiple functions, including the biogenesis of er-derived protein and oil bodies  <cit> , the maintenance of er morphology, the formation of cuticular wax  <cit> , peroxisome and chloroplast connections  <cit> .
one of the category  <dig> targets is repressor of silencing  which is cleaved by nb_mirc1_3p.
interestingly, the sequence of nb_mirc1_3p can also be found in nicotiana attenuata  <cit>  and its target site on ros <dig> is conserved in other nicotiana species as well.
ros <dig> encodes a nuclear dna glycosylase domain protein that catalyse the release of 5-methylcytosine  from dna via a base excision repair reaction  <cit> .
dna cytosine methylation is an important epigenetic mark conserved in many eukaryotes.
it has important roles in transposon silencing, genome organization, genomic imprinting and regulation of gene expression  <cit> .
during continuous adaptation to variable external and internal environments dna methylation patterns are dynamically controlled by methylation and demethylation reactions.
ros <dig> plays an important role in active dna demethylation and helps to maintain gene expression  <cit> .
the targeting of ros <dig> by a mirna provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of its activity.
the targeting of a dna glycosylase  by a mirna  was first predicted in arabidopsis  <cit>  and later it was shown that the expression of demeter-like protein <dig> mrna was down-regulated in mir402-overexpressing transgenic plants  <cit> .
the other category  <dig> target is a zinc-regulated transporter , iron-regulated transporter -like protein  <dig>  and is cleaved by nb-mirc12_5p.
zrt/irt-like protein family has been demonstrated to be involved in metal uptake and transport.
zrt/irt-like proteins generally contribute to metal ion homeostasis by transporting cations into the cytoplasm  <cit> .
it has been reported in several plant species that various mirnas are vital for maintaining nutrient homeostasis by regulating the expression of transporters that are involved in nutrient uptake and mobilization  <cit> .
other targets that are worthy to note are the wat1-related protein at5g40240-like gene targeted by nb_mirc7_3p; the cbl-interacting serine/threonine-protein kinase gene targeted by nb_mirc4_3p, proteasome subunit alpha type-2-a gene targeted by nb_mirc7_3p, suberization-associated anionic peroxidase targeted by nb_mirc10_3p and beta-amyrin synthase targeted by nb_mirc10_3p.
CONCLUSIONS
in this study we have confirmed the expression of known and new mirnas in various tissues of n. benthamiana using high-throughput sequencing and northern blot analysis.
as a result, this work represents a tissue specific atlas of known and new mirna expression of n. benthamiana.
in addition, we have experimentally identified many targets of both known and new mirnas using a high-throughput method for the global identification of mirnas targets.
this study provides a valuable resource of mirnas and their validated target mrnas to the plant research community that will be beneficial well into the future.

 nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. it is particularly popular among plant virologist as it is susceptible to a large number of different plant viruses. it is also widely used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. in addition, its susceptibility to pathogens has been used to transiently express proteins, using either engineered viruses or syringe-infiltration of agrobacterium tumefaciens. the syringe-infiltration method proved to be extremely powerful tool to identify and characterize many viral silencing suppressor proteins, pathogens effector proteins and also components of the plant nonsense-mediated mrna decay system. n. benthamiana is an herbaceous plant endemic to australia, belonging to the suaveolentes section of the nicotiana genus of solanaceae family. n. benthamiana is an allotetraploid species with chromosome pairs, resulting from the hybridization of two unknown progenitors. the estimated genome size of n. benthamiana is 3 gb. the draft genome sequence of the n. benthamiana nb- accession has been published recently. the origins of the n. benthamiana accessions used for research are generally not known. however it has been suggested that only a single accession of n. benthamiana, or a collection of closely related accessions, are being used by the plant research community today, since they are very similar in both susceptibility to pathogens and also in phenotype. the genomes of economically important close relatives of n. benthamiana, such as potato, tomato and pepper, genomes have been also sequenced recently. these species has either smaller or very similar genome size to n. benthamiana. the potato genome size is 844 mb, the tomato genome size is approximately 900 mb and both species has twelve chromosomes. other economically important members of the solanaceae family is the pepper and the eggplant. the widely cultivated c. annuum accession zunla- genome size is estimated to be gb, while the genome size of the eggplant is approximately  mb. gene expression is regulated at several layers to ensure optimal spatial and temporal accumulation of proteins. rna silencing is an evolutionarily