We have sequenced 12 sRNA libraries from three control and three V. nonalfalfae-treated roots of the susceptible cultivar-Celeia and the resistant cultivar-Wye Target. Using the miR-PReFER miRNA prediction pipeline, we identified 56 mature miRNAs belonging to 30 known plant miRNA families and 35 novel miRNA families represented by a different number of members. A genome-wide profiling study of miRNAs in Verticillium-treated roots of Gossypium barbadense (Verticullium-tolerant cultivar) and Gossypium hirsutum (Verticillium-susceptible cultivar) reported the identification of 215 known miRNA families and 14 novel miRNAs and more than 65 miRNA families showing the differences in expression between control and V. dahliae-treated samples in both cotton cultivars . In Populus beijingensis, Chen, Ren, Zhang, Xu, Zhang and Wang  identified 74 conserved miRNAs belonging to 37 miRNA families and 27 novel miRNAs from 35 MIR loci. P. beijingensis plants treated with a canker disease pathogen (Dothiorella gregaria) exhibited differential expression of 33 miRNAs. With the in silico analysis, they predicted nine target genes for three conserved miRNAs (pbe-miR477c, pbe-miR482b, and pbe-miR2111) and 63 target genes for 35 novel miRNAs. Targeted genes are involved in plant development, abiotic and biotic stress responses or encode leucine-rich repeats, which are targeted by different miRNAs and are involved in disease resistance in other plants [17, 19, 40, 41].
In our study, a significant upregulation in response to infection with V. nonalfalfae was observed for hlu-miR159c–d in both hop cultivars. Similarly, as reported, miR159 was upregulated in P. beijingensis treated with D. gregaria , P. trichocarpa treated with Botryosphaeria dothidea  and in Triticum aestivum during Puccinia graminis f.sp. tritici infection . Recently, Zhang, Zhao, Zhao, Wang, Jin, Chen, Fang, Hua, Ding and Guo  discovered that cotton plants treated with V. dahlia export miR159 to the fungal hyphae where it targets an isotrichodermin C-15 hydroxylase (HiC-15) which is responsible for fungal virulence. MIR159 family is conserved in many terrestrial plant species [45, 46] and its main role is a regulation of GAMYB or GAMYB-like transcription factors that possess highly conserved binding sites for miR159 . In silico miRNA target prediction revealed that hlu-miR159c–d (upregulated in both cultivars) and hlu-miR319c–f (upregulated in the resistant cultivar) bind to transcription factor GAMYB but have also other distinct targets. Besides, hlu-miR319c–f targets transcripts of hops teosinte branched 1 (A0A061GDP3) which belongs to the TCP transcription factor family. While both miRNAs regulate MYB TFs in Arabidopsis, miR319 acts predominantly on transcription factors of the TCP family and to a lesser extent on the expression of MYB, since the expression levels and domain of miR319 limit its regulation of MYB, while the sequence of miR159 prevents binding to TCP transcripts [48, 49], which is also observed in our study. Recently, Wu, Qi, Meng and Jin  showed that sly-miR319 acts as a positive regulator of tomato resistance to Botrytis cinerea infection by repressing the expression SlTCP29. Moreover, previous studies show that abscisic acid (ABA) induces the accumulation of miR159 in germinating Arabidopsis thaliana seeds, which in turn mediates the cleavage of MYB101 and MYB33 transcripts , and that miR159 is also involved in the signalling of the phytohormone gibberellin (GA) during floral development by targeting GAMYB-like transcripts . Hormone signalling is one of the key pathways that modulate plant responses to biotic stress .
