Identification of micro RNAs and their targets in defense response of hop (Humulus lupulus L.) after infection with fungus Verticillium nonalfalfae

V okviru raziskav doktorske disertacije smo izvedli umetno inokulacijo odpornega in občutljivega kultivarja hmelja s fitopatogeno glivo Verticillium nonalfalfae ter v različnih časovnih točkah po inokulaciji določili profil fenolnih spojin in glivne kolonizacije. Ugotovili smo, da se v koreninah občutljivega kultivarja pojavlja bimodalen vzorec kolonizacije, prav tako pa se je gliva razširila v stebla, medtem ko se je relativna količina glive v odpornem kultivarju tekom eksperimenta zmanjševala. V koreninah odpornega kultivarja smo tretji dan po inokulaciji zaznali statistično značilno povečanje skupnih flavanolov. V steblih je bila akumulacija fenolov v občutljivem kultivarju v primerjavi z odpornim višja, saj smo zaznali znatno povečanje flavonolov 3. in 15. dan ter hidroksicimetnih kislin 6. dan po inokulaciji. Študija identifikacije, karakterizacije, filogenetske analize in analize izražanja treh genov Argonavt ter dveh genov Dicerju-podobnih proteinov in dveh genov od RNA-odvisne RNA polimeraze je pokazala grozdenje z njihovimi ortologi iz ožje sorodnih vrst Cannabis sativa, Morus notabilis in Ziziphus jujuba. Ugotovili smo, da se tretji dan po inokulaciji znatno zniža izražanje AGO2 pri obeh kultivarjih, kar lahko povzroči zmanjšano post-transkripcijsko utišanje genov, ki ga posredujejo AGO2 vezane male RNA. Prav tako smo opazili zmanjšano izražanje AGO7 pri občutljivem kultivarju prvi dan po inokulaciji in zmanjšano izražanje RDR6 pri odpornem kultivarju tretji dan. Slednje nakazuje na morebitno zavrtje biogeneze trans-delujočih malih RNA. V študiji identifikacije in diferenčnega izražanja miRNA smo odkrili 56 znanih in 43 novih miRNA. Kot odziv na okužbo z V. nonalfalfae smo zaznali spremembe v izražanju petih znanih in dveh novih miRNA v občutljivem kultivarju ter šestih znanih miRNA v odpornem kultivarju. Diferenčno izražene miRNA predvidoma utišajo 49 transkriptov, ki so pri občutljivem kultivarju vključeni v lokalizacijo proteinov in sintezo pigmentov, medtem ko so pri odpornem kultivarju vključeni v regulacijo transkripcijskih faktorjev in hormonsko signalizacijo.In the studies conducted as part of the doctoral dissertation, a resistant and a susceptible hop cultivar were artificially inoculated with the phytopathogenic fungus Verticillium nonalfalfae and the profiles of phenolic compounds and fungal colonization were determined at different sampling times after inoculation. We observed a bimodal pattern of colonization in the roots of the susceptible cultivar and the fungus spread to the stems, while the relative amount of fungus in the resistant cultivar decreased over the course of the experiment. In the roots of the resistant cultivar, a significant increase in total flavanols was observed on the third day after inoculation. In the stems, the accumulation of phenolics was higher in the susceptible cultivar than in the resistant cultivar as a significant increase in flavonols was observed on days 3 and 15 and in hydroxycinnamic acids on day 6 after inoculation. A study on the identification, characterization, phylogenetic, and expression analyses of three Argonaute genes, two Dicer-like protein genes and two RNA-dependent RNA polymerase genes in hops showed that they clustered with their orthologues from Cannabis sativa, Morus notabilis and Ziziphus jujuba. We found that AGO2 was significantly downregulated in both cultivars at day 3 post-inoculation, which may result in reduced post-transcriptional gene silencing mediated by AGO2-bound small RNAs. We also observed decreased expression of AGO7 in the susceptible cultivar on day 1 post-inoculation and decreased expression of RDR6 in the resistant cultivar on day 3, the latter possibly indicating suppression of biogenesis of trans-acting siRNAs. In a study of miRNA in hop, we identified 56 known and 43 novel miRNAs. In response to V. nonalfalfae infection, we detected changes in the expression of five known and two novel miRNAs in the susceptible cultivar and six known miRNAs in the resistant cultivar. The differentially expressed miRNAs silence 49 transcripts involved in protein localization and pigment synthesis in the susceptible variety, whereas they are involved in transcription factor regulation and hormone signaling in the resistant cultivar

