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Gazzetti, Katia (2013) Involvement and contribution to virulence of Endo-Beta-1,4-Xilanases of Fusarium Graminearum during host infection. [Tesi di dottorato]

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Abstract (inglese)

Fusarium graminearum is the fungal pathogen mainly responsible for Fusarium head blight (FHB) of cereal crops, which attacks wheat spikes, reducing crop production and quality of grain by producing trichothecene mycotoxins.
Several cytohistological studies showed that spike infection is associated with the production of cell wall degrading enzymes. Wheat tissue, as in other commelinoid monocot plants, is particularly rich in xylan which can be hydrolyzed by fungal endo-β-1,4-xylanase.
The FGSG_03624 is one of the most expressed xylanase genes in wheat spikes 3 days after inoculation and was heterologously expressed in the yeast Pichia pastoris. The recombinant protein (22.7 kDa) possessed xylanase activity and induced cell death and hydrogen peroxide accumulation in wheat leaves infiltrated with 10 ng/μl or in wheat glume surface treated with 20 ng/μl. This effect reflects that observed with other described fungal xylanases (from Trichoderma reesei, T. viride and Botrytis cinerea) with which the FGSG_03624 protein shares a stretch of amino acids reported as essential for elicitation of necrotic responses.
Several F. graminearum mutants with the FGSG_03624 gene disrupted were obtained, and showed about 40% reduction of total xylanase activity in comparison to the wild type when grown in culture with xylan as carbon source.
Even if the FGSG_03624 xylanase is able to induce hypersensitive-like symptoms on a monocot plant, FGSG_03624 gene deletion mutants were fully virulent on wheat cvs. Bobwhite and Nandu, probably because of xylanase gene redundancy. Therefore the role of this xylanase during wheat infection remains elusive.
To get insight about the importance of xylanase activities produced by the pathogen during infection of host plants, Xyr1, a transcriptional regulator factor putatively regulating the expression of several xylanase genes has been deleted by targeted homologous recombination.
Deleted mutants of the FGSG_17662 gene, encoding Xyr1, were heavily impaired both in total secreted xylanase activity and in fungal biomass formation when grown in liquid culture with xylan as sole carbon source. In comparison to WT, the fungal dry weight was reduced, by 40% to 70% and the total xylanase activity was reduced by about 90%.
F. graminearum Xyr1 deletion mutants are now available to be submitted to expression analysis in vivo to verify the Xyr1 mediated regulation of the expression of genes encoding for xylanolitic enzymes during plant infection.
The Xyr1 disrupted mutants will be characterized for their virulence by inoculating Triticum aestivum spikes. If these mutants will be verified as impaired in virulence, in planta overexpression of xylanase inhibitors may be considered as a new tool to control FHB

Abstract (italiano)

Fusarium graminearum è un fungo filamentoso conosciuto come principale agente della fusariosi della spiga (FHB), una importante malattia che colpisce principalmente cereali da granella. Nel frumento il patogeno attacca la spiga in fase di fioritura, causando considerevoli perdite di raccolto e riducendo la qualità delle cariossidi a causa della contaminazione da micotossine tricoteceni, prodotte dal fungo durante il processo infettivo.
Diversi studi citologici hanno dimostrato una correlazione tra l'™infezione della spiga e la produzione da parte di F. graminearum di enzimi degradativi della parete cellulare vegetale. I tessuti del frumento, come quelli di altre piante monocotiledoni commelinoidi, sono particolarmente ricchi in xilano, il quale può essere idrolizzato dalle endo-β-1,4-xilanasi fungine.
FGSG_03624 è uno dei geni codificanti endo-xilanasi più espressi da F. graminearum in spiga di frumento al terzo giorno di infezione, ed è stato espresso eterologamente nel lievito Pichia pastoris. La proteina ricombinante ottenuta (22.7 kDa) possedeva attività xilanasica e induceva morte cellulare e accumulo di perossido di idrogeno in tessuti di frumento quali foglie infiltrate con 10 ng/μl o glume trattate con 20 ng/μl. Questo effetto riflette quanto osservato per altre xilanasi fungine di Trichoderma reesei, T. viride and Botrytis cinerea, con cui FGSG_03624 condivide uno stretch di aminoacidi riportato in letteratura come essenziale per l'™elicitazione della risposta necrotica.
Sono stati ottenuti diversi mutanti con il gene FGSG_03624 deleto, i quali mostravano circa il 40% di riduzione dell'attività xilanasica totale rispetto al ceppo wild type se cresciuti in una coltura contenente xilano come unica fonte di carbonio.
Anche se la xilanasi FGSG_03624 è in grado di indurre sintomi simili a quelli osservati nella risposta ipersensibile in tessuti di monocotiledone, i mutanti ottenuti dalla delezione del gene corrispondente sembravano possedere completa virulenza quando inoculati in spighe di frumento tenero delle cultivar Nandu e Bobwhite, probabilmente a causa della ridondanza dei geni codificanti xilanasi. Il ruolo della xilanasi FGSG_03624 durante l'™infezione del frumento rimane quindi elusivo.
Per ottenere informazioni circa l'™importanza dell'™attività xilanasica prodotta da F. graminearum durante l'™infezione della pianta ospite, il fattore trascrizionale Xyr1, che regola putativamente l'™espressione di diversi geni codificanti per enzimi xilanolitici, è stato deleto tramite ricombinazione omologa sito-specifica.
I mutanti derivati dalla delezione del gene FGSG_17662, codificante Xyr1, sono stati cresciuti in coltura liquida con xilano come unica fonte di carbonio e hanno mostrato una ridotta capacità di produrre biomassa ed una consistente diminuzione dell'™attività xilanasica totale rilevata nel mezzo di coltura: in particolare, l'™attività xilanasica prodotta dai mutanti era solo il 10% dell'™attività è secreta dal ceppo WT, e il peso secco dei mutanti risultava ridotto del 40-70%.
I mutanti di F. graminearum con gene xyr1 inattivato sono ora disponibili per l'™analisi trascrizionale in vivo, che consentirà di studiare la regolazione mediata da Xyr1 dell'™espressione di geni codificanti enzimi xilanolitici durante l'™infezione della pianta ospite.
I mutanti saranno inoltre caratterizzati per la loro virulenza mediante inoculo di spighe di Triticum aestivum. Se la virulenza di questi mutanti sarà dimostrata essere compromessa dalla delezione di xyr1, la sovrespressione di inibitori delle xilanasi in pianta potrà essere considerata come strumento per il controllo della fusariosi della spiga

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Tipo di EPrint:Tesi di dottorato
Relatore:Favaron, Francesco
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > SCIENZE DELLE PRODUZIONI VEGETALI > PROTEZIONE DELLE COLTURE
Data di deposito della tesi:29 Gennaio 2013
Anno di Pubblicazione:29 Gennaio 2013
Parole chiave (italiano / inglese):xylanase, Fusarium graminearum, necrotizing factor, gene disruption, Triticum aestivum, FBH, Xyr1, XlnR
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:5659
Depositato il:16 Ott 2013 08:27
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