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Giacomello, Francesca (2010) A Fusarium graminearum PG is required for virulence during wheat infection. [Tesi di dottorato]

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Abstract (inglese)

Fusarium graminearum is a relevant pathogen of monocot species and is the main causal agent of Fusarium Head Blight (FHB), a devastating disease which commonly affects cereals. Besides, this fungus produces in the infected grains significant levels of mycotoxins, such as the trichotecenes deoxinivalenol (DON). F. graminearum penetrates in the host tissue during anthesis and once inside the tissue is able to spread systemically through the vascular vessels. During penetration and colonization of wheat spike tissues, the fungus secrete several cell wall degrading enzymes (CWDE). In particular, endo-polygalacturonases (PGs) have been shown to be early secreted in wheat plants, but, their role during the infection process has not been ascertained yet.
Two pg encoding genes have been previously identified in the F. graminearum genome database and the isoforms encoded by these two genes (named PG1 and PG2) have been characterized both in vitro and in vivo.
Aim of the present work was to clarify the importance of these F. graminearum PGs during infection of host wheat plants through the knock-out of their corresponding encoding genes, obtained by targeted homologous recombination.
Two ∆PG1 mutants and two ∆PG2 mutants strains (producing only PG2 and PG1, respectively) were obtained and characterised both in vitro and during plant infection. The PG activity produced in liquid culture by the ΔPG1 mutants was negligible compared to that produced by WT and ΔPG2 mutants. However, no difference in dry weight was obtained when growing WT and mutant strains in the presence of pectin as the sole carbon source.
Infection experiments of wheat host plants were also performed. We observed that all the knock-out mutants tested maintained the capacity to infect the plant, but the ΔPG1 strains showed a significant reduction of virulence (about 75% less infected spikelets) compared to WT strain, while no reduction of virulence was observed with the ΔPG2 mutants.
We also quantified the amount of DON mycotoxin produced by the fungus during wheat infection: the ∆PG1 strains produced from 8 to 15 times less DON mycotoxin compared to WT and ΔPG2 strains, probably due to their reduced colonization of the plant tissue.
F.graminearum WT and a ∆PG1 mutant strain were also transformed to constitutively express the green fluorescent protein (GFP). The obtained mutants were used to perform a comparative study of the early events of wheat colonization. Compared to WT, the ΔPG1 mutant colonized more slowly the ovaries, tissues rich of pectin,, and also the colonization of the vascular vessels of the rachis by this mutant was strongly delayed compared to WT.
In particular, the retarded colonization of the ovary by the ΔPG1 mutant might be due to the reduced production of PG activity. The fungal growth in the infected wheat tissue could result therefore slowed down allowing the plant to initiate its defence reactions.
Taken together, these results seems to indicate that PG1 play an important role during pathogenesis and therefore can be considered a virulence factor.

Abstract (italiano)

F. graminearum è l’agente causale della fusariosi della spiga, una malattia descritta per la prima volta in Inghilterra nel 1884 ed ora una delle più studiate in quanto colpisce le tre principale risorse di cibo mondiale frumento, mais e riso. Questo fungo può causare la distruzione del raccolto in poche settimane determinando ingenti perdite economiche. In aggiunta, il prodotto ricavato dalle piante infette presenta elevati livelli di micotossine, in particolare tricoteceni quali il deossinivalenolo (DON). Questa tossina è un potente inibitore delle sintesi proteica negli eucarioti e risulta dannoso per la salute umana e animale.
F. graminearum penetra nell’ospite durante la fase di antesi attraverso l’ovario e colonizza la superficie interna di lemma e palea. Una volta penetrato esso è in grado di diffondere nella spiga in modo sistemico. Durante la penetrazione e la colonizzazione dei tessuti della spiga, il fungo produce e rilascia numerosi enzimi degradativi della parete cellulare; tra questi, le endo-poligalacturonasi (PGs) sono tra i primi enzimi secreti ma il loro ruolo durante l’infezione non è stato ancora chiarito.
Nel database genomico di F. graminearum sono stati identificati due putativi geni codificanti pg. Le isoforme codificate da questi geni (chiamate PG1 and PG2) sono state caratterizzate in vitro e in vivo (Tomassini et al., 2009). L’obiettivo del presente lavoro di tesi è chiarire l’importanza di queste PG prodotte durante l’infezione della pianta ospite attraverso l’ottenimento di mutanti knock-out ottenuti attraverso ricombinazione omologa target del gene target.
Due mutanti ∆PG1 e due mutanti ∆PG2 (producenti rispettivamente solo PG2 e PG1) sono stati ottenuti e caratterizzati in vitro e durante l’infezione della pianta ospite. In coltura liquida il mutante ΔPG1 è risultato produrre scarsa attività PG rispetto al fungo WT e al mutante ΔPG2. Tuttavia i mutanti analizzati non hanno mostrato differenze in peso secco dopo crescita in terreno liquido contenente pectina come unica fonte di carbonio.
I mutanti sono stati stati in seguito saggiati in esperimenti di infezione di piante di frumento. Si è osservato che tutti i mutanti testati mantenevano la loro capacità di infettare la spiga. Tuttvia, il mutante ΔPG1 presentava una ridotta virulenza (circa 75% in meno di spighe infettate) rispetto al WT mentre il mutante ΔPG2 non mostrava alcuna riduzione di virulenza.
E’ stata inoltre quantificata la micotossina DON prodotta dai mutanti durante l’infezione della pianta ospite: il mutante ∆PG1 produceva da 8 a 15 volte meno DON rispetto a WT e mutante ΔPG2, probabilmente a causa della ridotta colonizzazione della spighetta da parte di questo mutante.
Il ceppo WT e un mutante ∆PG1 sono stati inoltre trasformati in modo da esprimere costitutivamente la green fluorescent protein (GFP). I mutanti così ottenuti sono stati utilizzati per studi istologici delle prime fasi di infezione. Rispetto al WT il mutante ΔPG1 era in grado di colonizzare l’ovario, un tessuto ricco in pectina, molto più lentamente; anche la colonizzazione da parte del mutante del tessuto conduttore nel rachide risultava più lenta. Questo rallentamento nella crescita del fungo nella spiga potrebbe essere dovuto alla ridotta attività PG da parte del mutante ΔPG1 e potrebbe permettere alla pianta di attivare più efficacemente le risposte di difesa. Nel complesso i risultati ottenuti nel presente lavoro sembrano indicare che la PG1 di F. graminearum svolge un importante ruolo durante la patogenesi e può quindi essere considerato un fattore di virulenza.

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Tipo di EPrint:Tesi di dottorato
Relatore:Sella, Luca
Dottorato (corsi e scuole):Ciclo 22 > Scuole per il 22simo ciclo > SCIENZE DELLE PRODUZIONI VEGETALI > PROTEZIONE DELLE COLTURE
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:01 Febbraio 2010
Parole chiave (italiano / inglese):Fusarium graminearum, fusariosi della spiga, endo-poligalacturonasi, fattore di virulenza
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:2456
Depositato il:29 Set 2010 12:01
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