A significant downregulation was observed in our study for hlu-miR156e–f in the resistant hop cultivar, and in silico target prediction showed that it targets transcripts of 6 different SQUAMOSA PROMOTER-BINDING-LIKE proteins (SPB), protein SCO1 homolog 2 and LOB DOMAIN-CONTAINING protein. The interaction between hops’ miR156 and SPB15 was previously confirmed by 5' RLM-RACE analysis . Eleven SPB genes have been predicted to be targets of miR156 in Arabidopsis  and Oryza sativa , respectively. Furthermore, Bhogale, Mahajan, Natarajan, Rajabhoj, Thulasiram and Banerjee  validated the interaction between miR156 and StSPL3, StSPL6, StSPL9, StSPL13 in Solanum tuberosum ssp. andigena and observed that miR156 can be transported through the plants via the phloem. Upregulation of miR156 and SPB targeting was observed in the compatible interactions of P. beijingensis and P. trichocarpa treated with D. gregaria and B. dothidea, respectively [19, 42]. Proteins from SPB family are thought to be transcriptional activators and have roles in leaf development, vegetative phase change, flower and fruit development, plant architecture, shoot maturation, gibberellin signalling and response to fungal toxin [58, 59].
Another interesting target of hlu-miR156e-f that we identified in the miRNA target analysis is the transcript of LATERAL ORGAN BOUNDARIES (LOB) DOMAIN-CONTAINING protein (W9SE87). The latter protein family is involved in a positive feedback loop that promotes the expression of the NAC domain-containing protein 30 (NAC030)/VASCULAR-RELATED NAC-DOMAIN PROTEIN7 (VND7), which regulates genes associated with the differentiation of tracheary elements in Arabidopsis, e.g. genes involved in secondary wall biosynthesis, cell wall modifications such as xylan accumulation and programmed cell death . VASCULAR-RELATED NAC DOMAIN7 TF plays an important role in the response to infection with V. longisporum in Arabidopsis, as it induces de novo formation of functional xylem elements from bundle sheath cells, which subsequently leads to vein clearing and xylem hyperplasia within the vasculature of the roots as well as to enhanced drought tolerance . Additionally, LOB domain-containing proteins act as transcriptional activators that directly regulate EXPANSINA17, a gene encoding a cell wall-loosening factor that promotes lateral root emergence . A downregulation of miR156 and a significantly lower expression of miR164 (discussed below) in the resistant compared to the susceptible hop cultivar could indicate the role of these two miRNAs in the modulation of GA signalling and processes of root growth and xylem development in the resistant hop cultivar during the infection with V. nonalfalfae.
Hlu-miR160b, which is significantly upregulated in infected compared to control root samples of the resistant hop cultivar, is predicted to target transcripts of auxin response factors (ARFs), DNA binding proteins that bind to a specific sequence in promoters of auxin-responsive genes . Upregulation of miR160 and its regulation of ARFs has also been demonstrated during the pathogenesis of stem canker disease in P. trichocarpa  and in potato, where it targets StARF10, which binds to the promoter in the StGH3.6 gene, a mediator of salicylic acid–auxin cross-talk, and is thus associated with local defence and systemic acquired resistance to Phytophthora infestans . In A. thaliana, miR160 controls root cap formation by regulating the expression of ARF10 and ARF16. Disturbed miR160-directed regulation of ARF16 resulted in reduced fertility and fewer lateral roots . In addition, researchers observed defects in root growth in Arabidopsis plants expressing a miR160-resistant version of ARF17 .
In response to infection with V. nonalfalfae, the resistant cultivar showed significantly lower expression of hlu-miR164b, which targets transcripts of NAC domain-containing proteins. The hlu-miR164b cleavage site within the transcript of hops NAC domain-containing protein was confirmed by 5’ RLM-RACE analysis done by Mishra, Duraisamy, Matousek, Radisek, Javornik and Jakse . Hu, Lei, Wang, Liu, Tang, Zhang, Chen, Peng, Yang and Wu  observed a significant decrease of ghr-miR164 in the response of cotton plants to infection with V. dahliae. Additionally, the researchers showed that ghr-miR164 directly cleaves the mRNA of GhNAC100, which in turn increased the resistance of plants to the fungus. The decrease of miR164 was also observed in Oryza sativa upon infection with Magnaporthe oryzae strain Guy11, and rice plants with the dysfunctional miR164a/OsNAC60 regulatory module developed a significant susceptibility to infection with Guy11 . Auxin-induced expression of miR164 in wild-type Arabidopsis plants resulted in decreased levels of the NAC1 transcripts and reduced lateral root emergence. Additionally, Arabidopsis mir164a and mir164b mutants that express less miR164 show higher expression of NAC1 and have abundant lateral roots. This evidence may indicate that the auxin-miR164-NAC1 regulation provides a homeostatic mechanism that modulates auxin signalling during lateral root development .