conserved, important gene expression regulator of various cellular processes in eukaryotes, whereby 21-24 nt small rnas direct silencing of endogenous genes or pathogens. plant srnas have two major categories based on their biogenesis: small interfering rna processed from perfectly double stranded rna; and microrna derived from single stranded rna transcripts that form imperfectly double-stranded stem loop precursor structures. mir genes encode the precursor transcripts which are cleaved into mirnas by dicer-like, and subsequently loaded into an rna induced silencing complex, containing an argonaute protein. mirna guided riscs are able to inhibit expression of genes with at least partial homology to the mirna by either transcript cleavage or translational arrest. mirnas have important roles in different cellular processes including developmental/ physiological processes and abiotic/biotic stress. mirnas are classified into families, whereby mirnas with the same or very similar sequences are grouped into the same family. it has been shown that only a small number of mirna families are present across phylogenetically distant species. the majority of plant mirnas are evolutionarily young and are only present in one to few species and it is likely that mir loci in plant genomes are in a constant dynamic evolutionary state. although the draft genome of n. benthamiana is available and transcriptome-wide expression studies have been reported, the mirna profile of n. benthamiana has not been analysed. in this study, we used a comprehensive molecular and bioinformatic approach to detect and experimentally validate mirnas and their target mrnas from five different tissues of two n. benthamiana lines. in our study, we used illumina sequencing to identify small rnas, their cleaved target mrnas followed by northern blot validation of the mirnas. furthermore, we examined cleaved mirna targets from five different tissues of two aforementioned n. benthamiana accessions. we identified conserved and novel micrornas and validated their target mrnas in n. benthamiana with a genomic scale method. the targets were found to be involved in various biological processes including transcription, rna binding, dna modification, signal transduction, stress response and metabolic processes. we anticipate that this study will serve as a valuable resource of n. benthamiana mirnas and their validated target mrnas to the whole plant research community.  high-throughput sequencing of n. benthamiana small rnas to identify and comprehensively describe the expression of endogenous srnas in n. benthamiana, srnas containing 5′-phosphate and 3′-oh were cloned from roots, stems, leaves, flowers and small seedlings of n. benthamiana plants. the libraries were then sequenced by high-throughput illumina sequencing. by using biological replicates of five tissues, ten cdna libraries were made from the n. benthamiana line that is used in our laboratory. to compare the srna profile of our n. benthamiana line and the line, which has been sequenced, two additional srna libraries were prepared and sequenced from the leaves and small seedlings of nb- accession. these twelve cdna libraries yielded approximately million reads. sequences flanked by the 5′ and 3′ solexa adaptors, with a minimum and maximum length of and 28 nt, respectively, were compared with the draft n. benthamiana genome sequences. more than million non-redundant reads were perfectly matched to at least one locus, and were analysed further. the size distribution of sequence reads showed that the majority of srnas in our libraries belonged to the 24 nt size class and it was followed by the 21–22 nt size classes. our sequence data are in agreement with previous observations which show that in plants, this 24 nt size class is the overall most abundant class of srnas.table high-throughput sequencing statistics of n. benthamiana srnas fig. the size distribution of sequenced small rna reads. the size class distribution of redundant and non redundant small rna sequences in the twelve tissue samples. the percentage of the different size classes and the different tissues, which includes seedling, root, leaf, stem and flower with biological replicates and bti seedling and leaf cdna libraries represented in n. benthamiana identification of known and conserved mirnas in n. benthaniana mirnas are a well-studied class of regulatory srnas. they are present in all plant species studied so far and have important functions in plant development and stress responses. conserved families of mirnas were found in many plant species and analysis of data from high-throughput sequencing approaches identified eight deeply conserved mirna families that are present in all embryophytes. first we looked for known mirnas by comparing our libraries to known mirnas from other plant species and we identified known or conserved mirna families in the twelve srna libraries generated. as expected, all of the deeply conserved mirna families were present in our data sets. we analysed the number of reads for conserved mirnas and mir and were represented most frequently in the libraries. as expected mirna families expressed at very different levels and some mirna families