280 research outputs found
A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition
No full textEndo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP. © 2007 Elsevier Inc. All rights reserved
Analisi molecolare per l'endopoligalatturonasi in Pyrenophora graminea
No full textPyrenophora graminea Ito e Kuribayashi è un fungo ascomicete agente della striatura bruna dell’orzo e tipicamente trasmesso per seme. Le conoscenze sui meccanismi patogenetici di P.graminea sono relative esclusivamente alla produzione di composti fitotossici coinvolti nello sviluppo e nella intensità dei sintomi; sono noti, infatti, uno o più composti capaci di indurre sintomi tipici quando infiltrati in foglie di orzo (Haegi et al., 1994). Nell’ambito di una ricerca il cui intento è quello di fornire informazioni sulle basi genetico-molecolari della patogenicità, è stata intrapresa un’indagine sulla presenza ed espressione di geni per l’endo-poligalatturonasi in P.graminea. L’endo-poligalatturonasi (endo-PG) prodotta dai funghi svolge un ruolo importante nella degradazione delle pareti cellulari delle piante, digerendo i componenti pectici della lamella mediana e della parete primaria. La produzione di poligalatturonasi è generalmente sottoposta ad induzione da parte del substrato e a repressione da parte di fonti di carbonio preferenziali, quali ad esempio il glucosio. In alcuni casi però, come in Cochliobolus heterostrophus e in Aspergillus flavus, la sintesi di endo-PG non è repressa dal glucosio (Bateman et al., 1973; Whitehead, 1995). Questo enzima è spesso prodotto in forme molecolari multiple, sia in coltura che durante l’infezione della pianta, con differenti proprietà biochimiche e fisiologiche (Favaron e Marciano, 1992).
In prove preliminari, condotte con saggi enzimatici in piastra, era stata verificata la presenza di attività poligalatturonasica in diversi ceppi di P.graminea; per appurare un eventuale coinvolgimento delle poligalatturonasi nella patogenicità di questo fungo, si è deciso, quindi, di condurre un analisi molecolare di espressione utilizzando, come sonde, geni eterologhi dell’endo-PG clonati (Scott-Craig et al., 1990; Caprari et al., 1993). Come confronto è stato analizzato in alcuni esperimenti anche un ceppo di P.teres.
Le sonde pCC2 e pCC33, relative al gene dell’endo-PG rispettivamente di Fusarium moniliforme e di Cochliobolus carbonum, sono state inizialmente utilizzate sul DNA genomico di un ceppo di P.graminea (Pg2) e di uno di P.teres (Pt4); il risultato degli esperimenti di Southern blot ha dimostrato che le due sonde risconoscono una sequenza genomica in entrambe Pg2 e Pt4.
Si è quindi proceduto all’analisi dell’espressione dell’endo-PG nei due ceppi, mantenuti in coltura liquida in condizioni induttive e non (pectina; pectina e saccarosio in diverse proporzioni; saccarosio). Gli esperimenti di Northern blot hanno evidenziato in ambedue i ceppi un segnale di espressione, non molto intenso data l’eterologia della sonda, senza differenze significative tra i mezzi utilizzati.
Nel tentativo di isolare il gene omologo dell’endo-PG di P.graminea e di P.teres, sono stati effettuati esperimenti di PCR, utilizzando oligonucleotidi sintetizzati sulla base della sequenza genica clonata in F.moniliforme. Da entrambi i ceppi di Pyrenophora è stato ottenuto un prodotto di PCR con peso molecolare compatibile con quello atteso per il gene dell’endo-PG. I due frammenti (Pg2-PCR e Pt4-PCR) sono stati quindi riutilizzati come sonde omologhe in esperimenti di Southern blot e Northern blot.
i) Le due sonde omologhe (Pg2-PCR e Pt4-PCR) utilizzate in esperimenti di Southern-blot, effettuati su i due DNA genomici (Pg2 e Pt4) tagliati con enzimi di restrizione diversi, hanno presentato profili di ibridazione simili.
ii) Gli esperimenti di Northern-blot hanno evidenziato una espressione di endo-PG, apparentemente indipendente dai mezzi di coltura, il cui andamento è costante con entrambe le sonde.
