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21 publications mentioning sly-MIR482d

Open access articles that are associated with the species Solanum lycopersicum and mention the gene name MIR482d. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 342
To determine whether this down-regulation of ghr-miR482/miR482.2 caused up-regulation of their possible NBS-LRR targets, expression changes of 11 NBS-LRR genes that were predicted targets of ghr-miR482/miR482.2 were analysed. [score:13]
Furthermore we found that V. dahliae infection resulted in down-regulation of some members of the ghr-miR482 family and up-regulation of their NBS-LRR targets (Figures 6– 7). [score:9]
Four members of the ghr-miR482 family and several NBS-LRR genes were down-regulated and up-regulated in cotton seedlings infected with V. dahliae, respectively, implying that miR482 -mediated silencing of NBS-LRR genes is released in cotton upon fungal pathogen infection to activate disease defense. [score:9]
Expression of miR482 was found to be suppressed in tomato plants infected with viruses or bacteria, and consequently some of its disease resistant NBS-LRR target genes were induced two to three-folds [12]. [score:9]
Therefore, miR482-, miR2118- and nta-miR6019 -mediated cleavage of target disease resistance genes is expected to cause not only decay of their target mRNAs but also production of phased secondary small RNAs from the targeted R-genes. [score:9]
Expression Changes of miR482 and NBS-LRR Genes upon V. dahliae InfectionTo know whether, like in tomato plants infected with viruses or bacteria, the expression levels of miR482 were down-regulated in cotton plants infected with fungal pathogen V. dahliae by root dipping (Figure S6), we first performed small RNA northern blots using RNA isolated from the susceptible Sicot71 cultivar (G. hirsutum). [score:8]
Of these 36 candidate NBS-LRR targets, 16 were uniquely targeted by only one member of the gra-miR482 family and 20 were targeted by at least two members (Table S1). [score:7]
N = A or C or G or T; R = A or G; H = A or C or T; Y = C or T; M = A or C. Generation of phased Secondary Small RNAs in NBS-LRR GenesTo further confirm the authenticity of the predicted targets and to know whether phased secondary small RNAs were generated from the cleaved targets, we mapped four publically available cotton small RNA datasets (GSM699074-GSM699077 from V. dahliae-infected and mock -treated roots of G. hirsutum and G. barbadense) onto the G. raimondii genome and analysed the distribution of small RNAs across all the predicted targets of ghr-miR482/miR482.2 using the method previously described [17], [30]. [score:7]
miR482 and miR2118 are partially overlapping and both were predicted to target the conserved sequences encoding the P-loop motif of the NBS-LRR receptors [10], [11], [12], so would be expected to suppress the expression of large numbers of NBS-LRR defense genes. [score:7]
Of the 36 predicted NBS-LRR targets, only two are targets of ghr-miR482b and both are better targeted by other members of the ghr-miR482 family (Table S1). [score:7]
Identification of Gra-miR482 Targets in the G. raimondii Genome and Confirmation of ghr-miR482/miR482.2 -mediated Cleavage of NBS-LRR GenesPutative targets of gra-miR482/miR482.2 were first predicted based on the annotated G. raimondii transcripts [24] using psRNATarget (http://plantgrn. [score:7]
N = A or C or G or T; R = A or G; H = A or C or T; Y = C or T; M = A or C. To further confirm the authenticity of the predicted targets and to know whether phased secondary small RNAs were generated from the cleaved targets, we mapped four publically available cotton small RNA datasets (GSM699074-GSM699077 from V. dahliae-infected and mock -treated roots of G. hirsutum and G. barbadense) onto the G. raimondii genome and analysed the distribution of small RNAs across all the predicted targets of ghr-miR482/miR482.2 using the method previously described [17], [30]. [score:7]
These results suggest that the NBS-LRR target genes annotated in G. raimondii are conserved in G. hirsutum, and that an induced expression of NBS-LRR target genes in V. dahlia-infected G. hirsutum was a result of repression of ghr-miR482/miR482.2 biogenesis. [score:7]
NBS-LRR Defense Genes are Targets of miR482 in CottonIn tomato and M. truncatula, miR482 and miR2118 have been shown to regulate numerous NBS-LRR defense genes through targeting the conserved sequences encoding the P-loop motif of the NBS-LRR receptors [10], [12]. [score:6]
To know whether, like in tomato plants infected with viruses or bacteria, the expression levels of miR482 were down-regulated in cotton plants infected with fungal pathogen V. dahliae by root dipping (Figure S6), we first performed small RNA northern blots using RNA isolated from the susceptible Sicot71 cultivar (G. hirsutum). [score:6]
Plants have thus evolved the miR482- NBS-LRR regulatory loop as a counter mechanism to minimize the cost of over -expression of NBS-LRR genes in the absence of a pathogen, and to ensure rapid induction of disease resistance proteins upon pathogen attack. [score:6]
Of the 36 candidate NBS-LRR targets, the majority had small RNAs mapped to, and nine showed a phased distribution of those small RNAs (phase score >2) spreading downstream from the primary target site of ghr-miR482/miR482.2 (Figures 5, S5). [score:5]
Putative targets of gra-miR482/miR482.2 were first predicted based on the annotated G. raimondii transcripts [24] using psRNATarget (http://plantgrn. [score:5]
Plants infected with viruses or bacteria showed a reduced level of miR482 and an increased level of mRNAs of miR482 targets, suggesting that the miR482 -mediated silencing cascade is suppressed by pathogen attack and may be a defense response of plants [12]. [score:5]
In this study, we characterized the cotton miR482 family using published small RNA datasets [10], [26] and the genome sequence of G. raimondii [24], identified G. raimondii NBS-LRR genes potentially targeted by miR482/miR482.2, and analyzed V. dahliae -induced expression changes of miR482/miR482.2 and their predicted NBS-LRR targets in G. hirsutum. [score:5]
0084390.g007 Figure 7Expression analysis of NBS-LRR genes targeted by ghr-miR482/miR482.2. [score:5]
Expression analysis of NBS-LRR genes targeted by ghr-miR482/miR482.2. [score:5]
In conclusion, on the basis of characterization of the miR482 family and identification of NBS-LRR genes targeted by ghr-miR482/miR482.2, we demonstrated that V. dahliae infection represses certain members of the miR482 family and induces expression of specific disease resistance NBS-LRR genes in cotton. [score:5]
Based on this observation and on the experiment, in which N. benthamiana mRNAs encoding NBS-LRR proteins were found to be silenced by tomato miR482, the authors have proposed that plants are able to exploit virus- and bacterium-derived suppressors of RNA silencing to induce expression of defense-related genes to achieve non-race-specific resistance against viral and bacterial pathogens [12]. [score:5]
The miR482 family is thus expected to regulate the expression level of a number of NBS-LRR genes. [score:4]
In tomato and M. truncatula, miR482 and miR2118 have been shown to regulate numerous NBS-LRR defense genes through targeting the conserved sequences encoding the P-loop motif of the NBS-LRR receptors [10], [12]. [score:4]
On the other hand, the number of NBS-LRR genes regulated by miR482 in each species could be evolutionally determined by the balance between minimizing the disadvantageous effect of over -expression of NBS-LRR genes in the absence of a pathogen and maximizing the induction of NBS-LRR genes in the presence of a pathogen. [score:4]