showed clear tissue-specific expression changes. for example, the expression level of mir and mir families were high, while the expression of mir and mir families were similarly low in all tissues. however, for mir and mir low expression is not surprising because their expression level is generally low under normal growth conditions as they are induced by sulphur or phosphate deficiency respectively. a recent comprehensive atlas of mirnas from representative species across vascular plants identified known mirna families that were present across several terrestrial plants. these families formed eight groups depending on their distribution across lineages and species. mirnas belonging to group were ubiquitous and generally highly expressed across all terrestrial species while group mirna families were represented in all taxonomic lineages but could be absent or present at very low abundance levels in some species. most of the group and group mirna families were present in our data sets except the mir mir and mir families. similar to our result, these three mirna families were absent or their abundance was very low or in some cases 11– rpm) in srna libraries prepared from different solanaceae species such as petunia, pepper, potato, tomato and tobacco. furthermore, the remaining six mirna groups were distributed across species with diverse lineage enrichment and one of them was shown to be predominant in solanaceae species. as expected, in our libraries we identified several mirna families that belong to group such as mir mir mir and mir among the group mirna families the mir abundance level was the highest in our libraries similar to the tobacco libraries reported by chavez montes et al.. some of the group mirna families were missing from our libraries, however those mirna families also showed low abundance in the reported tobacco libraries.fig. conserved and other known mirnas in n. benthamiana. normalized read numbers of conserved and other known mirnas across the tissues included in this study. expression profiles are expressed in reads per million genome matching reads. heat map colours represents absolute normalized levels of mirna expression ranging from less than rpm to more than rpm as indicated in the colour key. we have identified known or conserved mirna families in the twelve srna libraries generated. all of the deeply conserved mirna families were present in our data sets. the expression levels of different mirna families were different and we also observed clear tissue-specific expressional changes within some mirna families as it was expected we also identified several mirna families which were identified previously as tobacco specific mirnas such as mir mir mir mir mir mir mir and mir. these mirna families generally showed low abundance across all tissues in our libraries with the exception of mir which was expressed at medium levels in all samples and mir which was relatively abundant in leaf and seedling samples. we checked the size distribution and starting nucleotide of the conserved and known mirna families in our libraries. in mirna families, 21 nt long mirnas were the most significant size class. in several cases they were accompanied by additional size classes, mostly nucleotide long sequences or in some cases nucleotide sequences were present in significant proportions. three mirna families exhibited a strong preference for nucleotide sequences and in seven mirna families the nucleotide long sequences were the most abundant size class. our observation that the mir has a predominant nucleotide size in our libraries is consistent with previous result and it might suggest a functional consequence since its size and sequence is conserved in terrestrial plant species. it was shown in several cases that size specificity has a functional consequence, like the nucleotide preference of phase initiating mirnas associated with the generation of tasirnas. we found seven mirna families with the nucleotide sequence dominance and two of them belongs to the described phase initiating mirnas. it is likely they are also involved in tasirna generation in n. benthamiana.fig. size distribution and starting nucleotide of the conserved and other known mirnas. the relative abundance of different size categories, from to nucleotides is shown for the conserved and other known mirnas presented in fig.  the relative abundance of the 5′-nucleotide is shown for the conserved and other known mirnas presented in fig.  in mirna families, 21 nt long mirnas were the most significant size class. in of the conserved mirna families, the majority of sequences started with uracyl at their 5′-end, although a portion of these have a different nucleotide composition at position to a variable degree. three mirna families exhibited a preference for an adenine at position 1 sorting of srnas into ago complexes is influenced by their 5′ terminal nucleotide. hence, we checked the 5′-nucleotide distribution of the conserved and known mirna families in our libraries, since it is an important feature that is correlating to their biogenesis and function. in of the conserved mirna families, the majority of sequences started with uracyl at their 5′-end, although a portion of