Botrytis cinerea expression profile and metabolism differs between noble and grey rot of grapes.

Botrytis cinerea, the pathogen causing grey rot (GR) with important economic losses in fruit crops, can also cause noble rot (NR) of grape berries under certain environmental conditions, leading to metabolic and physical changes necessary for producing highly regarded botrytized wines. The functional genes involved in biochemical processes in these harmful vs. beneficial berry rot types are still scarcely understood. We generated and analyzed transcriptomic data from healthy (H), NR and GR grape berries collected in the Tokaj wine region in Hungary. Our study shows that B. cinerea is most active in NR, followed by GR and H berries. In addition, expression profiles differed qualitatively between NR and GR, and to a smaller extent between months. Several functional genes expressed during NR were linked to well-known physico-chemical changes in botrytized grape berries, including berry skin degradation and the formation of metabolites favorable for botrytized wine production. In addition, we found that B. cinerea appeared to express genes involved in the biosynthesis of antimicrobials during NR, but not in GR, which likely contributes to the dominance of this fungus during NR.

A modified CTAB method for the extraction of high-quality RNA from mono-and dicotyledonous plants rich in secondary metabolites.

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.

Metatranscriptomic Analyses Reveal the Functional Role of Botrytis cinerea in Biochemical and Textural Changes during Noble Rot of Grapevines.

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.

Redox and Hormonal Changes in the Transcriptome of Grape (Vitis vinifera) Berries during Natural Noble Rot Development.

Noble rot is a favorable form of the interaction between grape (Vitis spp.) berries and the phytopathogenic fungus Botrytis cinerea. The transcriptome pattern of grapevine cells subject to natural noble rot development in the historic Hungarian Tokaj wine region has not been previously published. Furmint, a traditional white Tokaj variety suited to develop great quality noble rot was used in the experiments. Exploring a subset of the Furmint transcriptome redox and hormonal changes distinguishing between noble rot and bunch rot was revealed. Noble rot is defined by an early spike in abscisic acid (ABA) accumulation and a pronounced remodeling of ABA-related gene expression. Transcription of glutathione S-transferase isoforms is uniquely upregulated, whereas gene expression of some sectors of the antioxidative apparatus (e.g., catalases, carotenoid biosynthesis) is downregulated. These mRNA responses are lacking in berries exposed to bunch rot. Our results help to explain molecular details behind the fine and dynamic balance between noble rot and bunch rot development.

Physico-Chemical Characteristics and Culturable Microbial Communities of Grape Berries Change Strongly during Noble Rot Development.

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.