Furthermore, at least one of the secondary small RNAs generated from a miR482 targeted NBS-LRR gene has been shown to target mRNA encoding another defense-related protein [12], indicating that the secondary small RNAs generated from NBS-LRR genes can possess the characteristic of trans-acting siRNAs (ta-siRNAs) [16], [17]. [score:3]
Expression Changes of miR482 and NBS-LRR Genes upon V. dahliae Infection. [score:3]
All seven members of the ghr-miR482 family (Figure 1) are found in G. arboreum [10], suggesting that they may also be expressed from the A [t] genome of G. hirsutum. [score:3]
Further bioinformatic mining identified several miRNAs, including miR482 and miR2118, which target members of different R-gene families in tomato, potato, soybean and Medicago truncatula [10], [11]. [score:3]
Release of the G. raimondii genome sequence provided us the opportunity to estimate the number of NBS-LRR genes in cotton and how many are targets of miR482. [score:3]
2 in roots (Figures 6C, 6D, 6F, 6G), whereas the expression levels of ghr-miR482a, ghr-miR482d and ghr-miR482e. [score:3]
P-loops of the predicted targets of ghr-miR482/miR482.2 in cotton (G. raimondii). [score:3]
A) The P-loop sequence logo generated using all predicted NBS-LRR targets of ghr-miR482/miR482.2 in G. raimondii. [score:3]
This is supported by the observation that both the miR482 sequences and the predominant amino acid sequences of the P-loops of NBS-LRR proteins in cotton are different from those in tomato, soybean and M. truncatula [10], [12], a result of co-evolution of miR482 and their potential NBS-LRR targets in each species. [score:3]
Because of similar sequences between miR482 and miR482.2, we reasoned that the expression of miR482 and miR482.2 could not be unambiguously detected by the miR482 and the miR482.2 antisense probe, respectively; therefore, both probes were used together. [score:3]
B to H) Stem-loop qRT-PCR analysis of the expression level of individual members of the ghr-miR482 family. [score:3]
When we were performing northern blot analysis, we assumed that the overall expression level of all members of the ghr-miR482 family could be detected by using one representative member of ghr-miR482 and ghr-miR482.2 (ghr-miR482a and ghr-miR482e. [score:3]
Identification of Gra-miR482 Targets in the G. raimondii Genome and Confirmation of ghr-miR482/miR482.2 -mediated Cleavage of NBS-LRR Genes. [score:3]
The P-loop sequence of the NBS-LRR genes targeted by members of the ghr-miR482 family is shown in Figure 4A. [score:3]
In rice, miR2118 is highly expressed in developing inflorescence and triggers production of phased siRNAs [31], [32] but no miR482 has been reported. [score:3]
NBS-LRR Defense Genes are Targets of miR482 in Cotton. [score:3]
It is clear that the last three amino acids GKT that complement the first seven nucleotides of ghr-miR482 were completely conserved in all predicted NBS-LRR targets. [score:3]
We found that ∼12% of NBS-LRR genes in the G. raimondii genome are potential targets of the miR482 family, and that 10 of the 11 analysed could be induced upon V. dahliae infection. [score:3]
We found that 36 (12%) of these 300 NBS-LRR genes were potentially targeted by various numbers of the gra-miR482 (or ghr-miR482) family members. [score:3]
Expression analysis of ghr-miR482/miR482.2. [score:3]
One possibility is that the miR482-target sites of Gorai. [score:3]
According to the consensus nucleotide sequences of the top three P-loops that were found in the majority of NBS-LRR genes targeted by ghr-miR482, a variable nucleotide was always observed at the 3 [rd] position of a codon (Figure 4B). [score:3]
0084390.g004 Figure 4P-loops of the predicted targets of ghr-miR482/miR482.2 in cotton (G. raimondii). [score:3]
Four predicted targets of ghr-miR482/miR482.2 were selected for cleavage analysis using the approach of rapid amplification of cDNA ends or 5′ RACE. [score:3]
We then performed the more sensitive miRNA stem-loop qRT-PCR to analyze the expression levels and changes of individual members of the ghr-miR482 family in response to V. dahliae infection. [score:3]
We found that about 12% of the G. raimondii NBS-LRR genes are potential targets of miR482/miR482.2, and that miR482 -mediated cleavage of NBS-LRR genes triggers production of phased secondary small RNAs. [score:3]
In addition, several genes with a diverse function, including a gene encoding a MYB-domain containing protein, were also predicted to be targeted by member(s) of the miR482 family (Table S1). [score:3]
B) Top three types of consensus nucleotide sequences of the predicted ghr-miR482/miR482.2 targets and their alignments with ghr-miR482/miR482.2. [score:3]
It could be that these ghr-miR482d isoforms are expressed at very low levels or in tissues that have not been used in generating the small RNA data. [score:3]
Table S1 Predicted targets of miR482/miR482.2 and miRCan1 in G. raimondii. [score:3]
007G319800 was found to be targeted by ghr-miR482d. [score:3]
We confirmed some of the predicted targets were cleaved by ghr-miR482/miR482.2 (Figure 3) and found that miR482 -mediated cleavage of NBS-LRR genes triggers production of phased siRNAs (Figures 5, S5). [score:3]
009G033000 were confirmed to be targets of ghr-miR482 whereas Gorai. [score:3]
The target site (P-loop) of miR482 is one of the conserved motifs in the NBS-LRR proteins. [score:3]
Gain and loss of regulation of NBS-LRR genes by miR482 could be an on-going evolutionary event because for each type of P-loop the nucleotide variation was always found at the synonymous 3 [rd] position of a codon (Figure 4). [score:2]
This suggests that the miR482- NBS-LRR regulatory loop is part of the immune responses induced not only by viral and bacterial pathogens but also by fungal pathogens. [score:2]
These characteristics of the target sites suggest that gain and loss of regulation of NBS-LRR genes by ghr-miR482 is possible without changes to the protein sequence. [score:2]
Furthermore, diverse and finely controlled biogenesis of miR482/miR482.2 seems to play a role in the modulation of miR482 -mediated regulation of NBS-LRR genes. [score:2]
The miR482 -mediated regulation of NBS-LRR genes seems to be finely modulated in a few different ways. [score:2]
Identification of MIR482 in the G. raimondii Genome G. raimondii short sequences with 0–1 mismatch in comparison to their closest member of the miR482 family of G. hirsutum were first identified using each ghr-miR482 or ghr-miR482.2 as a query. [score:1]
The expected cleavage sites of ghr-miR482 and ghr-miR482.2 were indicated by pink and blue arrows, respectively. [score:1]
Three more members of this family, ghr-miR482a and ghr-miR482b in G. hirsutum and gra-miR482 in G. raimondii, were later reported [21]. [score:1]
Confirmation of ghr-miR482/miR482.2 -mediated cleavage of NBS-LRR genes was carried out using total RNA by 5′ RACE (rapid amplification of cDNA ends) as previously described [36]. [score:1]
2, suggesting that production of the phased small RNAs was triggered by ghr-miR482d -mediated cleavage. [score:1]
2 and ghr-miR482c in both leaves and roots, as well as for ghr-miR482d. [score:1]
B) Distribution pattern of small RNAs generated from the region immediately after the P-loop, which was cleaved by ghr-miR482d. [score:1]
We named these two as ghr-miR482d. [score:1]
009G033000, apart from the ghr-miR482 -mediated cleavage, additional cleavage sites located downstream and in phase with the cleavage sites of ghr-miR482d were found (Figure 3), further suggesting that these genes behave like TAS loci in Arabidopsis. [score:1]
