GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae.

KEY MESSAGE: GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae  with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

Genetic Structure and Core Collection of Olive Germplasm from Albania Revealed by Microsatellite Markers.

Olive is considered one of the oldest and the most important cultivated fruit trees in Albania. In the present study, the genetic diversity and structure of Albanian olive germplasm is represented by a set of 194 olive genotypes collected in-situ in their natural ecosystems and in the ex-situ collection. The study was conducted using 26 microsatellite markers (14 genomic SSR and 12 Expressed Sequence Tag microsatellites). The identity analysis revealed 183 unique genotypes. Genetic distance-based and model-based Bayesian analyses were used to investigate the genetic diversity, relatedness, and the partitioning of the genetic variability among the Albanian olive germplasm. The genetic distance-based analysis grouped olives into 12 clusters, with an average similarity of 50.9%. Albanian native olives clustered in one main group separated from introduced foreign cultivars, which was also supported by Principal Coordinate Analysis (PCoA) and model-based methods. A core collection of 57 genotypes representing all allelic richness found in Albanian germplasm was developed for the first time. Herein, we report the first extended genetic characterization and structure of olive germplasm in Albania. The findings suggest that Albanian olive germplasm is a unique gene pool and provides an interesting genetic basis for breeding programs.

Hop Polyphenols in Relation to Verticillium Wilt Resistance and Their Antifungal Activity.

(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.

New Male Specific Markers for Hop and Application in Breeding Program.

Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94-100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity.

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.

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion.

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

Genome Sequence of a Lethal Strain of Xylem-Invading Verticillium nonalfalfae.

Verticillium nonalfalfae, a soilborne vascular phytopathogenic fungus, causes wilt disease in several crop species. Of great concern are outbreaks of highly aggressive V. nonalfalfae strains, which cause a devastating wilt disease in European hops. We report here the genome sequence and annotation of V. nonalfalfae strain T2, providing genomic information that will allow better understanding of the molecular mechanisms underlying the development of highly aggressive strains.

Development of hop transcriptome to support research into host-viroid interactions.

Viroids, the smallest known pathogens, unable to encode any proteins, can cause severe diseases in their host plants. One of the proposed mechanisms of their pathogenicity includes silencing the host's genes via viroid-derived small RNAs, which are products of the host's immune response to the viroid's double stranded RNA. Humulus lupulus (hop) plants are hosts to several viroids; two of them, HLVd and CBCVd, are interesting models for studying host-viroid interactions, due to the symptomless infection of the former and severe stunting disease caused by the latter. To study these interactions, we constructed a deep hop NGS transcriptome based on 35 Gb paired-end sequencing data assembled into over 74 Mb of contigs. These transcripts were used for in-silico prediction of target transcripts of vd-sRNA of the two aforementioned viroids, using two different software tools. Prediction models revealed that 1062 and 1387 hop transcripts share nucleotide similarities with HLVd- and CBCVd-derived small RNAs, respectively, so they could be silenced in an RNA interference process. Furthermore, we selected 17 transcripts from 4 groups of targets involved in the metabolism of plant hormones, small RNA biogenesis, transcripts with high complementarity with viroid-derived small RNAs and transcripts targeted by CBCVd-derived small RNAs with high cellular concentrations. Their expression was monitored by reverse transcription quantitative PCR performed using leaf, flower and cone samples. Additionally, the expression of 5 pathogenesis related genes was monitored. Expression analysis confirmed high expression levels of four pathogenesis related genes in leaves of HLVd and CBCVd infected hop plants. Expression fluctuations were observed for the majority of targets, with possible evidence of downregulation of GATA transcription factor by CBCVd- and of linoleate 13S-lipoxygenase by HLVd-derived small RNAs. These results provide a deep transcriptome of hop and the first insights into complex viroid-hop plant interactions.

Comparative transcriptional analysis of hop responses to infection with Verticillium nonalfalfae.

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. 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.

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants.

Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant's xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections of hop plants and disease scoring are prerequisites for studying the pathogen's virulence/pathogenicity and its interaction with hop plants. In this protocol, we describe the root dipping inoculation method for conducting pathogenicity assay of V. nonalfalfae on hop plants.

Xylem Sap Extraction Method from Hop Plants.

Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many different virulence factors. Xylem sap of infected plants is therefore a rich source for investigating the molecules that are involved in molecular interactions of Verticillium - hop plants. This protocol provides instructions on how to infect hop plants with V. nonalfalfae artificially and how to obtain xylem sap from hop plants.

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor.

Plant-pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface-localized receptor Ve1 confers race-specific resistance against race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.

Identification and characterization of microRNAs in Humulus lupulus using high-throughput sequencing and their response to Citrus bark cracking viroid (CBCVd) infection.

BACKGROUND: Hop (Humulus lupulus L.) plants are grown primarily for the brewing industry and have been used as a traditional medicinal herb for a long time. Severe hop stunt disease caused by the recently discovered Citrus bark cracking viroid (CBCVd) is one of the most devastating diseases among other viroid infections in hop. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in gene expression regulation. To identify miRNAs in hop and their response to CBCVd-infection, two small RNA (sRNA) libraries were prepared from healthy and CBCVd-infected hop plants and were investigated by high throughput sequencing. RESULTS: A total of 67 conserved and 49 novel miRNAs were identified. Among them, 36 conserved and 37 novel miRNAs were found to be differentially recovered in response to CBCVd-infection. A total of 311 potential targets was predicted for conserved and novel miRNAs based on a sequence homology search using hop transcriptome data. The majority of predicted targets significantly belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. In addition, the identified miRNAs might also play an important roles in other cellular and metabolic processes, such as signal transduction, stress response and other physiological processes, including prenylflavonoid biosynthesis pathways. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs. CONCLUSIONS: Based on the results, we concluded that CBCVd-responsive miRNAs modulate several hormone pathways and transcriptional factors that play important roles in the regulation of metabolism, growth and development. These results provide a framework for further analysis of regulatory roles of sRNAs in plant defense mechanism including other hop infecting viroids in particular.