Size effects of microplastic on the bioavailability in selected crustacean species

Mikroplastika (MP) so po definiciji plastični delci manjši od 5 mm, ki se vse pogosteje pojavljajo v okolju in imajo nanj lahko negativne učinke. V naravno okolje pridejo bodisi direktno preko izpustov čistilnih naprav ali pa nastanejo pri mehaničnem preoblikovanju večjih plastičnih delov. Zaradi majhnosti teh delcev obstaja veliko tveganje, da imajo na organizme negativen učinek. Do danes obstaja še veliko neznank glede potencialne biodostopnosti MP za manjše vodne nevretenčarje. Ker je večina MP lažja od vode, obstaja vprašanje, ali je le-ta sploh dostopna za organizme in kako velikost MP vpliva na biodostopnost. Glavni cilj naloge je bil ugotoviti, ali je MP velikostnega razreda od 20-1000 &#956m dostopna za izbrane testne organizme vodne bolhe Daphnia magna in morske rakce Artemia franciscana. Kot drugo nas je zanimalo, kakšni so učinki MP na organizme po akutni izpostavitvi. Izbrane testne organizme smo izpostavljali različnim tipom MP za 48 ur pri koncentraciji 100 mg/L. Ugotovili smo, da so testni organizmi sposobni zaužitja vseh tipov testirane MP, vendar pa MP ne vpliva na preživetje. Naši rezultati nakazujejo, da je biodostopnost MP odvisna od njihove velikosti: manjši delci so bolj biodostopni kot večji. Prav tako smo dokazali vpliv MP na velikost osebkov.Microplastics (MP) are plastic particles &#88045 mm, which have become prevalent in the environment and have negative effects on it. They enter the environment either directly through discharges of sewage plants or resulting from mechanical transformation of large plastic parts. Due to their small size, they pose a risk to have negative effects on organisms. To date, there are still many unknowns regarding the potential bioavailability for small aquatic invertebrates. Since most of MP are lighter than water, we do not know whether it is accessible to all organisms and how the size of the MP affect the bioavailability. The aim of this study was to determine bioavailability and effects of the MP in size range from 20 – 1000 &#956m on model organisms Daphnia magna and Artemia franciscana. Selected model organisms were exposed for up to 48 hours or two weeks at concentration, 100 mg/L. Model organisms ingested all types of tested MP. However, MP does not affect the mortality. Our results show that bioavailability is related to the particle size: smaller particles were ingested more readily than larger. They also have a potential impact on the size of the organisms

The role of small RNA molecules in plant response to pathogen infection

Rastline imajo razvite raznolike in kompleksne mehanizme za regulacijo izražanja genov. Nedavno je bil odkrit nov mehanizem, imenovan RNK interferenca (RNKi). Osrednjo vlogo v RNKi imajo male nekodirajoče RNK (sRNK) dolge od 21-24 nukleotidov, ki z vezavo na komplementarna mesta v transkriptih preprečijo njihovo prevajanje v proteine. Ker sRNK definira izvor, strukturne lastnosti prekurzorjev ter sekvenčne lastnosti, jih delimo v več različnih razredov. mikroRNK (miRNK) ter sekundarne male interferenčne RNK (siRNK), med katere prištevamo tasiRNK in phasiRNK imajo pomembno vlogo v regulaciji izražanja genov v številnih bioloških procesih ter odzivu rastlin na biotske ali abiotske dejavnike stresa. Kljub številnim ohranjenim sRNK med rastlinskimi vrstami ter karakterizaciji njihovega delovanja, do danes še ni celovitega razumevanja njihove vloge v obrambnem odzivu rastlin pred fitopatogeni. Ta pregled povzema trenutno razumevanje patogeneze verticilijske uvelosti, obrambnega mehanizma rastlin pred fitopatogeni in biogeneze ter vloge miRNK, tasiRNK ter phasiRNK v obrambnem odzivu rastlin pred glivnimi patogeni. Nadaljnje raziskave rastlinskih sRNK in njihovo izražanje v odzivu rastlin na različne fitopatogene organizme so potrebne za jasno določitev njihove vloge. Novi pristopi sekvenciranja ter bioinformacijske analize in napovedovanja vloge miRNK tarč v času okužb nam lahko pri nemodelnih organizmih omogočijo razvoj novih načinov varstva rastlin.Plants have evolved diverse and complex mechanisms to regulate gene expression. Recently, a new mechanism called RNA interference (RNAi) has been discovered. At the core of RNAi are small non-coding RNAs (sRNAs), 21-24 nucleotides in length, that prevent the translation of transcripts into proteins by binding to complementary sites in transcripts. Because sRNAs are determined by origin, precursor structural properties, and sequence characteristics, they are classified into several classes like microRNAs (miRNAs) and secondary small interfering RNAs (siRNAs), which include tasiRNAs and phasiRNAs. They play important roles in regulating gene expression in a wide range of biological processes and in plant responses to biotic or abiotic stresses. Despite the numerous conserved sRNAs among plant species and the characterization of their function, there is still no comprehensive understanding of their role in plant defense responses against phytopathogens. This review summarizes the current understanding of Verticillium wilt pathogenesis, plant defense mechanisms against phytopathogens, and the biogenesis and roles of miRNAs, tasiRNAs, and phasiRNAs in plant defense responses against fungal pathogens. Further studies on plant sRNAs and their expression in response to various phytopathogens are needed to clearly define their roles. New sequencing approaches, bioinformatic analysis, and prediction of the role of miRNA targets during infection may allow us to develop new forms of plant protection in non-model organisms

Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9

Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 µg of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts

Core RNA interference genes involved in miRNA and ta-siRNA biogenesis in hops and their expression analysis after challenging with Verticillium nonalfalfae

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation

Identification and characterization of Verticillium nonalfalfae-responsive microRNAs in the roots of resistant and susceptible hop cultivars

MicroRNAs are 21- to 24-nucleotide-long, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They can modulate various biological processes, including plant response and resistance to fungal pathogens. Hops are grown for use in the brewing industry and, recently, also for the pharmaceutical industry. Severe Verticillium wilt caused by the phytopathogenic fungus Verticillium nonalfalfae, is the main factor in yield loss in many crops, including hops (Humulus lupulus L.). In our study, we identified 56 known and 43 novel miRNAs and their expression patterns in the roots of susceptible and resistant hop cultivars after inoculation with V. nonalfalfae. In response to inoculation with V. nonalfalfae, we found five known and two novel miRNAs that are differentially expressed in the susceptible cultivar and six known miRNAs in the resistant cultivar. Differentially expressed miRNAs target 49 transcripts involved in protein localization and pigment synthesis in the susceptible cultivar, whereas they are involved in transcription factor regulation and hormone signalling in the resistant cultivar. The results of our study suggest that the susceptible and resistant hop cultivars respond differently to V. nonalfalfae inoculation at the miRNA level and that miRNAs may contribute to the successful defence of the resistant cultivar

Changes in the phenolic compounds of hop (Humulus lupulus L.) induced by infection with Verticillium nonalfalfae, the causal agent of hop Verticillium wilt

Phenolic compounds are involved in plant responses to various biotic and abiotic stress factors, with many studies suggesting their role in defense mechanisms against fungal pathogens. Soilborne vascular pathogen Verticillium nonalfalfae causes severe wilting and consequent dieback in a wide range of economically important crops, including hops (Humulus lupulus L.). In this study, we investigated the differential accumulation of phenolics in the susceptible “Celeia” and resistant “Wye Target” hop cultivars during the pathogenesis of Verticillium wilt. Quantitative polymerase chain reaction showed that colonization in the roots of both cultivars was intensive, but decreased continuously throughout the experiment in the resistant cultivar, while the relative fungal amount continuously increased in the stems of the susceptible cultivar. In response to colonization in the roots of the resistant cultivar, a significant increase in total flavanols was detected at three days postinoculation (dpi), suggesting a possible role in preventing fungus spread into the stems. The accumulation of phenolic compounds was less pronounced in the stems of the resistant cultivar since, compared to the latter, significant increases in flavonols at 3 and 15 dpi and hydroxycinnamic acids at 6 dpi were observed in the stems of the susceptible cultivar

The potential of HTS approaches for accurate genotyping in grapevine (Vitis vinifera L.)

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price

The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.)

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price