2 and ghr-miR482d. [score:1]
When allowing one mismatch, we found five more loci each containing a gra-miR482d isoform and predicted to form a hairpin structure in the G. raimondii genome (Figure S2); however, these new gra-miR482d isoforms were not found in any of the published cotton (G. hirsutum, G. barbadense and G. arboreum) small RNA datasets so are not further analysed. [score:1]
According to the published cotton small RNA data [10], [20], [21], [22], [26] we analysed, the miR482 family of G. hirsutum has at least four members, and two members (ghr-miR482b and ghr-miR482d) have a corresponding ghr-miR482.2. [score:1]
Figure S1 Precursors of the members of the gra-miR482 family. [score:1]
2. One is identical to ghr-miR482d except for the last two nucleotides; another has no corresponding ghr-miR482 sequence found in G. hirsutum. [score:1]
0084390.g001 Figure 1Alignment of G. hirsutum miR482/miR482.2 members. [score:1]
011G075600 is shown in Figure 5. In this case, the 5' end of the first phased 21-nt small RNA was aligned with the expected cleavage site (the 10 [th] nucleotide counting from the 5' end) of ghr-miR482d rather than that of ghr-miR482d. [score:1]
Arabidopsis has no miR482 annotated, but its miR472 is closely related to miR482 found in other species. [score:1]
0084390.g003 Figure 3Ghr-miR482/miR482.2 -mediated cleavage of NBS-LRR genes. [score:1]
Distribution of small RNAs from the first four phases after the ghr-miR482d cleavage site in Gorai. [score:1]
Of the members of the gra-miR482 family, gra-miR482b/miR482b. [score:1]
The expected cleavage sites of ghr-miR482d and ghr-miR2118d are indicated by blue and pink arrow, respectively. [score:1]
Ghr-miR482/miR482.2 -mediated cleavage of NBS-LRR genes. [score:1]
In addition, we identified at least five precursors of new isoforms of ghr-miR482d in the G. raimondii genome, but these isoforms were not found in the G. hirsutum small RNA datasets we analysed. [score:1]
0084390.g005 Figure 5Ghr-miR482d trigged production of phased secondary small RNAs in Gorai. [score:1]
0084390.g006 Figure 6A) Northern blot detection of ghr-miR482/482.2 in leaves collected from V. dahliae-infected and mock -treated cotton plants at one day-post-inoculation (1 dpi). [score:1]
gov/geo/and used in identification of miR482 and miR482.2. [score:1]
The miR482 reported by Wang et al. [22] is a variant of ghr-miR482b because its 1 [st] – 20 [th] nucleotides are identical to the 3 [rd] – 22 [nd] nucleotides of ghr-miR482b (Figure 1). [score:1]
Interestingly, some MIR482 loci are able to produce a miR482.2 variant, whose 1 [st]–20 [th] nucleotides are overlapping with the 3 [rd]–22 [nd] nucleotides of miR482. [score:1]
The red and blue arrows indicate the expected cleavage position of ghr-miR482 and ghr-miR482.2, respectively. [score:1]
By searching the published cotton small RNA datasets, we found that gra-miR482 also exists in G. hirsutum; therefore, there are at least four miR482 members in the G. hirsutum genome. [score:1]
Although ghr-miR482b and ghr-miR482c each have a single match in the G. raimondii genome, ghr-miR482d has five matches (gra-miR482d, f-i), suggesting that ghr-miR482d could be generated from multiple loci in the D [t] genome of G. hirsutum. [score:1]
The sequences underneath the pink and blue lines are ghr-miR482d and ghr-miR2118d, respectively. [score:1]
There are three mismatches between ghr-miR482a and ghr-miR482b/miR482d, although only one mismatch between ghr-miR482a and ghr-miR482c. [score:1]
A) Northern blot detection of ghr-miR482/482.2 in leaves collected from V. dahliae-infected and mock -treated cotton plants at one day-post-inoculation (1 dpi). [score:1]
miR482 stem-loop qRT-PCR was performed according to the approach reported previously [38]. [score:1]
miR482 is a diversified miRNA family whose abundance varies significantly among different plant species. [score:1]
The Cotton miR482 Family. [score:1]
The first member of the cotton miR482 family was identified in G. hirsutum [20]. [score:1]
Identification of MIR482 in the G. raimondii Genome. [score:1]
007G320500, ghr-miR482 -mediated cleavage was also confirmed by cleavage analysis (Figure 3). [score:1]
2, gra-miR482d. [score:1]
They were named as gra-miR482d, f, g, h, and i (Figure 2A; Figure S1). [score:1]
Alignment of G. hirsutum miR482/miR482.2 members. [score:1]
Individual members of the miR482 family behaved differently in response to V. dahliae infection. [score:1]
Ghr-miR482d trigged production of phased secondary small RNAs in Gorai. [score:1]
To determine whether these G. hirsutum miRNAs exist in the G. raimondii genome, and how many loci are able to generate these miRNAs in the G. raimondii genome, we searched the G. raimondii genome for sequences that exactly match with ghr-miR482 and predicted hairpin structure using the genomic sequence surrounding the miRNA. [score:1]
The sequence identical to ghr-miR482d is detected at five loci that are able to form a stem-loop structure in the G. raimondii genome. [score:1]
21-nt small RNAs corresponding to the first phased register of ghr-miR482 were also observed in Gorai. [score:1]
miR482, miR2118 and nta-miR6019 belong to a specific type of miRNA that is 22-nt long and generated from pre-miRNAs containing asymmetric bulges in the miRNA/miRNA* duplex. [score:1]
By blasting the G. raimondii genome sequence, we found that ghr-miR482a has no homolog and thus could be a newly evolved ghr-miR482 member in G. hirsutum. [score:1]
Figure S2 Precursors of the isoforms of gra-miR482d and gra-miR482f-i (one mismatch with gra-miR482d, f-i). [score:1]
Some MIR482 loci generate both miR482 and its variant miR482.2, and some generate only either miR482 or miR482.2. [score:1]
The sequences of ghr-miR482d and ghr-miR482d. [score:1]
G. raimondii short sequences with 0–1 mismatch in comparison to their closest member of the miR482 family of G. hirsutum were first identified using each ghr-miR482 or ghr-miR482.2 as a query. [score:1]
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[+] score: 178
Other miRNAs from this paper: sly-MIR482e, sly-MIR482a, sly-MIR482b, sly-MIR482c, sly-MIR390a
While this does not exclude a plant -mediated downregulation of miR482/2118, the downregulation of the non- NBS-LRR regulating miRNAs (electronic supplementary material, figure S4), indicates that pathogen -mediated RNA silencing suppression may play a role here. [score:10]
Combining comparative expression analyses of members of the miR482/2118 family in three closely related tomato species (S. lycopersicum, Solanum pimpinellifolium and Solanum arcanum) and analyses of host resistance led to two observations: (i) the least resistant tomato, S. lycopersicum, showed downregulation of several miRNAs from 24 to 96 hours post-inoculation (hpi) relative to the mock control, while its more resistant wild relatives did not and (ii) downregulation of miR482a and miR482f during early time-points of infection (6 hpi) correlated with resistance to P. infestans. [score:9]
Although the expression of NBS-LRRs is undoubtedly regulated by multiple mechanisms in addition to negative regulation via miRNAs, we observe examples of strong co-regulation between members of miR482/2118 and their targets. [score:8]
Targeting of miR482/2118 leads either to the degradation of NBS-LRR mRNA or to an inhibition of the translation of the corresponding mRNAs [14]. [score:7]
These differences in co-regulation between wild and cultivated tomatoes could result from (i) differences in the evolutionary history of these plants (i. e. artificial versus natural selection) that brought about a more streamlined regulation of expression of miR482/2118 in S. lycopersicum or (ii) greater sensitivity to pathogen manipulation of host RNA silencing in S. lycopersicum, for example, due to pathogen-secreted RNA silencing suppressors. [score:7]
An effective plant resistance response during the necrotrophic phase may include the suppression of cell death-inducing proteins, such as R-proteins, perhaps through an upregulation of miR482/2118. [score:6]
Given that a third of the miR482/2118 potential targets in S. lycopersicum are associated with disease resistance to P. infestans in S. lycopersicum, we chose a subset of 11 NBS-LRRs associated with P. infestans resistance and Solyc02g036270.2.1 (as a positive control for cleavage, but a negative control in terms of P. infestans resistance) to study the co-regulation of NBS-LRRs and miR482/2118 in this interaction. [score:6]
By contrast, if pathogen -mediated RNA silencing suppression were effective at these later time-points, one would expect a downregulation of miRNAs, including miR482/2118. [score:6]
Such differences in co-regulation suggest that despite active targeting by miR482/2118 in S. lycopersicum, NBS-LRRs are likely to be regulated by other mechanisms in addition to the regulation by miR482/2118. [score:6]
Next, we evaluated how often pairs of miR482/2118 and NBS-LRRs are co-regulated and what type of co-regulation they are subjected to (i. e. negative co-regulation, positive co-regulation or no differential regulation of both miRNA and target). [score:6]
We found that co-regulation of miR482/2118 with their targets was time -dependent, and more prevalent at time-points critical for infection success and transitions in the pathogen's life cycle. [score:4]
cDNA libraries for mature miR482/2118 expression analyses were created using miScript Plant RT Kit (Qiagen, Hilden, Germany) using 250 ng total RNA and diluted 1 : 10 with nuclease-free H [2]O. cDNA libraries for all other expression analyses were created with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania) using 1000 ng total RNA and random hexamer primers and libraries were diluted 1 : 1 with nuclease-free H [2]O. cDNA libraries for the modified 5'RNA ligase -mediated rapid amplification of cDNA ends (5′RLM-RACE) were created using the GeneRacer Kit (Invitrogen, California, USA) using 50–100 ng mRNA from infections (24 and 48 hpi) and mock (48 hpi). [score:4]
One example of negative regulation of R-genes, specifically of nucleotide -binding site leucine-rich repeats (NBS-LRRs), is suppression by the microRNA (miRNA) family miR482/2118 [11– 13]. [score:4]
cDNA libraries for mature miR482/2118 expression analyses were created using miScript Plant RT Kit (Qiagen, Hilden, Germany) using 250 ng total RNA and diluted 1 : 10 with nuclease-free H [2]O. cDNA libraries for all other expression analyses were created with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania) using 1000 ng total RNA and random hexamer primers and libraries were diluted 1 : 1 with nuclease-free H [2]O. cDNA libraries for the modified 5'RNA ligase -mediated rapid amplification of cDNA ends (5′RLM-RACE) were created using the GeneRacer Kit (Invitrogen, California, USA) using 50–100 ng mRNA from infections (24 and 48 hpi) and mock (48 hpi). [score:4]
Relative expression (log2) of potential NBS-LRR targets of miR482/2118 in infected compared with mock-control plants of S. lycopersicum. [score:4]
Across-species comparisons of the gene expression of mature miR482/2118 and of the strength of resistance led to two main conclusions: (i) Co-evolution of P. infestans and S. lycopersicum may have resulted in a more efficient pathogen -mediated RNA silencing suppression compared with its more resistant sister species; and (ii) miR482a and miR482f could be identified as candidate miRNAs for mediating the resistance response of tomatoes to P. infestans. [score:4]
Compared with S. lycopersicum, expression between pairs of miRNAs was significantly less correlated in the wild tomatoes: in S. pimpinellifolium pairs of miR482/2118 members showed the same expression pattern at 2.2 ± 1.2 time-points (p-value = 0.012; figure 2 b) and in S. arcanum at 2.2 ± 0.9 time-points (p-value = 0.005; figure 2 c). [score:4]
Given the substantial co-regulation of miRNAs and their targets at 48 and 72 hpi, we evaluated the association of miR482/2118 expression with the life cycle progression of P. infestans on its hosts. [score:4]
Therefore, it has been hypothesized that the regulation of R-genes by miR482/2118 may have evolved into a pathogen detection mechanism, i. e. a counter-defence mechanism by which pathogen -mediated RNA silencing suppression activates the plant immune system [13]. [score:4]
Therefore, approximately 33% of the predicted direct NBS-LRR targets of miR482/2118 are associated with resistance to P. infestans. [score:4]
Previous studies have used to test whether the expression of NBS-LRRs is regulated by members of the miR482/2118 gene family [13– 14]. [score:4]
By contrast, the cultivated tomato appears to have a more global co-regulation of miR482/2118 expression. [score:4]
Co-regulation of members of miR482/2118 and their nucleotide -binding site leucine-rich repeats targets is time -dependent. [score:4]
We first identified miR482/2118 targets associated with P. infestans defence in S. lycopersicum. [score:3]
We screened for NBS-LRRs that are potential targets of miR482/2118 and classified as R-genes for P. infestans (electronic supplementary material, table S2). [score:3]
We observed that the direction of co-regulation is not static for every miR482/2118– NBS-LRR combination but can shift between time-points. [score:3]
Nucleotide -binding site leucine-rich repeats are targeted by miR482/2118 in Solanum lycopersicum during infection by Phytophthora infestans infection. [score:3]
In summary, we demonstrate that targeting of NBS-LRRs by miR482/2118 is effective in pathogen-challenged and unchallenged plants. [score:3]
In addition, we tested Solyc02g036270.2.1, because it is a functional miR482/2118 target [13] that is not associated with P. infestans resistance (figure 1; electronic supplementary material, table S2). [score:3]
Next, we examined the relationship between the expression of miR482/2118 miRNAs and the life cycle of P. infestans. [score:3]
Figure 1. Targeting of NBS-LRRs by miR482/2118 family members in S. lycopersicum. [score:3]
All of these members of miR482/2118 have targets associated with P. infestans defence in S. lycopersicum (electronic supplementary material, table S2). [score:3]
We determined at which time-points co-regulation was most prevalent, suggesting a potential influence of miR482/2118 on NBS-LRR-regulation. [score:3]
Figure 2. Expression of miR482/2118 family members in S. lycopersicum, S. pimpinellifolium and S. arcanum. [score:3]
We chose R-genes that were (i) predicted to be targeted by one or more members of miR482/2118 and (ii) associated with resistance to P. infestans. [score:3]
To test whether these NBS-LRRs are targeted by miR482/2118 in S. lycopersicum, we created libraries from S. lycopersicum infected with the pathogen and mock -treated (figure 1). [score:3]
The 52 potential miR482/2118 target genes [15] were used as queries for a BLASTn-search against the NCBI nr/nt database limited to S. lycopersicum. [score:3]
We quantified the expression of the seven members of miR482/2118 and 12 NBS-LRRs in infected and uninfected plants across five time-points (6 to 96 hpi) (figures  2 a and 3). [score:3]
Relative expression (log2) in infected compared with mock-control plants of S. lycopersicum (a), S. pimpinellifolium (b) and S. arcanum (c) of the seven miR482/2118 family members at 6, 24, 48, 72 and 96 hpi relative to mock control. [score:2]
