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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.

V okviru raziskav za izvedbo magistrskega dela smo analizirali izražanje nekaterih mikro RNA (miRNA) in njihovih tarč v odpornem in občutljivem kultivarju hmelja po okužbi z glivo Verticillium nonalfalfae v štirih različnih časovnih točkah ter eksperimentalno analizirali razrez tarčnih mRNA. Iz koreninskih in stebelnih vzorcev hmelja smo izolirali celotno RNA ter izvedli metodi RT-qPCR in 5' RLM-RACE. Ugotovili smo, da se izražanje med kontrolo in okuženo rastlino kot odziv na okužbo razlikuje v vsaj eni preverjeni časovni točki pri petih od sedmih analiziranih tarč in pri treh od petih analiziranih miRNA. Pri petih tarčah in pri eni miRNA so kot odziv na okužbo prisotne tudi razlike v izražanju med kultivarjema. Potrdili smo interakcijo mRNA dejavnika odziva na avksin z miR160. S pridobljenimi rezultati smo pridobili boljše razumevanje imunskega odziva hmelja in hmeljnih mehanizmov odpornosti, ki lahko v prihodnje pomagajo pri obvladovanju bolezni verticilijske uvelosti. In the research for this master thesis we have analysed the expression of microRNAs (miRNAs) and their targets in the resistant and susceptible cultivars of hops after infection with fungus Verticillium nonalfalfae in four different time points and experimentally analysed the cleavage of target mRNAs. We have isolated total RNA from root and stem samples of hops and conducted RT-qPCR and 5’RLM-RACE methods. We have determined that expression between control and inoculated plant is changed in at least one tested time point in five out of seven analysed mRNAs and in three out of five analysed miRNAs. Changes in expression between cultivars due to inoculation is present in five targets and one miRNA. We have confirmed the interaction of the auxin response factor and miR160. With the gathered results we have gained better understanding of immune response in hops and hop immunity mechanisms, which can help with verticillium wilt disease damage control in the future.

Navadni hmelj (Humulus lupulus L.) je dvodomna trajna dvokaličnica, ki pripada družini konopljevk. Njegov primarni škodljivec je fitopatogena gliva Verticillium nonalfalfae. Ločujemo blago in letalno obliko verticilijske uvelosti, ki vsako leto lahko uniči od 10 do 50 % vsega pridelka. Patogen z namenom kolonizacije rastlinskega gostitelja in preskrbe glive s hranili izloča efektorje, s katerimi vpliva na celično fiziologijo gostitelja (transkripcija, signalizacija, epigenetska regulacija, sinteza in izločanje proteinov), ali modulira njegov imunski odgovor (sinteza sekundarnih metabolitov, signalizacija). Poznavanje strukture, mehanizma translokacije in biološke funkcije glivnih efektorjev je ključno za razumevanje mehanizmov patogenosti glive in oblikovanje strategij za trajno zaščito pridelka v Sloveniji in po svetu. Cilj magistrske naloge je bil pridobiti očiščena, fluorescenčno označena proteina VnaSSP4.2 in Vna7.443 ter ju uporabiti v bioloških in biokemijskih testih z namenom razumevanja njune funkcije. S kloniranjem In-Fusion smo uspešno pripravili konstrukte Vna7.443::DsRed2, DsRed2::Vna7.443 ter reporter DsRed2, jih izrazili v E. coli ter proteine očistili in stabilizirali. Prav tako smo v E. coli izrazili konstrukt eGFP::VnaSSP4.2 in rekombinantni protein očistili. Rekombinantni fluorescenčni protein eGFP::VnaSSP4.2 v listih modelne rastline N. benthamiana povzroči kloroze in se v plazmalemi specifično lokalizira v membranskih mikrodomenah. Rekombinantni fluorescenčni protein DsRed2::Vna7.443 s prostim C- terminalnim koncem morda interagira z genomsko DNA gostiteljske rastline H. lupulus L. Protein Vna7.443 je strukturno podoben proteinom, ki vsebujejo MORN ponovitve. Potencialne vezave Vna7.443 na DNA in vivo, po nastanku por, ki jih oblikuje VnaSSP4.2, nismo preverili, smo pa delno potrdili hipotezo, da bi tak mehanizem lahko obstajal. Common hop (Humulus lupulus L.) is a dioecious perennial dicotyledonous plant that belongs to the Cannabaceae family. Its primary pest is the phytopathogenic fungus Verticillium nonalfalfae. We distinguish mild and lethal forms of verticillium wilt, which can destroy from 10 up to 50% of the entire crop every year. To colonize the plant host and supply the fungus with nutrients, the pathogen secretes effectors that modulate the host's cellular physiology (transcription, signalling, epigenetic regulation, protein synthesis and secretion), or the immune responses (synthesis of secondary metabolites, signalling). Unravelling the structure, translocation mechanism and biological function of fungal effectors is key to understanding the mechanisms of fungal pathogenicity and devising strategies for permanent crop protection in Slovenia, and around the world. The goal of the master's thesis was to obtain purified, fluorescently labelled VnaSSP4.2 and Vna7.443 proteins and to use them in biological and biochemical assays to unveil their functions. Using In-Fusion cloning, we successfully prepared the constructs for Vna7.443::DsRed2, DsRed2::Vna7.443 and the reporter DsRed2, expressed them in E. coli, and purified and stabilized the proteins. Likewise, we expressed eGFP::VnaSSP4.2 and purified it. The recombinant fluorescent protein eGFP::VnaSSP4.2, injected into the leaves of the model plant N. benthamiana, causes chlorosis and localizes in plasma membrane within specific membrane microdomains. The recombinant fluorescent protein DsRed2::Vna7.443 with a free C-terminal end may interact with the genomic DNA of the host plant H. lupulus L. The Vna7.443 protein is structurally similar to proteins containing MORN repeats. The potential binding of Vna7.443 to DNA in vivo, after the formation of the pore induced by VnaSSP4.2, was not performed, although we partially confirmed the hypothesis that such a mechanism could exist.

