Drill-assisted genomic DNA extraction from Botrytis cinerea

González, Celedonio; Noda, Judith; Espino, José J.; Brito, Nélida

doi:10.1007/s10529-008-9790-6

Cited by 7 publications

(6 citation statements)

References 8 publications

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“…Recombinant DNA methods were performed as described by Sambrook & Russell (2001). Genomic DNA from B. cinerea was extracted using a method previously developed in our laboratory (González et al. , 2008).…”

Section: Methodsmentioning

confidence: 99%

BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host

2011

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Summary• Proteins belonging to the cerato-platanin family are small proteins with phytotoxic activity. A member of this family, BcSpl1, is one of the most abundant proteins in the Botrytis cinerea secretome.• Expression analysis of the bcspl1 gene revealed that the transcript is present in every condition studied, showing the highest level in planta at the late stages of infection. Expression of a second cerato-platanin gene found in the B. cinerea genome, bcspl2, was not detected in any condition.• Two bcspl1 knock-out mutants were generated and both showed reduced virulence in a variety of hosts.• bcspl1 was expressed in Pichia pastoris and the recombinant protein was able to cause a fast and strong necrosis when infiltrated in tomato, tobacco and Arabidopsis leaves, in a dose-dependent manner. The BcSpl1-treated plant tissues showed symptoms of the hypersensitive response such as induction of reactive oxygen species, electrolyte leakage, cytoplasm shrinkage, and cell autofluorescence, as well as the induction of defense genes considered to be markers of the hypersensitive response. The Arabidopsis bak1 mutation partially prevented the induction of necrosis in this plant by BcSpl1. Two different BcSpl1-derived 40-amino acids peptides were also active in inducing necrosis.

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Section: Methodsmentioning

confidence: 99%

BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host

2011

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“…Primers (Table S2) were from Invitrogen (Paisley, Scotland). Genomic DNA from B. cinerea was extracted using a method previously developed in our laboratory [62]. The gene replacement strategy is outlined in Figure S3.…”

Section: Methodsmentioning

confidence: 99%

Botrytis cinerea Protein O-Mannosyltransferases Play Critical Roles in Morphogenesis, Growth, and Virulence

et al. 2013

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Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea.

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“…The method developed was compared with a DNA quantification method [33] for B. cinerea in 45 fruit samples (15 fruit samples of each kind: apple, table grape and pear). Concentrations of DNA were detected spectrophotometrically by measuring absorbance changes at 260 nm showed good integrity by the high molecular weight bands on electrophoresis (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…These results suggest that the sensitivity reached for this procedure allow determining very low level of B. cinerea antigens in apparently healthy fruit that can deteriorate suddenly due to the development of latent or quiescent infection into visible disease. Also, the DNA quantified by the method developed by González et al [33] from uninfected and infected fruit extracts samples was amplified by PCR, with the purpose of verify if the same correspond to specific DNA of B. cinerea [34]. …”

Section: Resultsmentioning

confidence: 99%

Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

et al. 2011

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BackgroundBotrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (William's) tissues.ResultsThe method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation.ConclusionsThe developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

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Drill-assisted genomic DNA extraction from Botrytis cinerea

Cited by 7 publications

References 8 publications

BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host

BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host

Botrytis cinerea Protein O-Mannosyltransferases Play Critical Roles in Morphogenesis, Growth, and Virulence

Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

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