All three tested genes revealed a cleavage site in the region complementary to the miR482/2118 sequences. [score:1]
Identification of miR482/2118 family members. [score:1]
The best hits in S. arcanum were aligned to the SlmiR482/2118 precursor sequences and the mature miR482/2118 sequences were determined. [score:1]
Members of miR482/2118 from S. arcanum were identified via a BLASTn against the S. arcanum genome using miR482/2118 precursor sequences of S. lycopersicum as query. [score:1]
Members of miR482/2118 from S. lycopersicum and S. pimpinellifolium have been previously identified in de Vries et al. [15]. [score:1]
This is a significantly lower co-regulation compared to that observed for miR482/2118 (p-value = 0.0002). [score:1]
Folding of S. arcanum miR482/2118 precursors into hairpins was predicted using RNAfold [34] (electronic supplementary material, figure S1). [score:1]
In this study, we investigated the expression of miR482/2118 during the infection of P. infestans on three different tomato species. [score:1]
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[+] score: 49
Therefore, the collective regulation by miR482, miR6024 as well as tasiRNAs triggered by them may control the expression level of I2 homologues in Solanaceae, and the down-regulation of the I2 homologues by these miRNAs may make the expansion of I2 homologues less costly in fitness. [score:7]
Therefore, the I2 homologues from tomato are targeted by miR6024 and may not be targeted by miR482. [score:5]
In addition to the miR482 family targeting I2 homologues in potato [33], we identified and confirmed that miR6024 targets I2 family in tomato. [score:5]
The miR482 family has high expression level in three tissues of potato (leaf, flower and stolon) (five members, with an average of 35,760 reads per million), while miR6024 family has a much lower expression in tomato (leaf, flower and fruit) (2,540 reads per million). [score:5]
We hypothesized that miR6024 and miR482 might have facilitated the expansion of the I2 family in Solanaceae species, since they can minimize their potential toxic effects by down -regulating their expression. [score:4]
Identification of miRNAs for the I2homologuesThe resistance gene R3a from potato was shown to be targeted by members of 22-nt miR482 family [33]. [score:3]
The resistance gene R3a from potato was shown to be targeted by members of 22-nt miR482 family [33]. [score:3]
Previous study showed that I2 homologues in potato were targeted by miR482. [score:3]
However, our data showed that I2 homologues in tomato were targeted by miR6024 rather than miR482. [score:3]
However, no cleavage product was detected at the predicted target site of miR482. [score:3]
Computational analysis showed that I2 homologues from tomato may also be targeted by miR482 family, which has about 380 reads per million from three tissues (leaf, flower and fruit) of tomato (miRbase release 20). [score:3]
Whether the I2 family from tomato is regulated by miR482 or other miRNAs remains unknown. [score:2]
However, no cleavage of I2 homologues by miR482 was confirmed in tomato though the miR482 does exist in tomato genome. [score:1]
MiR482 was shown to be an ancient miRNA [64]. [score:1]
The resistance gene R3a from potato was shown to be cleaved by 22-nt miR482 family and produce phasiRNA [33]. [score:1]
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[+] score: 46
Other miRNAs from this paper: sly-MIR482e, sly-MIR482a, sly-MIR482b, sly-MIR482c, sly-MIR398a
FRG3 gene-specific primers are listed in Table 1. Transient Expression of MicroRNAs and FRG3 in Nicotiana benthamiana and Target ValidationDNA fragments encoding sly-miR482d-3p and sly-miR482e-3p and the target gene FRG3 were inserted into vector GATEPEG100. [score:6]
Our previous results demonstrated that slmiR482f (slmiR482e-3p) and slmiR5300, two members of miR482/2118 superfamily, acted on several NBS-LRR targets at either the transcript stability or translational level in tomato (Ouyang et al., 2014). [score:5]
MiRBase21 has seven miR482 entries including miR482a, b, c, d-5p, d-3p, e-5p, and e-3p of which five correspond to miR482s targeting NBS-LRRs and two correspond to the complementary miR [∗] sequences (miRBase21, Kozomara and Griffiths-Jones, 2014). [score:3]
Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation. [score:3]
Moreover, transgenic expression of miR482 causes significant increases of nodule numbers in soybean (Li et al., 2010). [score:3]
The miR482/2118 superfamily has been demonstrated to suppress a wide range of R genes, conferring resistance to fungal, bacterial and viral pathogens. [score:3]
Evolutionarily dynamic, but robust, targeting of resistance genes by the miR482/2118 gene family in the Solanaceae. [score:3]
TABLE S1 Predicted targets of sly-miR482d family. [score:3]
Members of the miR482/2118 superfamily target the P-loop motif in NBS-LRR gene mRNAs (Shivaprasad et al., 2012). [score:3]
For example, many miRNA families, such as the miRNA482/2118 superfamily, target nucleotide -binding site and leucine-rich repeat domain containing proteins (NBS-LRRs) (Zhai et al., 2011; Li et al., 2012; Shivaprasad et al., 2012; Ouyang et al., 2014; Xia et al., 2015). [score:3]
The miR482 family was found to target I2 homologs in potato (Li et al., 2012). [score:3]
We only used those sequences provided by miRBase21with entries as miR482, distinguish between miR482 sequences and their complementary miR [∗] sequences and provide the results from the target prediction for each of the seven miRNAs published as miR482/2118 members in miRBase21 (Dai and Zhao, 2011). [score:3]
FRG3 gene-specific primers are listed in Table 1. DNA fragments encoding sly-miR482d-3p and sly-miR482e-3p and the target gene FRG3 were inserted into vector GATEPEG100. [score:2]
Our previous study reported that slmiR482f (referred to as slmiR482e-3p) and slmiR5300, two members of the miR482/2118 superfamily, regulate resistance to Fusarium oxysporum f. sp. [score:2]
However, no cleavage of I2 homologs by miR482 has been observed in tomato. [score:1]
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[+] score: 31
Three miRNAs (miR394, miR414, miR1917) were down regulated; five miRNAs (miR156, miR159, miR396, miR482, miRZ7) were up regulated, while one miRNA (miR828) whose target is EIN2 was not affected, by exogenous ethylene treatment (Figure 9). [score:5]
Three miRNA families (miR394, miR414 and miR1917) were down regulated, in the contrary, four miRNA families (miR156, miR159, miR396, miR482 and miRZ7) were up-regulated, and however, the miR828 had no obvious change. [score:5]
MiR156 and miR394 were down regulated in the fruit ripening, miR159 showed down regulation in the breaker stage, while, miR396 showed a obvious increase in the breaker stage, miR828 and miR1917 were down regulated in the red ripe stage, miR482 and miRZ7 showed down regulation in the red and softening ripe stage. [score:5]
In addition, targets of the conserved miRNAs include disease resistance proteins (miR472, miR482) which are related to pathogen resistance [33, 34]. [score:5]
Other conserved miRNAs targets include F-box protein (miR394, miR414), ATP sulfurylase (miR395), Pectate Lyase (miR482), endo-1, 4-beta- glucanase (miR396), Laccase (miR397), all of which are involved in regulation of metabolic processes. [score:4]
A target of miR482 is pectate lyase which is an important enzyme in fruit softening [80, 81]. [score:3]
Moreover, 3 miRNAs (miR394, miR414, and miR482) predicted to have targets related to fruit ripening and softening and ethylene response were validated (Figure 8). [score:3]