Odkrivanje funkcije kandidatnih sekretornih efektorskih proteinov (CSEPs) je ključno za razumevanje interakcije med fitopatogenimi organizmi in rastlinskimi gostitelji. Hemibiotrofna fitopatogena gliva vaskularnega venenja Verticillium nonalfalfae, ki primarno okužuje hmelj (Humulus lupulus L.) sintetizira štiri opisane apoplastne efektorske proteine: VnaPRX1.1277 (peroksidaza razreda II), VnaSSP4.2, VnaUn.279 in VnaChtBP (hitin vezavni protein), znanih citoplazemskih efektorskih proteinov pa nima. Cilj magistrske naloge je bil karakterizacija kandidatnega sekretornega efektorskega proteina Vna7.443, izbranega na osnovi predhodnih študij. Proteinu Vna7.443 smo z bioinformacijskimi pristopi predvidili homologne proteine, potencialne tarčne celične procese in osnovne opisne parametre. Subcelično lokalizacijo fuzijskih proteinov Vna7.443 z mRFP (monomerni rdeči fluorescenčni protein) označbo na C- in N-terminalnem koncu smo izvedli s konfokalno mikroskopijo na listnih izsečkih Nicotiana benthamiana, ki smo jih posredno transformirali z Agrobacterium tumefaciens. Ugotovili smo, da: 1) je protein Vna7.443 najverjetneje citoplazemski efektorski protein, ki se, v primeru ko je C-terminalni konec prost, lokalizira v jedro rastlinske celice, 2) ne sproži preobčuljivostnega odziva v modelni rastlini, 3) vsebuje osem tandemskih ponovitev WD40 in 4) ima ohranjene homologe znotraj rodov gliv Verticillium, Cercospora in Fusarium. Detection of candidate secreted effector proteins (CSEPs) function is key to understanding the interaction between phytopathogenic organisms and plant hosts. The hemibiotrophic phytopathogenic vascular wilt fungus Verticillium nonalfalfae, which primarily infects hops (Humulus lupulus L.), produces four known apoplastic effector proteins VnaPRX1.1277 (class II peroxidase), VnaSSP4.2, VnaUn.279 and VnaChtBP (chitin binding protein), however, it has no known cytoplasmic effector proteins. The aim of the master's thesis was to characterize the candidate secretory effector protein Vna7.443, selected on the basis of previous studies. Homologous proteins, potential target cell processes, and basic descriptive parameters were determined for the Vna7.443 protein by bioinformatics approaches. Subcellular localization of Vna7.443 fusion proteins with mRFP labeling at the C- and N-terminal end was performed by confocal microscopy on Agrobacterium tumefaciens-transformed Nicotiana benthamiana leaf sections. The results showed that Vna7.443 protein is likely a cytoplasmic effector protein that, when the C-terminal end is free, localizes to the plant cell nucleus, but does not trigger a hypersensitive response in planta. Vna7.443 contains eight tandem WD40 repeats and has preserved homologues within fungal genuses Verticillium, Cercospora and Fusarium.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