In the susceptible hop cultivar treated with V. nonalfalfae, we observed a significant downregulation of hlu-miR167a–d, hlu-miR167f, hlu-miR828a–b and two novel miRNAs, i.e. miRNA-363/miRNA-1427 and miRNA-898/miRNA-2452. All aforementioned miRNAs, except miRNA-898/miRNA-2452, had significantly higher expression in the resistant cultivar in response to infection with V. nonalfalfae. Contrary to our results MIR167 showed higher expression in interactions T. aestivum-P. graminis f. sp. tritici , P. beijingensis-D. gregaria  and Persicaria minor-Fusarium oxysporum . The observed differences in the expression pattern could be due to different sampling time, tissue or different functions of the same miRNA family in different species during infection with different pathogens. Similarly as in our study, a mediator of RNA polymerase II was predicted as a target of MIR167 in Persicaria minor . Compared to the susceptible hop cultivar, the resistant cultivar showed a significantly lower expression of MIR169. Similarly, Li, Zhao, Li, Hu, Wang, Cao, Xu, Zhao, Xiao, Yang, et al.  observed a higher expression of miR169 in the susceptible O. sativa cultivar, but not in the resistant cultivar when infected with M. oryzae. By in silico analysis of miRNA targets, we identified the nuclear transcription factor Y (NF-YA) as a target of miR169. In rice, miR169 suppresses the expression of NF-YA genes and thus acts as a negative regulator in rice immunity against blast fungus M. oryzae, since the transgenic lines that overexpress miR169 became hyper-susceptible to M. oryzae due to the reduced expression of defence-related genes . A significantly lower expression of miR169 in the resistant hop cultivar might thus contribute to hop resistance. Additionally, miR169 regulates NF-YA2 and NF-YA10 genes, which are involved in the control of primary root growth  and regulate leaf growth via auxin signalling in Arabidopsis .
The resistant hop cultivar shows a significantly lower expression of miR171 after infection with V. nonalfalfae compared to the susceptible hop cultivar. In our study we identified three miR171 targets that encode the proteins of the GRAS-domain family; Scarecrow-like protein 22 (SCR22), scarecrow-like protein 6 (SCR6) and GRAS domain-containing protein. The cleavage site of miR171 within the hop transcripts of the GRAS domain-containing protein was confirmed by Mishra, Duraisamy, Matousek, Radisek, Javornik and Jakse . Proteins of the GRAS family are involved in gibberellin (GA) signalling, which regulates various aspects of plant growth and development . In Arabidopsis, SCR protein regulates asymmetric cell division of the cortex/endodermal initial cells during root development . In addition, Arabidopsis miR171c negatively regulates shoot branching by targeting three different members of the GRAS family . We may assume that the resistant hop cultivar modulates GA hormone signalling via the miR171-GRAS regulatory pathway, which may contribute to the observed resistance to V. nonalfalfae.
The resistant hop cultivar shows also a significantly lower expression of miR390 after infection with V. nonalfalfae compared to the susceptible hop cultivar. In Arabidopsis, miR390 targets precursor RNAs of TAS3 and thus triggers the biogenesis of trans-acting small interfering RNA or tasiARFs that cleave the transcripts of ARF genes, thereby regulating plant growth and lateral root development . Although miRNA target analysis didn’t predict binding sites of hlu-miR390a in the transcripts of hop TAS3, we identified its four potential targets. One of the targets is involved in the regulation of the response to a stimulus and encodes proteins with successive leucine-rich repeat motifs. This hop protein might belong to the class of toll-like receptors which bind pathogen- and danger-associated molecular patterns .