The members of each family were different, the miR156, miR166 and miR171 had more than ten members, in the contrary, miR160, miR319, miR394, miR395, miR399, miR408, miR472, miR482, miR827 had only one member in their corresponding family. [score:1]
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[+] score: 29
We do not know of any examples where a miR482/2118 miRNA superfamily member regulates targets at the translational level. [score:6]
In particular, slmiR482a, a miR482/2118 superfamily member, targets the LRR1 mRNA as a siRNA -mediated secondary target [23]. [score:5]
To conclude, our results support the notion that the miR482/2118 superfamily -mediated reduction of gene expression involves multiple NB-domain-encoding genes, including tm-2, and occurs via mRNA cleavage and/or translational control mechanisms in tomato. [score:5]
miR482a, a member of the miR482/2118 superfamily, targets mRNAs for R proteins, with nucleotide -binding site (NB) and leucine-rich repeat (LRR) motifs, for degradation both directly and through generation of secondary small interfering RNAs (siRNAs) in Nicotiana benthamiana infected with Pst DC3000 [22], [23]. [score:4]
SlmiR482 and slmiR5300 are members of the miR482/2118 superfamily and members of this family have been shown to target the p-loop motif in the mRNA of the NB-LRR encoding R genes [23]. [score:3]
There is a precedence for regulation of mRNA cleavage by miR482 family members in tomato [23]. [score:2]
These two miRNAs belong to the miR482/2118 Superfamily in tomato [23], [48], [49]. [score:1]
Table S4 Sequence reads for members of the miRNA482 a-f family. [score:1]
miR5300 was first identified as a novel tomato miRNA [24] and later classified as a member of the miR482/2118 superfamily [23]. [score:1]
The miR482 family has six members, including miR482a-f [23], [45], [46], [47]. [score:1]
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[+] score: 22
A few miRNAs including miR393(i), miR482(i), miR1446(i) and variant_miR156(i) were also upregulated at stage 1 (Fig 3A and 3B), whereas, miR164(i), miR319(iv) and miR1446(i) were upregulated (P < 0.05) at stage 5 (Fig 3A). [score:7]
Using psRNAtarget Analysis server, conserved miRNAs including, miR164(i), miR171(i), miR159(i), miR394(i), miR156(i), miR482(ii), miR166(i), and miR168(i) were predicted to target genes including NAC, GRAS, GAMYB-like, Peroxiredoxin, SBP, Resistance protein, HB and AGO-1, respectively. [score:5]
0175178.g005 Fig 5Using psRNAtarget Analysis server, conserved miRNAs including, miR164(i), miR171(i), miR159(i), miR394(i), miR156(i), miR482(ii), miR166(i), and miR168(i) were predicted to target genes including NAC, GRAS, GAMYB-like, Peroxiredoxin, SBP, Resistance protein, HB and AGO-1, respectively. [score:5]
However, some of the conserved miRNAs including miR393(i), miR482(i), miR1446(i) and variant of miR156(i) and 4 novel miRNAs including Sly_miRNA667, Sly_miRNA996, Sly_miRNA1987 and Sly_miRNA2712 were also upregulated at stage 1 (invasion of J2s/ initiation of feeding sites). [score:4]
The cleavage site of genes including NAC (Solyc07g062840.2), HB (Solyc03g120910.2), AGO1 (Solyc06g072300.2), Resistance protein (Solyc02g036280.2) and GAMYB-like (Solyc06g073640.2, Solyc01g009070.2) was deciphered to lie between 10 [th] and 11 [th] base from 5’ end pairing of miR164(i), miR166(i), miR168(i), miR482(ii) and miR159(i), respectively (Fig 5). [score:1]
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[+] score: 18
In Solanaceae, miR482 was also found targeted to NBS–LRR genes 30, 31, 34. [score:3]
MiR482 regulates defense mechanisms of plant via targeting conserved sequences encoding the P-loop of NBS–LRR resistance proteins [38]. [score:3]
In cotton, ~12% of cotton NBS–LRR genes were predicted targets by gra-miR482 family [40]. [score:3]
Other miRNAs, such as miR482, miR6019, miR6020, miR6022, miR5300, miR1507, and others, are thought to suppress nucleotide -binding site and leucine-rich repeat (NBS– LRR) defense genes, which play critical roles in effector-triggered immunity (ETI), also a crucial component of plant immune system 29– 33. [score:3]
Previous studies have shown, miR482 is involved in the process of tomato, cotton, soybean, and peanut interacting with Potato spindle tuber viroid, Cucumber mosaic virus, Verticillium dahliae, soybean cyst nematode, and Ralstonia solanacearum 35– 39. [score:1]
MiR482, a class of miRNAs found in various plants, acts in plant–pathogen interaction [34]. [score:1]
In miR482-silencing lines, with the abundance of miR482b reduced significantly, plant resistance to P. infestans enhanced observably, which further proved miR482b could enhance the susceptibility of tomato to P. infestans. [score:1]
Using high-throughput sequencing and homology -based computational research, we have previously identified a number of tomato miRNAs involved in tomato– P. infestans interaction including miR482, miR172, miR6024, miR6026, miR6027, etc. [score:1]
Among these miRNA candidates, miR482 is one of the most important miRNAs involved in plant–pathogen interaction. [score:1]
Various letters indicate significant difference among samples, and letters shared in common between or among the groups indicate no significant difference (P < 0.05) To gain insight into the molecular mechanisms responsible for the late blight resistance, we previously used high-throughput sequencing to identify P. infestans -induced miRNAs in tomato, including miR169, miR398, miR482, miR6024, miR6026, and miR6027 25, 47, 48. [score:1]
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[+] score: 18
In our study, we found 44 target genes, but only the targets of 4 miRNAs (sly‐miR156, sly‐miR160, sly‐miR166 and sly‐miR482) were matched with their results (Table S4). [score:5]
In tomato degradome results, 15 target genes were related to stimulus response such as ARF and disease resistance proteins were identified in category 0 cleaved by sly‐miR160, sly‐miR168, sly‐miR172, sly‐miR396, sly‐miR482, sly‐miR6023 and sly‐miR6024 families (Figure  6, Table S4). [score:5]
The rest of 51 target genes were belonged to less significant categories 3 and 4. One of the stimulus response‐associated target PSII degraded by 16 miRNA families such as sly‐miR167, sly‐miR319, sly‐miR390, sly‐miR482, sly‐mir1919, sly‐miR5302 and sly‐miR9479. [score:5]
Three miRNAs (sly‐miR172a, sly‐miR482d‐3p and sly‐miR9472‐5p) were up‐regulated in qRT‐PCR analysis showing a positive correlation with deep sequencing results. [score:2]
We performed qRT‐PCR to validate the deep sequencing results with randomly selected eight miRNAs (sly‐miR156a‐5p, sly‐miR169e‐3p, sly‐miR172a, sly‐miR393a‐5p, sly‐miR399a‐5p, sly‐miR408b‐5p, sly‐miR482d‐3p and sly‐miR9472‐5p). [score:1]
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[+] score: 16