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.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

Verticillium spp. are soil-borne fungi containing many pathogenic species that affect a wide range of cultivated plants. In potatoes, Verticillium dahliae, V. nonalfalfae and V. albo-atrum cause Verticillium wilt or, together with other pathogens, are responsible for the occurrence of the potato early dying disease. Verticillium pathogens are notoriously difficult to control, and techniques proved to be efficient, such as chemical soil fumigation, are causing environmental concerns worldwide. Phosphites (Phi) represent a class of chemicals with a good environmental footprint, that are currently used to control oomycete pathogens, such as the potato late blight agent, Phytophtora infestans. To determine the potential inhibitory effects of Phi on Verticillium spp., isolates of V. dahliae and V. nonalfalfae were tested in vitro and the IC50 of Phi was found to vary between 60.9 and 481.9 μg/mL. The effects of Phi were further tested in field trials, carried for two years, using Phi-treated potato plants infected with a V. nonalfalfae isolate. Infection progression was assessed in both years by qPCR and was found to be significantly reduced in Phi-treated plants. The concentration of Phi in these plants, determined by high performance ion chromatography, was found to vary between 25.0 and 86.2 μg/mL in leaves and between 55.7 and 113.8 μg/mL in tubers. Even though the concentrations of Phi required to control Verticillium spp. were found to be relatively high compared to those needed to inhibit the development of oomycete pathogens, the results from this study indicate that Phi can limit the development of Verticillium spp.

Verticillium wilt caused by Verticillium spp., also called potato early dying disease, is one of the most serious soilborne diseases affecting potato production in China. The disease has been expanding into most potato production areas over the past few years. Information on host resistance against Verticillium wilt among the potato cultivars in China is scarce, but it is critical for sustainable management of the disease. This study, therefore, evaluated 30 commercially popular potato cultivars against Verticillium dahliae strain Vdp83 and Verticillium nonalfalfae strain Vnp24, two well-characterized strains causing Verticillium wilt of potato in China. Both strains were isolated from diseased potato plants, and they were previously proven to be highly virulent. Ten plants of each cultivar were inoculated with the V. dahliae strain and incubated on greenhouse benches. Symptoms were rated at weekly intervals, and the relative area under the disease progress curve was calculated. The experiment was repeated once, and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals. Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V. nonalfalfae strain. Cultivar responses to V. nonalfalfae were like those exhibited against V. dahliae, except for one cultivar. This study showed that resistance among potato cultivars exists in China against Verticillium spp. and that the resistance to V. dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions. Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study.

Verticillium dahliae and Verticillium nonalfalfae are soil-borne fungal pathogens with a ubiquitous geographical distribution affecting a wide range of plants of economic importance. Though Verticillium wilt represents a major problem for crop production in Nova Scotia, Canada, little is known about the distribution of these pathogens in the major agricultural areas from this province. To address the paucity of available data, a molecular-based survey of these Verticillium pathogens, documenting their distribution, incidence, and in-sample abundance, was carried out for two successive years, in several representative agricultural locations in this province. Soil and plant samples (potato and strawberry) were analyzed using real-time quantitative PCR targeting the Intergenic Spacer ribosomal DNA locus of the Verticillium pathogens. Molecular data revealed that V. dahliae has a wider distribution and a higher incidence than V. nonalfalfae (former V. albo-atrum group 1) in the tested fields while V. albo-atrum sensu stricto (former V. albo-atrum group 2) was not identified in any of the samples collected from this region. Also, V. dahliae was found to have a higher incidence in fields used to grow potatoes as compared to fields used to grow strawberries, irrespective of the rotating crops.