In the resistant cultivar, we observed a downregulation of hlu-miR408a–b and a significantly lower expression of the latter in response to infection with V. nonalfalfae compared to the susceptible hop cultivar. It is reported that miR408 targets laccases and cupredoxins and is involved in various abiotic stress responses in Arabidopsis , while its role in biotic stress responses has not yet been fully described. Yin, Ramachandran, Zhai, Bu, Pappu and Hulbert  observed an increased expression of miR408 in Arabidopsis plants that overexpressed the effector protein SR1-a of Puccinia graminis f. sp. tritici (Pgt), but the increase was not significantly higher in wheat leaves. In our study, the binding site of miR408 was found only in transcripts of the long-chain acyl-coenzyme synthetase. In A. thaliana, long-chain acyl-coenzyme synthetase activates C16 or C18 fatty acids, which represent a substrate for cutin and wax . The downregulation of miR408 may modulate the biosynthetic pathways of cutin and wax, which could lead to the accumulation of these compounds in the roots of resistant hop cultivar when treated with V. nonalfalfae.
In the susceptible hop cultivar, we observed a downregulation of miR828 and two novel miRNAs, namely miRNA-363/miRNA-1427 and miRNA-898/miRNA-2452. Recent studies showed that miR828 regulates phenylpropanoid biosynthesis either by direct cleavage of MYB TFs or by cleaving trans-acting siRNA gene 4 (TAS4) which result in the production of ta-siRNAs that silence the expression of MYB gene [81, 82]. In our study, a hlu-miR828a–b cleavage site was not predicted within the transcripts of MYB or TAS4 transcripts, however, it targets transcripts of proteins with RNA pol II transcription regulator recruiting activity. The latter protein contains DNA-binding domains of MYB-related proteins, as well as the SANT domain family which are found in nuclear receptor co-repressors and subunits of chromatin-remodelling complexes [83, 84]. Serine/threonine-protein phosphatase and 3-hydroxyisobutyryl-CoA hydrolase are potential targets of hlu-miR828a–b that were also significant in GO enrichment analysis of molecular functions and biological processes in the susceptible hop cultivar.
Cleavage sites of novel miRNA-363/miRNA-1427 were identified in transcripts of polyphenol oxidase, a protein from the family of the ER lumen retaining receptors and in dynamin-related protein 4C. In previous studies, novel miRNAs targeting polyphenol oxidase were identified in P. trichocarpa , Salvia miltiorrhiza , Solanum tuberosum  and Vitis vinifera  however, they differ in sequence from miRNA-363/miRNA-1427 identified in our study. The other two potential targets, the protein of the ER lumen retaining receptor family and dynamin-related protein 4C, are both involved in cellular localization or transport.
Additionally, novel miRNA miRNA-898/miRNA-2452 potentially targets transcripts of vacuolar protein sorting-associated protein, which is involved in protein transport between endosomes and the trans-Golgi network .
The resistant cultivar showed a significantly lower expression of novel miRNA-617 than the susceptible one after infection. The cleavage site of miRNA-617 was predicted in transcripts of wall-associated receptor kinase from receptor-like kinases (RLK) protein family, which are involved in the detection and signal transduction during pathogen attacks .
With RT-qPCR based on the SYBR green approach, we have shown that all targeted miRNAs are amplified in vitro, but with this approach, we cannot directly compare expression patterns. There may be many reasons for the differences in the results obtained with different approaches, as has been shown previously . In our case, the templates for miRNA sequencing were enriched samples for small RNAs, whereas we used total RNA for RT-qPCR analysis. Studies have already shown that the library preparation steps create bias in sequencing results [88, 89]. Thus, the differences in the results of DE analysis may be due to different approaches of sample processing and reverse transcription. One of the important impacts could also be differential expression analysis. While relative quantification by the ΔΔCt method is used for qPCR analysis , normalization of read counts by the DESeq2’s median of ratios is used in the analysis of RNA-Seq data . However, it is beyond the scope of this study to comment on a possible bias arising from either method and the appropriateness of comparing the results of the two methods.