Other miRNAs from this paper: osa-MIR156a, osa-MIR156b, osa-MIR156c, osa-MIR156d, osa-MIR156e, osa-MIR156f, osa-MIR156g, osa-MIR156h, osa-MIR156i, osa-MIR156j, osa-MIR166a, osa-MIR166b, osa-MIR166c, osa-MIR166d, osa-MIR166e, osa-MIR166f, osa-MIR167a, osa-MIR167b, osa-MIR167c, osa-MIR169a, osa-MIR171a, osa-MIR393a, osa-MIR396a, osa-MIR396b, osa-MIR396c, osa-MIR397a, osa-MIR397b, osa-MIR398a, osa-MIR398b, osa-MIR399a, osa-MIR399b, osa-MIR399c, osa-MIR399d, osa-MIR399e, osa-MIR399f, osa-MIR399g, osa-MIR399h, osa-MIR399i, osa-MIR399j, osa-MIR399k, osa-MIR156k, osa-MIR156l, osa-MIR166k, osa-MIR166l, osa-MIR167d, osa-MIR167e, osa-MIR167f, osa-MIR167g, osa-MIR167h, osa-MIR167i, osa-MIR168a, osa-MIR168b, osa-MIR169b, osa-MIR169c, osa-MIR169d, osa-MIR169e, osa-MIR169f, osa-MIR169g, osa-MIR169h, osa-MIR169i, osa-MIR169j, osa-MIR169k, osa-MIR169l, osa-MIR169m, osa-MIR169n, osa-MIR169o, osa-MIR169p, osa-MIR169q, osa-MIR171b, osa-MIR171c, osa-MIR171d, osa-MIR171e, osa-MIR171f, osa-MIR171g, osa-MIR172a, osa-MIR172b, osa-MIR172c, osa-MIR166g, osa-MIR166h, osa-MIR166i, osa-MIR171h, osa-MIR393b, osa-MIR408, osa-MIR172d, osa-MIR171i, osa-MIR167j, osa-MIR166m, osa-MIR166j, osa-MIR396e, mtr-MIR166a, mtr-MIR169a, mtr-MIR399b, mtr-MIR399d, mtr-MIR393a, mtr-MIR399c, mtr-MIR399a, mtr-MIR399e, mtr-MIR156a, mtr-MIR171a, mtr-MIR156b, mtr-MIR167a, mtr-MIR166b, mtr-MIR169c, mtr-MIR169d, mtr-MIR169e, mtr-MIR171b, mtr-MIR166c, mtr-MIR166d, mtr-MIR169f, mtr-MIR156c, mtr-MIR156d, mtr-MIR399f, mtr-MIR399g, mtr-MIR399h, mtr-MIR399i, mtr-MIR399j, mtr-MIR399k, mtr-MIR166e, mtr-MIR156e, mtr-MIR171c, mtr-MIR398a, mtr-MIR172a, mtr-MIR393b, mtr-MIR398b, mtr-MIR168a, mtr-MIR169g, mtr-MIR156f, mtr-MIR399l, mtr-MIR156g, mtr-MIR399m, mtr-MIR399n, mtr-MIR399o, mtr-MIR398c, mtr-MIR156h, mtr-MIR166f, mtr-MIR166g, mtr-MIR171d, mtr-MIR171e, mtr-MIR396a, mtr-MIR396b, mtr-MIR169h, mtr-MIR169b, mtr-MIR156i, mtr-MIR171f, mtr-MIR399p, osa-MIR169r, sly-MIR166a, sly-MIR166b, sly-MIR167a, sly-MIR169a, sly-MIR169b, sly-MIR169c, sly-MIR169d, sly-MIR171a, sly-MIR171b, sly-MIR171c, sly-MIR171d, sly-MIR397, sly-MIR156a, sly-MIR156b, sly-MIR156c, sly-MIR172a, sly-MIR172b, sly-MIR399, osa-MIR827, osa-MIR396f, mtr-MIR2118, osa-MIR2118a, osa-MIR2118b, osa-MIR2118c, osa-MIR2118d, osa-MIR2118e, osa-MIR2118f, osa-MIR2118g, osa-MIR2118h, osa-MIR2118i, osa-MIR2118j, osa-MIR2118k, osa-MIR2118l, osa-MIR2118m, osa-MIR2118n, osa-MIR2118o, osa-MIR2118p, osa-MIR2118q, osa-MIR2118r, mtr-MIR169k, mtr-MIR169j, mtr-MIR399q, osa-MIR396g, osa-MIR396h, osa-MIR396d, osa-MIR5072, mtr-MIR4414a, mtr-MIR4414b, mtr-MIR482, mtr-MIR172b, mtr-MIR172c, mtr-MIR171h, mtr-MIR168b, mtr-MIR399r, mtr-MIR156j, sly-MIR482e, sly-MIR482a, mtr-MIR167b, mtr-MIR168c, mtr-MIR408, mtr-MIR396c, mtr-MIR171g, stu-MIR6024, sly-MIR6024, stu-MIR482c, stu-MIR482b, stu-MIR482a, stu-MIR482d, stu-MIR482e, sly-MIR482b, sly-MIR482c, stu-MIR6025, stu-MIR6026, sly-MIR6026, sly-MIR168a, sly-MIR168b, mtr-MIR169i, mtr-MIR172d, mtr-MIR397, mtr-MIR169l, mtr-MIR399s, mtr-MIR399t, stu-MIR7980a, stu-MIR7983, stu-MIR8007a, stu-MIR8007b, stu-MIR7980b, stu-MIR399a, stu-MIR399b, stu-MIR399c, stu-MIR399d, stu-MIR399e, stu-MIR399f, stu-MIR399g, stu-MIR399h, stu-MIR3627, stu-MIR171b, stu-MIR166a, stu-MIR166b, stu-MIR166c, stu-MIR166d, stu-MIR171a, stu-MIR171c, stu-MIR399i, stu-MIR827, stu-MIR172b, stu-MIR172c, stu-MIR172a, stu-MIR172d, stu-MIR172e, stu-MIR156a, stu-MIR156b, stu-MIR156c, stu-MIR156d, stu-MIR171d, stu-MIR167c, stu-MIR167b, stu-MIR167a, stu-MIR167d, stu-MIR399j, stu-MIR399k, stu-MIR399l, stu-MIR399m, stu-MIR399n, stu-MIR399o, stu-MIR393, stu-MIR398a, stu-MIR398b, stu-MIR396, stu-MIR408a, stu-MIR408b, stu-MIR397, stu-MIR171e, stu-MIR156e, stu-MIR156f, stu-MIR156g, stu-MIR156h, stu-MIR156i, stu-MIR156j, stu-MIR156k, stu-MIR169a, stu-MIR169b, stu-MIR169c, stu-MIR169d, stu-MIR169e, stu-MIR169f, stu-MIR169g, stu-MIR169h, sly-MIR403, sly-MIR166c, sly-MIR156d, sly-MIR156e, sly-MIR396a, sly-MIR167b, sly-MIR169e, sly-MIR396b, sly-MIR171e, sly-MIR172c, sly-MIR408, sly-MIR172d, sly-MIR827, sly-MIR393, sly-MIR398a, sly-MIR399b, sly-MIR6025, sly-MIR169f, sly-MIR171f
Five of these NBS-LRR genes were targeted by miR482, while another NBS-LRR gene (Solyc10g008240.2.1) was targeted by miR6025. [score:5]
The miR482/2118 superfamily is a conserved phasiRNA trigger that regulates NBS-LRR gene expression across different plants (Zhai et al., 2011; Shivaprasad et al., 2012). [score:4]
Five miR482 triggering and one miR6025 triggering NBS-LRR loci were identified in our dataset, which further supports the phasiRNA based NBS-LRR gene expression in tomato (Shivaprasad et al., 2012). [score:3]
Interestingly, the expression of miR482 is not compromised upon the infection of R. irregularis. [score:3]
Six miR482-triggering NBS-LRR PHAS loci were identified in tomato (Shivaprasad et al., 2012). [score:1]
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[+] score: 11
In the network, it could be clearly seen that miR6024 had 11 targets and among the targets two of them were also the targets of miR482 (Figure 5). [score:7]
On the basis of the conservation of the TAS genes in plants, three TAS5 gene family members: TAS5, TAS5b, and TAS5d (TAS5b and TAS5d were found in our previous study; Zuo et al., 2016), all miR482 targets, were identified (Table 3). [score:3]
For instance, miR156 and miR482 were the largest ones with seven members in the families in this study. [score:1]
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[+] score: 7
Three miRNA-PHAS regulation cascades (miR482d-PHAS, miR482e-tasLRR, and miR6024-tasLRR) were also identified and confirmed in response to B. cinerea infection. [score:2]
Moreover, the tasNB-LRR regulator cascades triggered by miR482 family members are conserved pathways in many plants, particularly in Solanaceae species 11, 12. [score:2]
The miR482 family is well-known as a trigger miRNA involved in phasiRNA biogenesis 11, 19, 30. [score:1]
From the analysis of phasiRNAs responsive to B. cinerea infection, five phasiRNAs triggered by four conserved miRNAs (miR6024, miR482d, miR482e, and miR390a) were also found. [score:1]
Otherwise, the remaining four phasiRNAs triggered by conserved miRNAs (siR07-2 triggered by miR482e, siR71-2 triggered by miR482d, and both siR23-4 and siR43-7 triggered by miR6024) had increased abundance in both B. cinerea-infected leaves and B. cinerea-infected fruits (Fig.   4). [score:1]
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[+] score: 6
For conserved miRNA families, the miR167, miR396, and miR482 families were predominantly expressed in the range of 2000 transcripts per million (TPM) clean tags to 14,000 TPM in the stamen libraries, whereas the miR159, miR166, and miR482 families were predominantly in the range of 1500 TPM to 4500 TPM in the pistil libraries. [score:3]
Whereas two miRNAs, miR397–5p and miR398b-3p, were common in the stamen and pistil libraries at 2 d after heat-stress treatment, 12 known miRNAs belonging to the miR159, miR160, miR319, miR393, miR482, miR1918, miR6026, and miR6027 families (Fig.   5a, Additional file 1: Table S5) were common at 12 d after heat-stress treatment. [score:1]
In the stamen library, the miRNA167 and miRNA396 families were the most abundant, whereas in the pistil library, the miRNA159 and miRNA482 families were the most abundant (Fig.   4). [score:1]
The majority of the 26 miRNA families contained more than one member, and miR156, miR171, miR172, miR319, miR396, and miR482 had more than seven members. [score:1]
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Proceedings of the National Academy of Sciences, USA 109, 1790– 1795 Li H Deng Y Wu T Subramanian S Yu O 2010 Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation. [score:3]
In the case of sly-miR5301, the predicted miRNA was found to be a close homologue of miR482. [score:1]
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The members of different  R-gene families in tomato, potato, soybean, and  Medicago truncatula are targeted by miR482 and miR2118 miRNAs [12, 13]. [score:3]
Several miRNA families, including miR157, miR159, miR162, miR164, miR167, miR171, miR172, miR390, miR396, and miR482, were moderately abundant (Figure  2A). [score:1]
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MiR6027, miR171a, miR482, miR319, and miR1919a were moderately expressed and had a TPM between 10 and 100. [score:3]