Hop (Humulus lupulus L.) response mechanisms in drought stress: Proteomic analysis with physiology.

Drought is one of the major environmental devastating stressors that impair the growth and productivity of crop plants. Despite the relevance of drought stress, changes in physiology and resistance mechanisms are not completely understood for certain crops, including hop (Humulus lupulus L.). In this research the drought response of hop was studied using a conventional physiological approach (gas exchange techniques, fluorescence, relative water content measurements) and proteomic analysis (2D-DIGE). Plants of two cultivars (Aurora and Savinjski golding) were exposed to progressive drought in a pot experiment and analysed at different stress stages (mild, moderate and severe). Measurements of relative water content revealed a hydrostable water balance of hop. Photosynthesis was decreased due to stomatal and non-stomatal limitation to the same extent in both cultivars. Of 28 identified differentially abundant proteins, the majority were down regulated and included in photosynthetic (41%) and sugar metabolism (33%). Fifteen % of identified proteins were classified into the nitrogen metabolism, 4% were related to a ROS related pathway and 7% to other functions.

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae.

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found-a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker.

Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants.

Different Gene Expressions of Resistant and Susceptible Hop Cultivars in Response to Infection with a Highly Aggressive Strain of Verticillium albo-atrum.

Verticillium wilt has become a serious threat to hop production in Europe due to outbreaks of lethal wilt caused by a highly virulent strain of Verticillium albo-atrum. In order to enhance our understanding of resistance mechanisms, the fungal colonization patterns and interactions of resistant and susceptible hop cultivars infected with V. albo-atrum were analysed in time course experiments. Quantification of fungal DNA showed marked differences in spatial and temporal fungal colonization patterns in the two cultivars. Two differential display methods obtained 217 transcripts with altered expression, of which 84 showed similarity to plant proteins and 8 to fungal proteins. Gene ontology categorised them into cellular and metabolic processes, response to stimuli, biological regulation, biogenesis and localization. The expression patterns of 17 transcripts with possible implication in plant immunity were examined by real-time PCR (RT-qPCR). Our results showed strong expression of genes encoding pathogenesis-related (PR) proteins in susceptible plants and strong upregulation of genes implicated in ubiquitination and vesicle trafficking in the incompatible interaction and their downregulation in susceptible plants, suggesting the involvement of these processes in the hop resistance reaction. In the resistant cultivar, the RT-qPCR expression patterns of most genes showed their peak at 20 dpi and declined towards 30 dpi, comparable to the gene expression pattern of in planta detected fungal protein and coinciding with the highest fungal biomass in plants at 15 dpi. These expression patterns suggest that the defence response in the resistant cultivar is strong enough at 20 dpi to restrict further fungus colonization.

New microsatellite markers for Campanula pyramidalis (Campanulaceae) and cross-amplification in closely related species.

PREMISE OF THE STUDY: Microsatellite markers were identified and characterized to study the genetic diversity and structure, conservation status, taxonomy, and biogeography of subspecific taxa and populations of Campanula pyramidalis (Campanulaceae). METHODS AND RESULTS: Eleven microsatellite markers were developed from genomic libraries enriched for di- and trinucleotide repeats. A total of 80 alleles were observed in the tested natural population. The number of alleles per locus, observed heterozygosity, and expected heterozygosity ranged from four to 13, 0.217 to 0.913, and 0.521 to 0.895, respectively. CONCLUSIONS: The new microsatellite markers will be useful for studying genetic diversity and structure as well as for better assessing the conservation status of subspecific taxa and populations of C. pyramidalis. Furthermore, a set of seven loci was successfully cross-amplified in C. secundiflora and C. versicolor and will be of great value for addressing unsolved taxonomic and biogeographic issues within the C. pyramidalis species complex.

The secretome of vascular wilt pathogen Verticillium albo-atrum in simulated xylem fluid.

Verticillium albo-atrum is a vascular wilt pathogen capable of infecting many important dicotyledonous plant species. Fungal isolates from hop differ in aggressiveness, causing either mild or lethal symptoms in infected plants. As in other plant pathogenic fungi, extracellular proteins, such as cell wall-degrading enzymes and effectors, are thought to be crucial in the pathogenesis process. In this study, mild and lethal isolates from three countries were grown in simulated xylem medium and secretome analysis by 2D-DIGE showed low qualitative and high quantitative variability among the isolates. Functional classification of 194 identified proteins representing 100 unique protein accessions revealed an arsenal of cell wall-degrading enzymes and potential effectors. The set of proteins that were more abundant in at least two lethal isolates included enzymes acetylcholinesterases, lipases, polygalacturonases, pectate lyase, rhamnogalacturonan acetylesterases, acetylxylan esterase, endoglucanase, xylanases, mannosidases, and a protein similar to alginate lyase and also potential effectors necrosis- and ethylene-inducing protein, small basic 14 kDa hypothetical protein and 79 kDa hypothetical proteins. Other proteins associated with virulence showed different expression profiles between mild and lethal isolates. The results suggest that the increased virulence of lethal isolates has little background shared by all three lethal isolates and that upregulation of isolate specific sets of proteins may be most important.

Evaluation of reference genes for RT-qPCR expression studies in hop (Humulus lupulus L.) during infection with vascular pathogen verticillium albo-atrum.

Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses.