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Pais da Cunha, Adérito Tomás (2011) Overcome of grape chemical barriers
by the fungal pathogen Botrytis cinerea.
[Tesi di dottorato]

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Abstract (inglese)

Botrytis cinerea in contact with mature grape berries encounters an environment particularly rich in polyphenols and PR proteins, where the stilbenic phytoalexin trans-resveratrol may accumulate. To mimic conditions similar to those found in grape berries, B. cinerea was grown in vitro with grape PR proteins and polyphenols extracted from mature grapes and with trans-resveratrol. Results showed that in the presence of highly toxic amounts of trans-resveratrol, grape polyphenols allowed total recovery of fungal growth, and proteins allowed partial recovery. These resveratrol-polyphenol or resveratrol-protein combinations also induced a strong release into the medium of laccase activity, which is likely to be involved in trans-resveratrol detoxification. The grape protein pattern changed during fungal growth; most grape proteins quickly disappeared from the culture when polyphenols and trans-resveratrol were present together. Similar protein patterns were obtained in vitro by incubating grape proteins with grape polyphenols and/or trans-resveratrol with a purified B. cinerea laccase. Under these conditions, most proteins became insoluble. The grape protein pattern obtained from grape berries infected by B. cinerea strongly resembled that obtained in vitro by incubating grape proteins and polyphenols with fungal laccase. It seems that B. cinerea, through laccase secretion and activity and by exploiting the berry polyphenols, easily neutralizes the toxicity of grape stilbenic phytoalexins and makes the grape pathogenesis-related proteins insoluble. The effect of laccase, resveratrol and polyphenols on fungal spore germination was also studied. Results showed that resveratrol alone initially does not inhibit the spore germination. But the inhibition was completely relieved by the presence of grape polyphenols. Instead, the pre-incubation of resveratrol with laccase completely inhibited the spore germination. In addition, we investigate the involvement of B. cinerea proteases in the degradation of grape PR proteins. An aspartyl and a tripeptidyl protease were purified from B. cinerea in vitro culture. The purified proteases activities partially degraded PR proteins. The expression analysis of tripeptidyl and aspartic protease gene families revealed that several members of these families are expressed in the presence of grape PR proteins.
In conclusion, our results support that in a grape berry environment characterized by an abundance of polyphenols, B. cinerea laccase not only detoxify the trans-resveratrol but also modifies the solubility of grape proteins and this environment may facilitate the fungal protease to degrade grape PR proteins.

Abstract (italiano)

Durante l’infezione dell’uva il fungo fitopatogeno Botrytis cinerea incontra tessuti particolarmente ricchi di polifenoli e proteine PR e dove si accumula la fitoalessina trans-resveratrolo. Per simulare condizioni simili a quelli trovati negli acini d'uva, B. cinerea è stato coltivato in vitro con proteine e polifenoli estratti da uve mature, e con trans-resveratrolo. I risultati hanno dimostrato che in presenza di livelli tossici di trans-resveratrolo, i polifenoli dell'uva favoriscono una normale crescita del patogeno mentre le proteine consentono un parziale recupero della crescita.
Le combinazioni polifenoli-resveratrolo o resveratrolo-proteina inducevano il rilascio di una forte attività laccasica nel mezzo, che sembra essere coinvolta nella disintossicazione del trans-resveratrolo. I risultati hanno di mostrato anche che il pattern delle proteine dell’uva era alterato durante la crescita del fungo. Infatti, le proteine dell’uva scomparivano rapidamente dalla coltura nella quale polifenoli e trans-resveratrolo erano presenti simultaneamente. Profili proteici simili sono stati ottenuti in vitro, incubando proteine con polifenoli dell'uva e /o trans-resveratrolo con le laccasi purificata di B. cinerea. In queste condizioni, la maggior parte delle proteine diventava insolubile. Questo pattern era molto simile a quello osservato negli acini infettati da B. cinerea. Pertanto, B. cinerea, attraverso la secrezione di attività laccasica e sfruttando i polifenoli, neutralizza facilmente la tossicità delle fitoalessine stilbeniche e rende le proteine PR insolubili. L'effetto di laccasi è stato studiato anche sulla germinazione delle spore di B. cinerea. Il resveratrolo da solo inizialmente non inibiva la germinazione delle spore, invece, la sua pre-incubazione con le laccasi, inducendo la formazione di trans-ε-viniferina, ne inibiva la germinazione. Invece se nel mezzo erano presenti anche i polifenoli non si osservava alcuna inibizione della germinazione. Successivamente è stato indagato il coinvolgimento delle proteasi di B. cinerea nella degradazione delle proteine d’uva. Del mezzo coltura è stata purificata una aspartyl e una tripeptidyl proteasi. Queste proteasi sono state in grado di degradare parzialmente le proteine d’uva. Un’analisi di espressione dei geni delle famiglie di tripeptidyl e aspartyl proteasi ha dimostrato anche altri membri di queste famiglie erano espresse in presenza di proteine PR dell’uva. I risultati permettono di concludere che nell’acino d’uva, caratterizzato d’una grande varietà di polifenoli, la laccasi non solo anulla la tossicità del trans-resveratrolo, ma modifica anche la solubilità delle proteine dell'uva. Questo effetto potrebbe facilitare l’azione proteasica del fungo verso le proteine PR d’uva.

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Tipo di EPrint:Tesi di dottorato
Relatore:Favaron, Francesco
Correlatore:Sella, Luca
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > SCIENZE DELLE PRODUZIONI VEGETALI > PROTEZIONE DELLE COLTURE
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:30 Gennaio 2011
Parole chiave (italiano / inglese):stilbenic phytoalexins, PR proteins, polyphenols, grey mould, B. cinerea
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > AGR/12 Patologia vegetale
Struttura di riferimento:Dipartimenti > Dipartimento Territorio e Sistemi Agro-Forestali
Codice ID:3814
Depositato il:01 Ago 2011 09:39
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