these have a different nucleotide composition at position to a variable degree. three mirna families exhibited a preference for an adenine at position although one of them has a different nucleotide composition at position to a variable degree. in three mirna families the most frequently found first nucleotide at their 5′-end was cytosine. mirnas mostly are incorporated into the ago effector complex to give sequence specificity to either slice or translationally repress their target mrnas. ago harbours mirnas that favour a 5′ terminal uridine. in line with this observation, most of the conserved and known mirna families in our libraries started with uracyl at their 5′-end. we also found several mirnas that start with either adenine or cytosine. it was reported in many different plant species that mirnas belonging to these mirna families are starting with the same nucleotide at the 5′-position. many plant mirnas show tissue-specific and developmental-stage-specific expression patterns and they have important roles during development and stress adaptation. to validate the tissue-specific expression patterns of selected conserved and known mirnas we carried out northern blot analysis using seedling, root, leaf, stem and flower samples isolated from our n. benthamiana line; and leaf and seedling samples from n. benthamiana nb- accession that were obtained from the boyce thompson institute for plant research. northern analysis usually was in broad agreement with the sequencing data as in the cases of mir mir mir mir mir mir mir and mir. in certain cases we observed a discrepancy between the northern blot and sequencing data, as in the cases of mir mir mir mir and mir. however, these differences are not unexpected as it has been reported that northern analysis are not always in full agreement with the sequencing data, presumably due to the inherent biases in next-generation sequencing technologies. theoretically, the oligoprobes used for northern analysis might have detected heterogeneous mirnas. however, we expect that northern analysis should reveal more accurate in vivo expression patterns. we also performed rt-qpcr analysis to validate our northern results by checking the expression pattern of mir and mir in n. benthamiana tissues. we found that the results of northern analysis and rt-qpcr were identical. furthermore, comparing the northern analysis data of our n. benthamiana line to the nb- accession to detect tissue specific expression pattern of mirnas, we were not able to confirm the differences occasionally observed in the mirna read numbers at certain mirna families. our mirna expression data therefore might indicate that these n. benthamiana lines are closely related based on their very similar mirna expression patterns.fig. expression analysis of selected conserved and other known mirnas in different n. benthamiana tissues. total rna was extracted from different tissues including, seedling, root, leaf, stem, flower from n. benthamiana plants used in our laboratory and from plants from boyce thompson institute. the rna was separated on page and transferred to nylon membranes for northern blot analysis of the mirnas. oligonucleotide probes were used to detect specific mirnas, and an u6-specific probe was used to detect u rna as a loading control for each membrane targets of conserved and known mirnas to generate a mirna cleaved target library from n. benthamiana we applied a high-throughput experimental approach that can identify mrnas targeted by srnas. the poly-a fraction of total rna extracted from seedling, root, leaf, stem, flower of n. benthamiana plants; and leaf and seedling of the nb- accession were analysed for the identification of target transcripts of conserved, other known, and new mirnas. we obtained a total number of million short sequencing reads representing the 5′ ends of uncapped, poly-adenylated rnas. after initial processing, equal numbers of 20- and 21-nt sequence reads were obtained, and 48 % of the short sequencing reads could be mapped to the n. bentamiana transcriptome. in plants, mirna mediated mrna cleavage is highly specific and mirnas have been shown to bind with near perfect complementarity to their mrna targets, which generally leads to the slicing of the mrna between positions and of the ago bound mirna. as a consequence, the cleaved mrna targets should have distinct peaks in the degradome sequence tags at the predicted cleavage site relative to the other regions of the transcript. in our analysis we have applied the paresnip pipeline to identify cleaved targets for both known and new mirnas in n. benthamiana. abundance of the sequenced tags was plotted on each transcript and the results are shown on additional file 3: figure s the cleaved target transcripts have been categorized into five classes as it was defined previously in cleaveland. in our target list we kept high-confidence mirna-target gene interactions and only category targets as low–confidence mirna-target pairs. category targets are defined as only one raw read at the expected cleavage position, however it might also be the result of the degradation of the target rna, therefore they were omitted from our target list. this approach identified a