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

(1) Background: Verticillium wilt (VW) of hop is a devastating disease caused by the soil-borne fungi Verticillium nonalfalfae and Verticillium dahliae. As suggested by quantitative trait locus (QTL) mapping and RNA-Seq analyses, the underlying molecular mechanisms of resistance in hop are complex, consisting of preformed and induced defense responses, including the synthesis of various phenolic compounds. (2) Methods: We determined the total polyphenolic content at two phenological stages in roots and stems of 14 hop varieties differing in VW resistance, examined the changes in the total polyphenols of VW resistant variety Wye Target (WT) and susceptible Celeia (CE) on infection with V. nonalfalfae, and assessed the antifungal activity of six commercial phenolic compounds and total polyphenolic extracts from roots and stems of VW resistant WT and susceptible CE on the growth of two different V. nonalfalfae hop pathotypes. (3) Results: Generally, total polyphenols were higher in roots than stems and increased with maturation of the hop. Before flowering, the majority of VW resistant varieties had a significantly higher content of total polyphenols in stems than susceptible varieties. At the symptomatic stage of VW disease, total polyphenols decreased in VW resistant WT and susceptible CE plants in both roots and stems. The antifungal activity of total polyphenolic extracts against V. nonalfalfae was higher in hop extracts from stems than those from roots. Among the tested phenolic compounds, only p-coumaric acid and tyrosol markedly restricted fungal growth. (4) Conclusions: Although the correlation between VW resistance and total polyphenols content is not straightforward, higher levels of total polyphenols in the stems of the majority of VW resistant hop varieties at early phenological stages probably contribute to fast and efficient activation of signaling pathways, leading to successful defense against V. nonalfalfae infection.

RNA-interferenca (RNAi) je ohranjen mehanizem regulacije izražanja genov, prisoten v živalih, rastlinah in glivah. Ravno v kraljestvu gliv, na modelnem organizmu Neurospora crassa, je bil mehanizem RNAi intenzivno preučevan, študije pa so vodile v odkritje glavnih komponent mehanizma, ki vodijo proces utišanja, to so proteini od RNA odvisna polimeraza RNA (RdRP), Dicer (DCL) ter Argonavt (AGO). Pri filamentoznih patogenih glivah, kamor uvrščamo tudi glivo Verticillium nonalfalfae (V. nonalfalfae), ki je povzročiteljica vaskularne bolezni hmelja, je bil mehanizem RNAi izpostavljen kot eden izmed mehanizmov patogenosti gliv. Male in mikro molekulam RNA podobne RNA (milRNA) so bile pri glivah vpletene ne samo v regulacijo izražanja endogenih genov, temveč izpostavljene kot efektorji, ki jih glive transportirajo v rastlinske gostitelje, kjer regulirajo izražanje gostiteljevih obrambnih mehanizmov. Zaradi pomembnega doprinosa mehanizma RNAi k patogenosti gliv smo v doktorski disertaciji okarakterizirali mehanizem RNAi pri glivi V. nonalfalfae z identifikacijo ključnih komponent sinteze malih RNA in milRNA ter komponente utišanja tarčnih genov. Potrdili smo obstoj in izražanje dveh proteinov RdRP, dveh DCL in dveh AGO. Nadaljnje smo identificirali ter potrdili izražanje devetih milRNA glive ter potrdili aktivnost mehanizma RNAi z eksperimentalno analizo razreza tarčnih mRNA, napovedanih v anotiranem genomu glive. Z raziskavami v okviru doktorske disertacije smo prvič poročali o obstoju mehanizma RNAi pri glivi V. nonalfalfae, kar bo bistveno doprineslo k razvoju novih strategij zatiranja škodljivca ter nadzora verticilijske bolezni hmelja. RNA interference (RNAi) is a conserved mechanism for regulating gene expression in animals, plants and fungi. The RNAi mechanism was studied extensively in the model fungus Neurospora crassa, and these studies led to the identification of the core components of the mechanism, the RNA-dependant RNA polymerase (RdRP), Dicer (DCL) and Argonaut (AGO), that mediate silencing events. In filamentous fungi, including Verticillium nonalfalfae (V. nonalfalfae), the causal agent of wilting disease of hops, the RNAi mechanism was identified as one of the pathogenic mechanisms of these fungi. Small and microRNA-like RNAs (milRNAs) were implicated not only in regulating endogenous genes but also as effectors that fungi can export into host plants, where they regulate the expression of host defence mechanisms. Due to the important contribution of RNAi to the pathogenicity of fungi, in this doctoral dissertation, we characterised the RNAi mecahanism of V. nonalfalfae by identifying the core components of small and milRNA synthesis and the components required for gene silencing. We confirmed the existence and expression of two RdRP, two DCL and two AGO proteins. Furthermore, we identified and validated the expression of nine fungal milRNAs and confirmed the activity of the RNAi by experimental analysis of the target cleavage sites of the target gene models identified in silico in the annotated fungal genome. With the studies conducted in the scope of the doctoral dissertation, we reported on the existence of RNAi in V. nonalfalfae for the first time, and the obtained knowledge will help in the development of new strategies for the management of verticillium wilt disease in hops.

