RESUMEN
The complexity of the interaction between the necrotrophic pathogen Botrytis cinerea and grape berries (Vitis vinifera spp.) can result in the formation of either the preferred noble rot (NR) or the loss-making grey rot (GR), depending on the prevailing climatic conditions. In this study, we focus on the functional gene set of V. vinifera by performing multidimensional scaling followed by differential expression and enrichment analyses. The aim of this study is to identify the differences in gene expression between grape berries in the phases of grey rot, noble rot, and developing rot (DR, in its early stages) phases. The grapevine transcriptome at the NR phase was found to exhibit significant differences from that at the DR and GR stages, which displayed strong similarities. Similarly, several plant defence-related pathways, including plant-pathogen interactions as hypersensitive plant responses were found to be enriched. The results of the analyses identified a potential plant stress response pathway (SGT1 activated hypersensitive response) that was found to be upregulated in the GR berry but downregulated in the NR berry. The study revealed a decrease in defence-related in V. vinifera genes during the NR stages, with a high degree of variability in functions, particularly in enriched pathways. This indicates that the plant is not actively defending itself against Botrytis cinerea, which is otherwise present on its surface with high biomass. This discrepancy underscores the notion that during the NR phase, the grapevine and the pathogenic fungi interact in a state of equilibrium. Conversely the initial stages of botrytis infection manifest as a virulent fungus-plant interaction, irrespective of whether the outcome is grey or noble rot.
RESUMEN
BACKGROUND: High-quality RNA extraction from woody plants is difficult because of the presence of polysaccharides and polyphenolics that bind or co-precipitate with the RNA. The CTAB (cetyl trimethylammonium bromide) based method is widely used for the isolation of nucleic acids from polysaccharide-rich plants. Despite the widespread use of the CTAB method, it is necessary to adapt it to particular plant species, tissues and organs. Here we described a simple and generalized method for RNA isolation from mature leaf tissues of several economically important woody (17) and herbaceous plants (2) rich in secondary metabolites. High yields were achieved from small amount (up to 50 mg) of plant material. Two main modifications were applied to the basic protocol: an increase in ß-mercaptoethanol concentration (to 10%v/v) and the use of an effective DNase treatment. As opposed to similar studies, we tried to describe a more detailed protocol for isolating RNA, including the exact quantity and concentration of the reagents were used. RESULTS: Our modified CTAB method is proved to be efficient in extracting the total RNA from a broad range of woody and herbaceous species. The RNA yield was ranged from 2.37 to 91.33 µg/µl. The A260:A280 and A260:A230 absorbance ratios were measured from 1.77 to 2.13 and from 1.81 to 2.22. The RIN value (RNA Integrity Number) of the samples fell between 7.1 and 8.1, which indicated that a small degree of RNA degradation occurred during extraction. The presence of a single peak in the melt curve analyses and low standard errors of the Ct values of replicated measurements indicated the specificity of the primers to bind to the cDNA. CONCLUSIONS: Our RNA isolation method, with fine-tuned and detailed instructions, can produce high quality RNA from a small amount of starting plant material that is suitable for use in downstream transcriptional analyses. The use of an increased concentration of the reducing agent ß-mercaptoethanol in the extraction buffer, as well as the application of DNaseI-treatment resulted in a method suitable for a wide range of plants without the need of further optimalization, especially in Rhus typhina (Staghorn sumac), for which molecular-genetic studies have not yet been sufficiently explored.
RESUMEN
Botrytis cinerea, can lead to the formation of noble rot (NR) of grape berries under certain environmental conditions, resulting in favored metabolic and physical changes necessary for producing highly regarded botrytized wines. The functional genes involved in the textural and biochemical processes are still poorly characterized. We generated and analyzed metatranscriptomic data from healthy (H) berries and from berries representing the four stages of NR from the Tokaj wine region in Hungary over three months. A weighted gene co-expression network analysis (WGCNA) was conducted to link B. cinerea functional genes to grape berry physical parameters berry hardness (BH), berry skin break force (F_sk), berry skin elasticity (E_sk), and the skin break energy (W_sk). Clustered modules showed that genes involved in carbohydrate and protein metabolism were significantly enriched in NR, highlighting their importance in the grape berry structural integrity. Carbohydrate active enzymes were particularly up-regulated at the onset of NR (during the transition from phase I to II) suggesting that the major structural changes occur early in the NR process. In addition, we identified genes expressed throughout the NR process belonging to enriched pathways that allow B. cinerea to dominate and proliferate during this state, including sulphate metabolizing genes and genes involved in the synthesis of antimicrobials.
RESUMEN
Botrytis cinerea is a well-known pathogen of grapevine. However, under certain microclimatic conditions, Botrytis infection results in noble rot, an essential process in the production of the world-known Tokaji aszú wines in Hungary. We investigated the physico-chemical characteristics and culturable microorganisms associated with grape berries through several noble rot phases in the two main cultivars grown in Tokaj: Vitisvinifera cv. "Furmint" and "Hárslevelu". We measured physical and analytical parameters routinely tested in viticulture and analyzed the ITS rDNA sequence data of fungi isolated from the sampled berries. We observed significant differences in the physico-chemical parameters among the noble rot phases as well as sampling dates. The greatest variation in berry texture and microbial structure was observed in the initial phases, with variables converging as the noble rot progressed. By finding a bijection between the examined chemical properties and the factorial parameters (e.g., noble rot phase, collection time, cultivar), an appropriate sweet winemaking material can be designed. Fungal community differed significantly among cultivars, with higher number of species observed in Hárslevelu. Our results reveal that there is more to noble rot than only Botrytiscinerea and other microorganisms may play important roles in the aszú process.
RESUMEN
Plastic deformation of micron-scale crystalline materials differs considerably from bulk samples as it is characterized by stochastic strain bursts. To obtain a detailed picture of the intermittent deformation phenomena, numerous micron-sized specimens must be fabricated and tested. An improved focused ion beam fabrication method is proposed to prepare non-tapered micropillars with excellent control over their shape. Moreover, the fabrication time is less compared with other methods. The in situ compression device developed in our laboratory allows high-accuracy sample positioning and force/displacement measurements with high data sampling rates. The collective avalanche-like motion of the dislocations is observed as stress decreases on the stress-strain curves. An acoustic emission (AE) technique was employed for the first time to study the deformation behavior of micropillars. The AE technique provides important additional in situ information about the underlying processes during plastic deformation and is especially sensitive to the collective avalanche-like motion of the dislocations observed as the stress decreases on the deformation curves.