total of target mrnas, for conserved and other known mirnas.table targets of conserved and other known mirnas in nicotiana benthamiana a a the abundance for the degradome fragment indicating cleavage at that position/total number of fragments x,000 b new target in n. benthamiana for conserved and other known mirnas altogether we found targets for conserved mirnas, from which were classed as category six as category and fifteen as category for other known mirnas we have found targets, from which one was classed as category two as category and eight as category we identified targets for conserved mirna families out of some of the conserved mirnas without any identified targets were expressed at a low level in all of the srna libraries, which may explain the lack of detection of cleaved targets. however, both mir and mir were expressed at a considerable level in at least some of the tissues without mrna targets identified in the degradome libraries. as an alternative explanation these mirnas might be engaged in translational repression, however in different plant species cleaved target mrnas were identified as their target which makes this explanation less plausible. we have confirmed conserved targets of conserved mirnas already identified in different plants. many of the identified conserved targets are members of different families of transcription factors, such as squamosal promoter-binding, myb, arf, nac, hd-zipiii, ccaat-binding, ap and tcp. in addition to these targets we also identified new targets in n. benthamiana for conserved mirnas. we found one target of the non-conserved mir which was already identified in tomato. we also identified nine new targets of seven non-conserved mirnas. most of the non-conserved mirna families for which no target had been identified were expressed at a very low level in all tissues, except mir mir mir and mir. n. benthamiana specific mirnas we used strict criteria to identify new mirnas. briefly, the srna reads were mapped to the n. benthamiana genome and secondary structures were predicted for each locus. based on the hairpin prediction, the presence of mirna* strands in the srna libraries and validated target mrna in the degradome libraries we found new mirna candidates. nb_mirc1_3p was the most abundant nicotiana specific mirna in our srna libraries and it expressed in all of the tissues. the sequence of nb_mirc1_3p was also reported previously from a large scale srna dataset as endogenous small rna of nicotiana attenuata, however it was not further investigated which small rna class it belongs to. the second most abundant mirna was the nb_mirc2_3p and it was also present in all of the small rna libraries. for the remaining n. benthamiana specific mirnas the relative number of reads in all tissues was low. however, this result is in line with the observation shown previously, that mirna abundance decreases as the conservation of the sequence decreases.fig. novel mirnas in n. benthamiana. normalized read numbers of novel mirnas across the tissues included in this study. expression profiles are expressed in reads per million genome matching reads. heat map colours represents absolute normalized levels of mirna expression ranging from less than rpm to more than rpm as indicated in the colour key as a next step, we checked the size distribution and starting nucleotide of the new n. benthamiana specific mirnas in our libraries. the 21 nt long mirnas were the most significant size class and half of the new mirnas belong to this group. in several cases they were accompanied by additional size class, nucleotide long sequences were present in significant proportions. three mirnas exhibited a strong preference for nucleotide sequences and in five mirna families the nucleotide long sequences were the most abundant size class. we found that two mirna families showed the coexistence of 20– nucleotide variants with the nucleotides being the most abundant variants.fig. size distribution and starting nucleotide of the novel n. benthamiana specific mirnas. the relative abundance of different size categories, from to nucleotides is shown for the novel mirnas presented in fig.  the relative abundance of the 5′-nucleotide is shown for the novel mirnas presented in fig. 5 next we checked the 5′-nucleotide distribution of the new mirna families in our libraries. seven started with uracil at their 5′-end. eight mirna families exhibited a preference for an adenine at position although six of these have a different nucleotide composition at position to a variable degree. in three mirna families the most frequently found first nucleotide at their 5′-end was cytosine. one mirna family exhibited a preference for a guanine at position although a significant fraction has uracil at the 5′-position. although the majority of plant mirnas have a uracil at position it is not unusual that some mirna families contain adenine, cytosine and even guanine in significant proportions at the 5′-position. young mirnas are often weakly expressed, processed imprecisely or lack targets. our data fits well with these previous observations since most of the n. benthamiana specific mirnas showed low relative abundance in our libraries and in several cases