Verticillium nonalfalfae, a soilborne vascular fungus, shows promise for biocontrol of highly invasive Ailanthus altissima strains. This announcement provides draft genome sequences of the aggressive isolate G1/5 (wild-type strain), the highly aggressive isolate Vert56 (improved strain), and the mildly aggressive isolate I3/2, all obtained from symptomatic A. altissima trees in Austria.

The naturally occurring Verticillium nonalfalfae shows promise for biocontrol of the highly invasive Tree of Heaven (Ailanthus altissima), but might also bear a risk for non-target tree species. In this study, we conducted inoculations on potted seedlings of A. altissima as well as on eight indigenous and two invasive tree species associated with Tree of Heaven in Austria. Although vascular discolourations developed in all inoculated tree species, V. nonalfalfae was reisolated from Ailanthus and eight of the ten non-target-species, whereas typical disease symptoms and mortality only occurred on A. altissima. Results confirmed high susceptibility (S) of A. altissima to V. nonalfalfae but indicated tolerance (T) of Acer campestre, Acer pseudoplatanus and Quercus robur, possible resistance (PR) of Fraxinus excelsior, Populus nigra, Tilia cordata, Ulmus laevis and Ulmus minor and resistance (R) of Fraxinus pennsylvanica and Robinia pseudoacacia to this potential biocontrol agent. Results from seedling inoculations were confirmed by cursory field observations in Ailanthus-inoculated forest stands, where admixed A. campestre, A. pseudoplatanus, F. excelsior, Populus alba, R. pseudoacacia and U. laevis canopy trees remained asymptomatic, while mortality was induced in Ailanthus.

Ailanthus altissima (Mill.) swingle is a highly invasive tree that has become established worldwide, especially in the Mediterranean Basin because of its good drought resistance. Ailanthus altissima is included in the list of Invasive Alien Species of the EU, so measures for eradication and management are required. Assessment for potential biological control agents is of great interest to manage this invasive tree in natural ecosystems. Verticillium dahliae Kleb. and Verticillium nonalfalfae Inderb. et al. (formerly V. albo-atrum Reinke & Berthold) have been reported as the causal agents of Verticillium wilt and mortality of ailanthus (Shall and Davis 2009; Rebbeck et al., 2013; Snyder et al., 2013; Brooks et al. 2020). Ailanthus trees with Verticillium wilt symptoms (wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality) were detected for the first time in 2007 in Celra (42.040466N, 2.864436E) (Catalonia, Northeastern Spain), then spread to neighboring ailanthus populations. In 2018, ailanthus trees in a 570 km2 area in Catalonia were surveyed for disease symptoms. The incidence of wilt disease in ailanthus trees in forest ecosystems ranged from 50 to 90%, and the severity, 60 to 92%. One hundred and fifty branch samples showing wilt symptoms were collected and disinfected by immersion in 1% sodium hypochlorite for 2 min, then cut into 5mm pieces. These were placed onto PDA plates and incubated at 22.5 °C and 12 h light photoperiod for 7-10 days. Eighty-four tentative Verticillium sp. isolates were recovered and subcultured on modified water agar (WA-p) and PDA for identification (Inderbitzin et al. 2011, 2013). The majority of isolates (77 %) were identified as V. dahliae based on morphology; production of brown-pigmented microsclerotia and conidia features and dimensions (5.7 ± 0.9 μm long). Sequencing of mycelial DNA using primer pair ITS1-F and ITS4, resulted in consensus sequences of 503 bp. BLASTn analysis of ITS sequence of native isolate VdGi688 gave 100% identity to the ITS sequences of V. dahliae type strain PD322 (92% coverage) and Vd16_9 (100% coverage). In addition, 23% isolates morphologically corresponded to V. albo-atrum or V. nonalfalfae; melanized resting mycelia and round to oval-shaped conidia (5.2 ± 0.9 μm × 2.2 ± 0.5 μm). The ITS consensus sequence (544 bp) of native isolate VaaGi02 gave 99% identity (90-100 % coverage) to V. albo-atrum isolates CBS 127169, PSU 140, Vaa_TN1 and to V. nonalfalfae type PD592, CBS5451.88 and Vert 18. Sequences from isolates VdGi688 and VaaGi02 were deposited in GenBank as MW624723 and MW624724, respectively. Koch's postulates for seven V. dahliae isolates and eight V. albo-atrum isolates were fulfilled by injection of 1 mL of 1 x 107 conidia/mL suspension into the stem of A. altissima seedlings under greenhouse conditions. Six plants were inoculated per isolate in two independent experiments. Control plants were inoculated with sterile distilled water. All isolates caused leaf chlorosis, defoliation, and apical stem death, as well as internal necrosis and vascular discoloration. Control plants remained asymptomatic. The pathogens were re-isolated from internal symptomatic tissues of inoculated plants. To our knowledge, this is the first report of V. dahliae and V. albo-atrum sensu lato causing Verticillium wilt on A. altissima in Spain. The study suggests the potential of native isolates of Verticillum spp. in the biological control of ailanthus in the Mediterranean Basin. This work was funded by the Diputacio de Girona (Spain) (2017/8719, 2019/3091, 2020/7565, and 2021/1468).