In conclusione i prodotti di PCR, ottenuti dai genomi dei due ceppi Pg2 e Pt4, rappresentano una parte del gene dell’endo-PG di Pyrenophora; essi costituiscono, così, uno strumento più sensibile per una analisi fine di espressione con sonde omologhe, ma soprattutto, consentiranno uno studio strutturale del gene dell’endo-PG in P.graminea e in P.teres.
Al momento sono in corso esperimenti che estendono nel tempo l’analisi di espressione (campioni mantenuti in coltura su tre mezzi diversi: pectina 1% e saccarosio 0,2%; pectina 1% e saccarosio 1,2%; saccarosio 1,2%; tutti con 6 tempi d’induzione) ed in parallelo, sui filtrati colturali degli stessi campioni esaminati, vengono effettuati saggi di attività enzimatica.
Le ricerche proseguiranno per accertare la reale importanza dell’endo-PG nell’interazione P.graminea/orzo, confrontando l’andamento dell’espressione osservato su mezzi di crescita contenenti pectina commerciale con quello ottenuto per colture cresciute su estratti di pianta e con quello osservabile direttamente nella pianta infetta
The Fusarium graminearum Xyr1 transcription factor regulates xylanase expression but is not essential for fungal virulence
No full textThe fungal pathogen Fusarium graminearum is the causal agent of the Fusarium head blight disease of wheat and other small grain cereals. This fungus is known to produce high amount of cell wall degrading enzymes during infection of wheat spikes; besides, wheat tissue is particularly rich in xylan which can be hydrolyzed by fungal xylanases. In order to establish the role of F. graminearum xylanase activity in pathogenicity, we performed a targeted gene disruption of the F. graminearum xyr1 gene, encoding the major regulator of xylanase genes expression. The Δxyr1 mutant resulted strongly affected in growing on wheat cell walls and its xylanase activity was dramatically reduced compared to wild-type; besides, the disruption of the xyr1 gene heavily reduced the expression of xylanase encoding genes both in vitro and during wheat spike infection, thus confirming the involvement of the F. graminearum Xyr1 in the regulation of genes controlling xylan degradation. However, despite of the deep impact caused by xyr1 gene disruption on the expression of xylanase genes and on total xylanase activity, the virulence of the Δxyr1 mutant appeared not affected on Triticum aestivum and T. durum spikes and on soybean seedlings. In conclusion, although a possible role of the xylanase activity in the virulence of F. graminearum cannot be conclusively excluded, our results seem to question the importance of a large amount of xylanase activity during the infection process
Gel detection of Allium porrum polygalacturonase-inhibiting protein reveals a high number of isoforms
No full textPolygalacturonase inhibiting proteins PGI Ps are leucine-rich repeat glycoproteins. localized in the cell wall of most plant species, capable of countering the activity of endo-polygalacturonases (endo-PGs) produced by phytopathogenic fungi. The PGIP from Allium porrum leaves was analysed to ascertain the presence of different molecular forms of PGIP. Leek PGIP was separated into two fractions: a soluble and an ionically wall-bound PGIP, each of which was then purified by cation-exchange chromatography. Two and three peaks of PGIP activity were obtained, respectively. PGIP isoforms contained in each peak were separated by isoelectrofocusing (IEF) on a polyacrylamide gel. Following the separation, the gel was first overlaid with sodium polygalacturonate and then treated with the endo-PG from either Sclerotinia sclerotiorum, Fusarium moniliforme or Botrytis aclada. The endo-PG(s) hydrolyse the overlaid substrate except where active inhibitors are present. The presence of PGIPs is revealed by ruthenium red staining of the nonhydrolysed substrate. Each PGIP peak following IEF separation revealed several PGIP isoforms with pIs between 5.0 and 7.0. More than 20 isoforms were detected in total. with considerable differences in their inhibitory activity. While similar PGIP isoform patterns were obtained by developing the IEF gels with the endo-PGs of S. sclerotiorum and B. aclada, less intense PGIP bands were observed with the endo-PG from B. aclada, consistent with inhibition assays performed in solution. The endo-PG from F. moniliforme, which is not inhibited at all by leek PGIP in solution, consistently showed no PGIP band on the gel assay
The xylanase inhibitor TAXI-III limits cell death induced by a xylanase secreted by Fusarium graminearum during wheat infection.
