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.
Verticillium wilt (VW) can cause substantial yield loss in hop particularly with the outbreaks of the lethal strain of Verticillium albo-atrum. To elucidate genetic control of VW resistance in hop, ...an F₁ mapping population derived from a cross of cultivar Wye Target, with the predicted genetic basis of resistance, and susceptible male breeding line BL2/1 was developed to assess wilting symptoms and to perform QTL mapping. The genetic linkage map, constructed with 203 markers of various types using a pseudo-testcross strategy, formed ten major linkage groups (LG) of the maternal and paternal maps, covering 552.98 and 441.1 cM, respectively. A significant QTL for VW resistance was detected at LOD 7 on a single chromosomal region on LG03 of both parental maps, accounting for 24.2–26.0 % of the phenotypic variance. QTL analysis for alpha-acid content and yield parameters was also performed on this map. QTLs for these traits were also detected and confirmed our previously detected QTLs in a different pedigree and environment. The work provides the basis for exploration of QTL flanking markers for possible use in marker-assisted selection.
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.
Olive (Olea europaea L.) is an evergreen Mediterranean oil fruit tree with great economic, cultural and historical importance. For accurate gene expression studies of specific genes, reverse ...transcriptase–quantitative polymerase chain reaction (RT-qPCR) is often the method of choice, using suitable reference genes (RGs). This study identified RGs for RT-qPCR studies of developing olive fruit from 29 RG candidates. We used 12 sampling points to cover the five stages of olive fruit development. According to the results of the geNorm algorithm, the two best RGs were TIP41-like family protein (TIP41) and TATA binding protein (TBP), while several classical RGs proved not to be suitable. Using the two new RGs, four genes involved in the metabolism of fatty acids were studied and showed distinct expression patterns associated with mesocarp development and ripening stages. In addition to identifying two RGs for future analysis of gene expression in olive fruit, our results also provide a list of potential RGs that can be easily tested in other studies of olive gene expression in different developmental stages or in biologically challenged olive tissues. The results are also valuable for future research on genes that influence the synthesis and accumulation of olive fruit metabolites.
Summary
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.
•CHO cell line response to increased pCO2 at two commonly used culture temperatures was studied by 2D-DIGE.•Increased pCO2 caused reduced metabolic activity and less efficient glucose metabolism, ...particularly at the low culture temperature.•Increased pCO2 at reduced culture temperature induced anti-oxidative mechanisms and altered glucose metabolism toward anaerobic pathways.•Our results provide better understanding of CHO cell line response to increased pCO2 at a lower culture temperature.
The correlation between dissolved carbon dioxide (pCO2) and cell growth, cell metabolism, productivity and product quality has often been reported. However, since pCO2 values in bioprocesses always vary concurrently with other bioprocess variables, it is very difficult to distinguish only the effect of pCO2. The aim of our work was to investigate further the specific effect of pCO2 and cell response on a proteome level. Proteome responses of three different CHO-Der3 cell lines in the exponential growth phase at normal (37°C) and reduced (33°C) culture temperatures, with normal (10%) and increased (20%) pCO2, were studied by comparative proteomic analysis (2D-DIGE). Cell viability and cell density, and the concentration of glucose, glutamine and lactate monitored over 72-h cultures showed that elevated pCO2 did not affect cell viability or productivity at either culture temperature, while metabolic activity was reduced. The specific metabolic profile also indicated altered glucose metabolism toward a less efficient anaerobic metabolism. Two-way ANOVA of proteomic data discriminated many more pCO2-specific changes in protein abundance (p<0.01) at 33°C than at 37°C and PCA analysis was able to distinguish clusters distinguishing cell lines and culture conditions at low temperature and elevated pCO2, indicating substantial proteome changes under these culture conditions. Cell sensitivity to increased pCO2 at the lower temperature was further confirmed by a significantly increased abundance of twelve proteins involved in anti- oxidative mechanisms and increased abundance of six proteins involved in glycolysis, including L-lactate dehydrogenase. Proteomic results support the metabolic data and the proposed pCO2 invoked metabolic switch toward anaerobic pathways. Anti- oxidative mechanisms, together with the anaerobic metabolism, allow the cells to detoxify while maintaining sufficient energy levels to preserve their vitality and functionality. This study provides further insight into the proteome responses of CHO cell lines to increased pCO2 at the two culture temperatures.
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.
Field assessments of resistance to powdery mildew of 103 hop cultivars, analyses of hop essential oil and correlation between the score for powdery mildew and the relative percentage of essential oil ...compounds were performed over three years. Seven susceptibility markers (peaks 29 (methyl-5-methyl-hexanoate), 30 (myrcene), 34 (iso-amyl-iso-butyrate), 38 (1-8-cineole), 56 (methyl-octanoate), 88 (methyl decanoate) and 122 (undetermined peak)) and seven resistance markers (peaks 112 (santalene), 114 (germacrene-D), 118 (alpha-selinene), 138 (cariophylene epoxide), 26, 135 and 158 (undetermined peaks)) were selected from peaks with a positive or negative correlation between powdery mildew scores and their presence in the essential oil of extremely susceptible or resistance cultivars. The number and value of resistance/susceptibility markers decreased with an increase in the level of cultivar susceptibility/resistance. Susceptible cultivars mainly appeared to contain North American germplasm, while more resistant cultivars belong to European hops. Analysis of the presence/absence of the selected markers showed that the absence of susceptibility markers, particularly 30, 34 and 38, can be of practical value in resistance hop breeding.
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 signalling pathways. We report here that
, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a CBM18 (carbohydrate binding motif 18)-chitin binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the
gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however,
knockout mutants exhibit wilting symptoms similar to the wild type fungus, suggesting that
activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP binds chitin and chitin oligomers in vitro with submicromolar affinity and protects 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 modelling 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.
PDF