BackgroundMicro RNAs are 21- to 24-nucleotide-long, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They can modulate various biochemical and physiological processes, including plant response and resistance to fungal pathogens, by regulating the expression of numerous genes. Hops are grown for use in the brewing industry and have recently attracted the attention of the pharmaceutical industry. Severe Verticillium wilt disease caused by the phytopathogenic fungus Verticillium nonalfalfae is the main factor in yield loss in many crops, including hops (Humulus lupulus L.). ResultsIn our study, we identified miRNAs in hops and their expression patterns in the roots of susceptible and resistant hop cultivars in the early stages of infection with the fungus V. nonalfalfae. In total 56 known and 43 novel miRNAs were predicted. In response to infection 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 and their gene ontology enrichment analysis showed that the susceptible cultivar responds by modulating the processes of protein localization and pigment synthesis, whereas the resistant cultivar responds by modulating transcription factors and hormone signalling.ConclusionThe results of our study suggest that the susceptible and the resistant cultivar respond differently at the miRNA level to infection with V. nonalfalfae and that miRNAs may contribute to the successful defence of the resistant cultivar.

Key Message Dynamic transcriptome profiling revealed excessive, yet ineffective, immune response to V. nonalfalfae infection in susceptible hop, global gene downregulation in shoots of resistant hop and only a few infection-associated genes in roots. Abstract Hop (Humulus lupulus L.) production is hampered by Verticillium wilt, a disease predominantly caused by the soil-borne fungus Verticillium nonalfalfae. Only a few hop cultivars exhibit resistance towards it and mechanisms of this resistance have not been discovered. In this study, we compared global transcriptional responses in roots and shoots of resistant and susceptible hop plants infected by a lethal strain of V. nonalfalfae. Time-series differential gene expression profiles between infected and mock inoculated plants were determined and subjected to network-based analysis of functional enrichment. In the resistant hop cultivar, a remarkably low number of genes were differentially expressed in roots in response to V. nonalfalfae infection, while the majority of differentially expressed genes were down-regulated in shoots. The most significantly affected genes were related to cutin biosynthesis, cell wall biogenesis, lateral root development and terpenoid biosynthesis. On the other hand, susceptible hop exhibited a strong defence response in shoots and roots, including increased expression of genes associated with plant responses, such as innate immunity, wounding, jasmonic acid pathway and chitinase activity. Strong induction of defence-associated genes in susceptible hop and a low number of infection-responsive genes in the roots of resistant hop are consistent with previous findings, confirming the pattern of excessive response of the susceptible cultivar, which ultimately fails to protect the plant from V. nonalfalfae. This research offers a multifaceted overview of transcriptional responses of susceptible and resistant hop cultivars to V. nonalfalfae infection and represents a valuable resource in the study of this plant-pathogen interaction. Electronic supplementary material The online version of this article (doi:10.1007/s00299-017-2177-1) contains supplementary material, which is available to authorized users.

Hop (Humulus lupulus L.) is grown mostly as flavouring and bittering ingredient for beer and is also appreciated in the herbal and cosmetic industry, as well as in pharmacology. Among several diseases that damage hop growing, the most devastating in European hop production is verticillium wilt, caused by the soil-borne fungal pathogen Verticillium nonalfalfae. Colonization pattern and differential expression of selected genes after artificial infection of susceptible and resistant hop cultivars with V. nonalfalafae in stems and roots have been analysed recently Švara et al., 2019. Here, we present the dataset related to verification of plant samples infections after artificial inoculation (fungi- and mock-inoculated). After inoculation plant samples were tested for the positive infection by PCR amplification of the V. nonalfalfae ITS DNA region with species specific primers developed and optimised for this purpose. For more insight please see the article “Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae”. Keywords: Verticilium nonalfalfae, Hop, Artificial infection rate, Molecular markers, ITS primers

Pri hmelju (Humulus lupulus L.) je poznana letalna oblika verticilijske uvelosti, ki se odraža z bolezenskimi znaki uvelosti, klorozami, nekrozami in končnim propadom rastline. Bolezen povzroča fitopatogena gliva Verticillium nonalfalfae (Vna), ki za uspešno kolonizacijo rastline izloča efektorske proteine. Ti jo ščitijo pred obrambnim odzivom rastline, modulirajo imunski odziv ali vplivajo na fiziologijo rastline. Razumevanje funkcije kandidatnih sekretornih efektorskih proteinov (CSEPs) in poznavanje molekul gostitelja, s katerimi določen CSEP interagira, je ključno pri načrtovanju novih strategij zatiranja verticiljske uvelosti in lahko pospeši žlahtnjenje kultivarjev hmelja odpornih na Vna. V okviru te doktorske disertacije smo okarakterizirali kandidatna efektorska proteina VnaChtBP in VnaSSP4.2, ki smo ju izbrali na osnovi predhodnih študij. S konfokalno mikroskopijo smo pri modelni rastlini Nicotiana benthamiana ugotovili nukleo-citosolno subcelično lokalizacijo obeh izbranih CSEPs. Z infiltracijo rekombinantnih proteinov VnaChtBP in VnaSSP4.2 v različne rastline nismo uspeli potrditi hipoteze o njihovi vpletenosti v PTI ali ETI odziv. Ugotovili smo, da se efektor VnaChtBP specifično veže na hitin in ščiti glivo pred razgradnjo z rastlinskimi hitinazami. Pokazali smo, da se rekombinantni protein VnaSSP4.2 veže na molekule PIP, kardiolipin in sulfatid, ki so opisani kot modulatorji imunskega odziva. Outbreaks of a lethal type of Verticillium wilt of hops (Humulus lupulus L.) are reported yearly. This disease is caused by a phytopathogenic fungus Verticillium nonalfalfae (Vna) and presents in wilting, chlorosis, necrosis, and plant death. In order to successfully colonize its host, Vna secrets effector proteins, which protect the fungus and modulate the plant immune system or plant physiology. Understanding the function of candidate secreted effector proteins (CSEPs) and knowledge of host interacting molecules is a crucial contribution to development of effective strategies for pest control and can accelerate breeding of Vna resistant cultivars. In the presented doctoral thesis, we characterized candidate effector proteins VnaChtBP and VnaSSP4.2, which were selected based on previous studies. Using confocal microscopy, a nucleo-cytosolic subcellular localization was determined for both effectors expressed in model plant Nicotiana benthamiana. Infiltration of recombinant proteins to various plants did not confirm the hypothesis of VnaChtBP or VnaSSP4.2 involvement in PTI or ETI responses. We were able to show that the recombinant protein VnaSSP4.2 binds to PIP, cadiolipin and sulfatide molecules, all known modulators of plant immunity. We determined that the effector VnaChtBP binds specifically to chitin and protects the fungus from degradation by plant chitinases.

Extensive, unprecedented wilt and mortality of the highly invasive tree-of-heaven (Ailanthus altissima) occurred recently within mixed-hardwood forests in south-central Pennsylvania. Until this study, the cause of the epidemic was unknown. Verticillium albo-atrum was consistently isolated from symptomatic Ailanthus seedlings and trees in areas having high levels of mortality, whereas V. dahliae was isolated from small scattered patches of diseased Ailanthus. Inoculations of potted Ailanthus seedlings in the greenhouse, as well as canopy trees in the field, revealed that both V. albo-atrum and V. dahliae were capable of infecting Ailanthus, but V. albo-atrum was more pathogenic. All Ailanthus seedlings and canopy trees inoculated with V. albo-atrum died within 3 months. In contrast, Ailanthus seedlings and canopy trees inoculated with V. dahliae became symptomatic, but 84% were still living 1 year following inoculation. We conclude that the major cause of Ailanthus wilt within forests of south-central Pennsylvania is V. albo-atrum. Pending further studies, including risk analysis, V. albo-atrum may be considered as a possible biocontrol agent for invasive Ailanthus.