they were accompanied by additional size classes for various degrees. furthermore, some of the new mirnas identified in our srna libraries are produced from long hairpin precursors that may also indicate the young evolutionary origin. this situation is well observable on the nb_mirc precursor where two mirna duplexes were both identified in the srna libraries and detected by northern blots. however, we did not identify mirnas without target mrnas in our data set, since we used the presence of cleaved target mrna as a criterion to select new mirnas. we used northern blot analysis to check the expression level of n. benthamiana specific mirnas in five tissues. the mirnas that gave signal on the northern blot, in most cases were detectable in all of the tissues tested. some of the new mirnas were abundant in specific tissues, and many of them showed differential expression in the tissues analysed. the relative number of reads in all tissues was very low for most of the mirnas and this was also reflected in the northern blot analysis, since we did not obtain signal for six mirnas. the expression profiles obtained by northern blot analysis were different from the sequencing data for nb_mirc3_5p, nb_mirc6_3p, nb_mirc8_3pa, nb_mirc11_5p and nb_mirc17_5p. however, for the other new mirnas, sequencing data and expression profiles obtained by northern blot analysis showed good correlation. although the relative read numbers for most of the new mirnas were low, we were able to confirm the presence of 13 n. benthamiana specific mirnas by northern blot.fig. expression patterns of novel mirnas found in n. benthamiana. total rna was extracted from different tissues including, seedling, root, leaf, stem, flower from n. benthamiana plants used in our laboratory and from plants from boyce thompson institute. the rna was separated on page and transferred to nylon membranes for northern blot analysis of the novel mirnas. oligonucleotide probes were used to detect specific mirnas, and an u6-specific probe was used to detect u rna as a loading control for each membrane targets of n. benthamiana specific mirnas we identified targets for new mirnas using our degradome libraries. interestingly, in the case of nb_mirc we found target for both the mature and the star strand of the new mirna. based on the degradome data, among the identified target mrnas of the specific, new n. benthamiana mirnas, three were classed as category two as category twenty-two as category and five as category comparing the target list of the known and new mirnas the most obvious difference between them is that the known mirna targets mostly belong to category and. based on their molecular function both groups regulate various genes that are included in transcriptional regulation or enzymatic activity.table targets of novel mirnas found in nicotiana benthamiana a the abundance for the degradome fragment indicating cleavage at that position/total number of fragments x,000 the new mirnas target different genes with a wide variety of predicted functions. one of the three identified category targets of the new mirnas is the mrna of the heterotrimeric gtp binding protein alpha subunit. g proteins are highly conserved in eukaryotes. plant g proteins play an important regulatory role in multiple physiological processes. they are involved in signal transduction from hormone receptors, including the plant hormones gibberellin and abscisic acid that regulate gene expression; secretion, defence, stomata movements, channel regulation, sugar sensing and cell death. the heterotrimeric g protein complex is minimally composed of three subunits: alpha, beta and gamma. plants have only a small number of genes encoding the g proteins subunits. arabidopsis has only one canonical g protein alpha subunit, one beta subunit and three gamma subunit. plant alpha subunits are highly conserved between species, it binds and hydrolyse gtp and relay signals by interacting with downstream effector proteins. interestingly, during convergent evolution human g protein alpha- expression is also regulated by mirna in breast cancer cells. another category target is the mrna of peroxisome biogenesis factor. it is cleaved by nb_mirc5_3p and has been demonstrated to be a component of the peroxisomal matrix protein import machinery. peroxisomes are eukaryotic organelles and they are involved in a wide range of processes. plant peroxisomes are important in primary and secondary metabolism, development, and responses to abiotic and biotic stresses. previous studies identified pex genes in the arabidopsis genome and demonstrated that they play a pivotal role in the import of peroxisomal matrix proteins. pex perform multiple functions, including the biogenesis of er-derived protein and oil bodies, the maintenance of er morphology, the formation of cuticular wax, peroxisome and chloroplast connections. one of the category targets is repressor of silencing which is cleaved by nb_mirc1_3p. interestingly, the sequence of nb_mirc1_3p can also be found in nicotiana attenuata and its target site on ros is conserved in other nicotiana species as well. ros encodes a nuclear dna glycosylase domain protein that catalyse the release of 5-methylcytosine from dna via a base excision repair reaction. dna cytosine methylation is an important epigenetic mark conserved in many eukaryotes. it has important roles in transposon silencing, genome organization, genomic imprinting and regulation of gene expression. during continuous adaptation to variable external and internal environments dna methylation patterns are dynamically controlled by methylation and demethylation reactions. ros plays an important role in active dna demethylation and helps to maintain gene expression. the targeting of ros by a mirna provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of its activity. the targeting of a dna glycosylase by a mirna was first predicted in arabidopsis and later it was shown that the expression of demeter-like protein mrna was down-regulated in mir402-overexpressing transgenic plants. the other category target is a zinc-regulated transporter, iron-regulated transporter -like protein and is cleaved by nb-mirc12_5p. zrt/irt-like protein family has been demonstrated to be involved in metal uptake and transport. zrt/irt-like proteins generally contribute to metal ion homeostasis by transporting cations into the cytoplasm. it has been reported in several plant species that various mirnas are vital for maintaining nutrient homeostasis by regulating the expression of transporters that are involved in nutrient uptake and mobilization. other targets that are worthy to note are the wat1-related protein at5g40240-like gene targeted by nb_mirc7_3p; the cbl-interacting serine/threonine-protein kinase gene targeted by nb_mirc4_3p, proteasome subunit alpha type-2-a gene targeted by nb_mirc7_3p, suberization-associated anionic peroxidase targeted by nb_mirc10_3p and beta-amyrin synthase targeted by nb_mirc10_3p.  in this study we have confirmed the expression of known and new mirnas in various tissues of n. benthamiana using high-throughput sequencing and northern blot analysis. as a result, this work represents a tissue specific atlas of known and new mirna expression of n. benthamiana. in addition, we have experimentally identified many targets of both known and new mirnas using a high-throughput method for the global identification of mirnas targets. this study provides a valuable resource of mirnas and their validated target mrnas to the plant research community that will be beneficial well into the future. 
the conserved and novel mirna targets were found to be involved in various biological processes including transcription, rna binding, dna modification, signal transduction, stress response and metabolic process. it can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. to discover and characterize the mirnas and their cleaved target mrnas in n. benthamiana, we sequenced small rna transcriptomes and degradomes of two n. benthamiana accessions and validated them by northern blots. regulation of ros by a mirna provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ros activity. the identified conserved and novel mirnas along with their target mrnas also provides a tissue specific atlas of known and new mirna expression and their cleaved target mrnas of n. benthamiana. nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. we used a comprehensive molecular approach to detect and to experimentally validate n. benthamiana mirnas and their target mrnas from various tissues. we identified conserved mirna families and novel microrna candidates and validated their target mrnas with a genomic scale approach. the accumulation of thirteen novel mirnas was confirmed by northern blot analysis. nicotiana benthamianamicrornanon-coding rnassmall rnadegradomehigh throughput sequencingissue-copyright-statement© the author 2015 thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.
 nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. it can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. to discover and characterize the mirnas and their cleaved target mrnas in n. benthamiana, we sequenced small rna transcriptomes and degradomes of two n. benthamiana accessions and validated them by northern blots.  we used a comprehensive molecular approach to detect and to experimentally validate n. benthamiana mirnas and their target mrnas from various tissues. we identified conserved mirna families and novel microrna candidates and validated their target mrnas with a genomic scale approach. the accumulation of thirteen novel mirnas was confirmed by northern blot analysis. the conserved and novel mirna targets were found to be involved in various biological processes including transcription, rna binding, dna modification, signal transduction, stress response and metabolic process. among the novel mirna targets we found the mrna of repressor of silencing. regulation of ros by a mirna provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ros activity.  the identified conserved and novel mirnas along with their target mrnas also provides a tissue specific atlas of known and new mirna expression and their cleaved target mrnas of n. benthamiana. thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nicotiana benthamianamicrornanon-coding rnassmall rnadegradomehigh throughput sequencingissue-copyright-statement© the author 2015 