No full textCereals contain xylanase inhibitor proteins (XIs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. In wheat, three types of XIs have been identified: Triticum aestivum XI (TAXI), xylanase inhibitor protein (XIP) and thaumatin-like XI (TLXI). These inhibitors are considered part of the defence mechanisms that plants use to counteract microbial pathogens and recently we provided in planta evidences for the protective role of TAXI-III, a member of the TAXI type XIs. To elucidate the molecular mechanism underlying the capacity of the transgenic plants expressing Taxi-III to limit Fusarium Head Blight (FHB) disease symptoms caused by F. graminearum, we performed infiltration experiments on wheat tissues with a xylanase strongly expressed by F. graminearum during wheat spike infection which we have previously demonstrated to induce cell death and hydrogen peroxide accumulation. Experiments performed on glumes of flowering wheat spikes showed that the presence of TAXI-III significantly decreased cell death and hydrogen peroxide accumulation. Most interestingly, similar results were also obtained by infiltrating the same xylanase on glumes of transgenic wheat plants expressing TAXI-III. These results suggest that the reduced FHB symptoms on transgenic TAXI-III plants can be due to the direct inhibition of xylanase activity secreted by the pathogen but also to the capacity of TAXI-III to prevent the recognition of xylanase by a plant receptor possibly involved in cell death elicitation
Identification of amino acid residues of Fusarium verticillioides endo-polygalacturonase required to escape the inhibition by host plant PGIP
No full textEndopolygalacturonases (Endo-PGs) play an important role
in fungal infection since they depolymerize the pectin component
of plant cell wall. Most plant have evolved defence mechanisms
to contrast the action of endo-PGs. A direct mechanism is the interaction
with plant cell wall polygalacturonase-inhibiting proteins
(PGIPs) that block polygalacturonase activity. However,
fungal pathogens can elude the inhibition by plant PGIP, producing
PGs refractory to inhibition. Fusarium verticillioides endo-PG
is unaffected by host PGIPs (asparagus and leek) as well as by
Phaseolus vulgaris PGIP, that is the most efficient PGIP so far
characterized. Recently, we have identified a specific PG amino
acid residue responsible for the lack of recognition by bean
PGIP. Now we have mutated few amino acids of the F. verticillioides
endo-PG which allow the recognition by host PGIP. This
finding highlights the structural features of the enzyme to escape
PGIP inhibition. Moreover, this modified PG could be used to
replace native F. verticillioides PG in order to better clarify the
role of constitutive PGIP in plant defense
Recovering a leading coefficient and a memory kernel in first-order integro-differential operator equations
No full textAbstractWe are concerned with the identification of the scalar functions a and k in the convolution first-order integro-differential equation u′(t)−a(t)Au(t)−k∗Bu(t)=f(t), 0⩽t⩽T, k∗v(t)=∫0tk(t−s)v(s)ds, in a Banach space X, where A and B are linear closed operators in X, A being the generator of an analytic semigroup of linear bounded operators. Taking advantage of two pieces of additional information, we can recover, under suitable assumptions and locally in time, both the unknown functions a and k. The results so obtained are applied to an n-dimensional integro-differential identification problem in a bounded domain in Rn
Tissue-Specific Modulation of the Mitochondrial Calcium Uniporter by Magnesium Ions
No full textThis paper analyzes the kinetics of the Ca2+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Ca2+ concentrations near the steady-state value (1-4 microM). Heart mitochondria exhibit the lowest activity, and physiological Mg2+ concentrations inhibit the mitochondrial Ca2+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Ca2+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment
Role of Polygalacturonase-Inhibiting Proteins in the Arabidopsis thaliana-Fusarium graminearum interaction.
No full textComunicazione oral
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