Verticillium albo-atrum causes unprecedented wilt and mortality of the invasive tree-of-heaven (Ailanthus altissima) within forests of south-central Pennsylvania. Stem inoculation of potted Ailanthus seedlings in the greenhouse and canopy Ailanthus trees in the field with V. albo-atrum resulted in 100% mortality. Stem inoculation of understory striped maple saplings in the field also resulted in 100% mortality. However, the high susceptibility of striped maple was not observed in naturally infected stands, where only 1% of striped maple saplings exhibited Verticillium wilt. Inoculation of chestnut oak, northern red oak, red maple, sugar maple, white ash, and yellow-poplar seedlings or canopy trees with V. albo-atrum did not induce wilt symptoms. Non-Ailanthus tree species growing adjacent to dead and dying Ailanthus trees in the field were asymptomatic. Pending further studies, V. albo-atrum should be considered as a potential biocontrol agent for invasive Ailanthus.

Ailanthus altissima (Mill.) Swingle, commonly known as tree-of-heaven, is an invasive tree species that has spread throughout the United States since its introduction in 1784 (2). During a survey in July 2009, approximately 1,100 A. altissima trees were observed at two locations in western Virginia (a roadside in Montgomery Co. and a wooded area adjacent to a railroad in Bedford Co.) exhibiting foliar wilt symptoms, defoliation, yellowish vascular discoloration, or death at an incidence of ~77%. Similar symptoms on A. altissima were reported in Roanoke, VA in the early 1930s and after 2005 in Pennsylvania, attributed to a Verticillium sp. (1,2). To identify the causal agent, discolored xylem tissue samples were excised from 10 symptomatic A. altissima trees at both locations, soaked in 1% NaOCl for 2 min, rinsed with sterilized distilled water for 5 min, and placed onto plum extract agar. Cultures were incubated in the dark at 22°C for 7 to 14 days. The resultant colonies (three to four per location) were subcultured and identified putatively as a Verticillium sp. closely related to Verticillium albo-atrum Reinke and Berthold (3), based on melanized, thick-walled, resting mycelia and phialides arranged in verticillate whorls that amassed round, oval-shaped conidia (5.1 ± 1.2 μm × 2.8 ± 0.4 μm, n = 100). Molecular identification of two fungal isolates (one per location) was determined by amplification of the protein coding genes elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS), using PCR primers developed recently for Verticillium (3). A BLAST search on the edited contigs revealed 100% sequence similarity for all three protein coding genes among the two isolates and reference sequences of isolates PD592 (GenBank Accessions JN188227, JN188163, and JN188035 for EF, GPD, and TS, respectively) and VnAaPA140 (KC307764, KC307766, and KC307768 for EF, GPD, and TS, respectively) of the newly-proposed species, V. nonalfalfae (formerly V. albo-atrum). Aligned sequences from one representative isolate, VnAaVA2 (Bedford Co.), were deposited into GenBank as KC307758 (EF), KC307759 (GPD), and KC307760 (TS). To confirm pathogenicity to A. altissima, the two molecularly characterized isolates (one per location) were inoculated into 18 10-week old A. altissima stems that were grown in an environmental chamber at 24°C, 60% RH, and a 12-h photoperiod from seeds collected in Blacksburg, VA. A conidial suspension of each isolate was injected into each stem (0.1 ml of 1 × 108 CFU/ml/stem). All 36 seedlings inoculated with the proposed V. nonalfalfae isolates developed wilting of leaflets within 2 weeks post-inoculation (WPI), defoliation of leaflets by 6 WPI, and were dead by 9 WPI. Eighteen control seedlings were inoculated similarly with distilled water, and remained asymptomatic. Fungi resembling the proposed species V. nonalfalfae were reisolated from all inoculated stems except the control plants, and the species confirmed morphologically as described above. V. nonalfalfae is a recently proposed species that can infect a variety of plant species (3). To our knowledge, this is the first report of this proposed species on A. altissima in Virginia. New state reports of this pathogen on A. altissima are important for regulatory issues associated with using this pathogen as a potential biological control agent. References: (1) G. F. Gravatt and R. B. Clapper. Plant Dis. Rep. 16:96, 1932. (2) M. J. Schall and D. D. Davis. Plant Dis. 93:747, 2009. (3) P. Inderbitzin et al. PLoS ONE, 6, e28341, 2011.