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have shown that the abundance of miRNA families like MIR159, MIR166, MIR6022, MIR162, MIR482, and some other were consistently higher throughout the libraries. [score:1]
Micro RNA families MIR156, MIR172, and MIR5303 contained highest five members while 11 families viz, MIR162, MIR166, MIR167, MIR168, MIR171, MIR1919, MIR319, MIR398, MIR482, MIR6024, and MIR7997 contained several members (2–4). [score:1]
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The numbers of miRNAs varied in different miRNA families, with the most members (seven) in sly-miR156 and sly-miR482, followed with five miRNAs in the family of sly-miR171. [score:1]
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Other miRNAs from this paper: osa-MIR156a, osa-MIR156b, osa-MIR156c, osa-MIR156d, osa-MIR156e, osa-MIR156f, osa-MIR156g, osa-MIR156h, osa-MIR156i, osa-MIR156j, osa-MIR160a, osa-MIR160b, osa-MIR160c, osa-MIR160d, osa-MIR162a, osa-MIR164a, osa-MIR164b, osa-MIR166a, osa-MIR166b, osa-MIR166c, osa-MIR166d, osa-MIR166e, osa-MIR166f, osa-MIR167a, osa-MIR167b, osa-MIR167c, osa-MIR169a, osa-MIR171a, osa-MIR394, osa-MIR395b, osa-MIR395d, osa-MIR395e, osa-MIR395g, osa-MIR395h, osa-MIR395i, osa-MIR395j, osa-MIR395k, osa-MIR395l, osa-MIR395s, osa-MIR395t, osa-MIR395c, osa-MIR395a, osa-MIR395f, osa-MIR395u, osa-MIR396a, osa-MIR396b, osa-MIR396c, osa-MIR156k, osa-MIR156l, osa-MIR159a, osa-MIR159b, osa-MIR159c, osa-MIR159d, osa-MIR159e, osa-MIR159f, osa-MIR160e, osa-MIR160f, osa-MIR162b, osa-MIR164c, osa-MIR164d, osa-MIR164e, osa-MIR166k, osa-MIR166l, osa-MIR167d, osa-MIR167e, osa-MIR167f, osa-MIR167g, osa-MIR167h, osa-MIR167i, osa-MIR169b, osa-MIR169c, osa-MIR169d, osa-MIR169e, osa-MIR169f, osa-MIR169g, osa-MIR169h, osa-MIR169i, osa-MIR169j, osa-MIR169k, osa-MIR169l, osa-MIR169m, osa-MIR169n, osa-MIR169o, osa-MIR169p, osa-MIR169q, osa-MIR171b, osa-MIR171c, osa-MIR171d, osa-MIR171e, osa-MIR171f, osa-MIR171g, osa-MIR172a, osa-MIR172b, osa-MIR172c, osa-MIR166g, osa-MIR166h, osa-MIR166i, osa-MIR171h, osa-MIR408, osa-MIR172d, osa-MIR171i, osa-MIR167j, osa-MIR166m, osa-MIR166j, osa-MIR164f, osa-MIR396e, gma-MIR156d, gma-MIR156e, gma-MIR156c, gma-MIR159a, gma-MIR160a, gma-MIR166a, gma-MIR166b, gma-MIR167a, gma-MIR167b, gma-MIR172a, gma-MIR172b, gma-MIR156a, gma-MIR396a, gma-MIR396b, gma-MIR156b, gma-MIR169a, osa-MIR395m, osa-MIR395n, osa-MIR395o, osa-MIR395p, osa-MIR395q, osa-MIR395v, osa-MIR395w, osa-MIR395r, osa-MIR169r, gma-MIR159b, gma-MIR159c, gma-MIR162a, gma-MIR164a, gma-MIR167c, gma-MIR169b, gma-MIR169c, gma-MIR171a, gma-MIR171b, gma-MIR482a, sly-MIR160a, sly-MIR166a, sly-MIR166b, sly-MIR167a, sly-MIR169a, sly-MIR169b, sly-MIR169c, sly-MIR169d, sly-MIR171a, sly-MIR171b, sly-MIR171c, sly-MIR171d, sly-MIR395a, sly-MIR395b, sly-MIR156a, sly-MIR156b, sly-MIR156c, sly-MIR159, sly-MIR162, sly-MIR172a, sly-MIR172b, osa-MIR396f, gma-MIR167d, gma-MIR396c, mdm-MIR482a, gma-MIR167e, gma-MIR167f, gma-MIR172c, gma-MIR172d, gma-MIR172e, osa-MIR396g, osa-MIR396h, osa-MIR396d, osa-MIR395x, osa-MIR395y, gma-MIR396d, gma-MIR482b, gma-MIR167g, gma-MIR156f, gma-MIR169d, gma-MIR172f, gma-MIR171c, gma-MIR169e, gma-MIR394b, gma-MIR156g, gma-MIR159d, gma-MIR394a, gma-MIR396e, gma-MIR156h, gma-MIR156i, gma-MIR160b, gma-MIR160c, gma-MIR160d, gma-MIR160e, gma-MIR162b, gma-MIR164b, gma-MIR164c, gma-MIR164d, gma-MIR166c, gma-MIR166d, gma-MIR166e, gma-MIR166f, gma-MIR166g, gma-MIR166h, gma-MIR169f, gma-MIR169g, gma-MIR171d, gma-MIR171e, gma-MIR171f, gma-MIR171g, gma-MIR394c, gma-MIR408d, gma-MIR482c, gma-MIR171h, gma-MIR171i, gma-MIR169h, gma-MIR167h, gma-MIR169i, gma-MIR396f, gma-MIR396g, gma-MIR167i, sly-MIR482e, sly-MIR482a, gma-MIR171j, gma-MIR395a, gma-MIR395b, gma-MIR395c, gma-MIR408a, gma-MIR408b, gma-MIR408c, gma-MIR156j, gma-MIR156k, gma-MIR156l, gma-MIR156m, gma-MIR156n, gma-MIR156o, gma-MIR159e, gma-MIR159f, gma-MIR162c, gma-MIR166i, gma-MIR166j, gma-MIR169j, gma-MIR169k, gma-MIR169l, gma-MIR169m, gma-MIR169n, gma-MIR171k, gma-MIR172g, gma-MIR172h, gma-MIR172i, gma-MIR172j, gma-MIR396h, gma-MIR396i, gma-MIR482d, gma-MIR167j, gma-MIR171l, gma-MIR156p, gma-MIR171m, gma-MIR172k, gma-MIR171n, gma-MIR156q, gma-MIR171o, gma-MIR172l, gma-MIR169o, gma-MIR171p, gma-MIR394d, gma-MIR169p, gma-MIR156r, gma-MIR396j, gma-MIR171q, gma-MIR156s, gma-MIR169r, gma-MIR169s, gma-MIR396k, gma-MIR166k, gma-MIR156t, gma-MIR482e, gma-MIR171r, gma-MIR394e, gma-MIR169t, gma-MIR171s, gma-MIR166l, gma-MIR171t, gma-MIR394f, gma-MIR171u, gma-MIR395d, gma-MIR395e, gma-MIR395f, gma-MIR395g, gma-MIR166m, gma-MIR169u, sly-MIR482b, sly-MIR482c, gma-MIR156u, gma-MIR156v, gma-MIR156w, gma-MIR156x, gma-MIR156y, gma-MIR156z, gma-MIR156aa, gma-MIR156ab, gma-MIR160f, gma-MIR164e, gma-MIR164f, gma-MIR164g, gma-MIR164h, gma-MIR164i, gma-MIR164j, gma-MIR164k, gma-MIR166n, gma-MIR166o, gma-MIR166p, gma-MIR166q, gma-MIR166r, gma-MIR166s, gma-MIR166t, gma-MIR166u, gma-MIR169v, gma-MIR394g, gma-MIR395h, gma-MIR395i, gma-MIR395j, gma-MIR395k, gma-MIR395l, gma-MIR395m, mdm-MIR156a, mdm-MIR156b, mdm-MIR156c, mdm-MIR156d, mdm-MIR156e, mdm-MIR156f, mdm-MIR156g, mdm-MIR156h, mdm-MIR156i, mdm-MIR156j, mdm-MIR156k, mdm-MIR156l, mdm-MIR156m, mdm-MIR156n, mdm-MIR156o, mdm-MIR156p, mdm-MIR156q, mdm-MIR156r, mdm-MIR156s, mdm-MIR156t, mdm-MIR156u, mdm-MIR156v, mdm-MIR156w, mdm-MIR156x, mdm-MIR156y, mdm-MIR156z, mdm-MIR156aa, mdm-MIR156ab, mdm-MIR156ac, mdm-MIR156ad, mdm-MIR156ae, mdm-MIR159a, mdm-MIR159b, mdm-MIR160a, mdm-MIR160b, mdm-MIR160c, mdm-MIR160d, mdm-MIR160e, mdm-MIR162a, mdm-MIR162b, mdm-MIR164a, mdm-MIR164b, mdm-MIR164c, mdm-MIR164d, mdm-MIR164e, mdm-MIR164f, mdm-MIR166a, mdm-MIR166b, mdm-MIR166c, mdm-MIR166d, mdm-MIR166e, mdm-MIR166f, mdm-MIR166g, mdm-MIR166h, mdm-MIR166i, mdm-MIR167a, mdm-MIR167b, mdm-MIR167c, mdm-MIR167d, mdm-MIR167e, mdm-MIR167f, mdm-MIR167g, mdm-MIR167h, mdm-MIR167i, mdm-MIR167j, mdm-MIR169a, mdm-MIR169b, mdm-MIR169c, mdm-MIR169d, mdm-MIR171a, mdm-MIR171b, mdm-MIR171c, mdm-MIR171d, mdm-MIR171e, mdm-MIR171f, mdm-MIR171g, mdm-MIR171h, mdm-MIR171i, mdm-MIR171j, mdm-MIR171k, mdm-MIR171l, mdm-MIR171m, mdm-MIR171n, mdm-MIR172a, mdm-MIR172b, mdm-MIR172c, mdm-MIR172d, mdm-MIR172e, mdm-MIR172f, mdm-MIR172g, mdm-MIR172h, mdm-MIR172i, mdm-MIR172j, mdm-MIR172k, mdm-MIR172l, mdm-MIR172m, mdm-MIR172n, mdm-MIR172o, mdm-MIR394a, mdm-MIR394b, mdm-MIR395a, mdm-MIR395b, mdm-MIR395c, mdm-MIR395d, mdm-MIR395e, mdm-MIR395f, mdm-MIR395g, mdm-MIR395h, mdm-MIR395i, mdm-MIR396a, mdm-MIR396b, mdm-MIR396c, mdm-MIR396d, mdm-MIR396e, mdm-MIR396f, mdm-MIR396g, mdm-MIR408a, mdm-MIR482b, mdm-MIR482c, mdm-MIR408b, mdm-MIR408c, mdm-MIR408d, mdm-MIR482d, mdm-MIR159c, mdm-MIR171o, mdm-MIR169e, mdm-MIR169f, sly-MIR164a, sly-MIR164b, sly-MIR394, sly-MIR166c, sly-MIR156d, sly-MIR156e, sly-MIR396a, sly-MIR167b, sly-MIR169e, sly-MIR396b, sly-MIR171e, gma-MIR167k, gma-MIR167l, gma-MIR169w, sly-MIR172c, sly-MIR408, sly-MIR172d, sly-MIR169f, sly-MIR171f, mdm-MIR159d, mdm-MIR159e, mdm-MIR159f, mdm-MIR166j, mdm-MIR395j, mdm-MIR169g, mdm-MIR169h, mdm-MIR169i, mdm-MIR169j, mdm-MIR171p, mdm-MIR395k, mdm-MIR171q, mdm-MIR169k, mdm-MIR169l, mdm-MIR169m, mdm-MIR169n, mdm-MIR172p, mdm-MIR395l, mdm-MIR169o
Of 21 miRNA families across the seven plant species, only family - miR482 in Arabidopsis was not detected (Figure 1 and Table S2). [score:1]
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Recently, a fifth TAS family (TAS5) was identified from tomato, which is triggered by miR482 [19]. [score:1]
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We also observed Solanaceae-specific miR482 and miR6022 among the most abundant miRNAs in the non-infested phloem PC and LC sRNAs (Table S4). [score:1]
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