Antifungal Activity of α‑Sarcin against <i>Penicillium digitatum</i>: Proposal of a New Role for Fungal Ribotoxins

Among the putative defense proteins that occur in fungi, one of the best studied is α-sarcin, produced by the mold <i>Aspergillus giganteus</i>. This protein is the most significant member of the ribotoxin family, which consists of extracellular rRNA ribonucleases that display cytotoxic activity toward animal cells. Ribotoxins are rRNA endonucleases that catalyze the hydrolysis of the phosphodiester bond between G4325 and A4326 from the rat 28S rRNA. The results of several experimental approaches have led to propose ribotoxins as insecticidal agents. In this work, we report that α-sarcin displays a strong antifungal activity against <i>Penicillium digitatum</i>, being able to enter into the cytosol where it inactivates the ribosomes, thus killing the cells and arresting the growth of the fungus. This is the first time that a ribotoxin has been found to display antifungal activity. Therefore, this protein could play, besides the already proposed insecticidal function, a role in nature as an antifungal agent

Multiple sequence alignments showing similarity and conserved domains identified by the Interpro resource available at http://www.ebi.ac.uk/interpro/in A) Pep-13 transglutaminase peptide elicitors (according to Brunner et al., 2002 [52]), B) berberin-like proteins (Pfam PF08031), C) carbonic anhydrases (PROSITE PS51144; Pfam PF00194) and D) pectinesterase/pectate lyases (Pfam PF01095/SUPERFAMILY SSF51126).

<p>Signal sequences at N-terminus (SignP) and the consensus sequences are also reported.</p

List of effectors identified in the <i>P. plurivora</i> secretome by high resolution LC MS/MS.

<p>*Sequences containing the RXLR motif.</p><p>**Nucleotide sequence submitted to the EMBL/GenBank/DDBJ databases.</p><p>The secretion prediction according to signal peptide probability of Signal P 4.1 server is reported. Y and N indicate the presence or absence of the signal peptide for secretion, respectively.</p><p>List of effectors identified in the <i>P. plurivora</i> secretome by high resolution LC MS/MS.</p

<i>Fagus sylvatica</i> root exudate characterization.

<p>A) Representative photograph shoving the ability of <i>Fagus sylvatica</i> root exudate to attract <i>P. plurivora</i> zoospores. B) ¹H NMR spectrum of <i>F. sylvatica</i> root exudate acquired at 300.03 MHz in methanol-d4-buffer phosphate 1∶1. Protons responsible for NMR signals of molecules are highlighted in red in the structures. Signals of anomeric protons are marked with asterisks. C) Free amino acid profile of <i>F. sylvatica</i> root exudate (lower panel) compared to standards (upper panel). D) Bar chart showing the amount (nmol/mg of root exudate) of free amino acids detected in the <i>F. sylvatica</i> root exudate.</p

Characterization of purified recombinant (P)GKY20 peptide.

<p>(A) Mass spectrum of purified (P)GKY20 peptide. The measured molecular weight (2609.47 Da) is consistent with the theoretical value (2609.1 Da). (B) Reverse-phase HPLC chromatogram recorded at 280 nm wavelength. Purified peptide was applied to a C18 column (Jupiter 5u C18 300Å, 250 x 4.6 mm) and eluted with a linear gradient from 5% to 95% acetonitrile containing 0.05% trifluoroacetic acid, over 60 min at flow rate of 1 mL/min.</p

Schematic representation of expression vectors and recombinant proteins.

<p>Fusion proteins without (A) and with (B) His₆-Tag. The main restriction enzyme sites, <i>NdeI</i>, <i>EcoRI</i>, <i>KpnI</i>, <i>BamHI</i> and <i>SacI</i> were reported. MCS: multicloning site. Onconase: carrier protein (grey). Linker: DNA sequence coding for GTGDP amino acid residues (red). GKY20: human Thrombin derived peptide (blue).</p

Cyanogen bromide cleavage of ONC-DCless-H6-(PM)GKY20 fusion protein.

<p>Samples were analyzed on 20% SDS-PAGE. Lane M: marker (14.3 kDa). Lane 1, purified fusion protein. Lanes 2, 3: cleaved recombinant protein in 0.2 M HCl with 100- (lane 2) and 400-fold (lane 3) molar excess of CNBr over methionine residues. Sample were incubated in the dark at room temperature for 24 h. Onc-P: Onconase/Peptide fusion protein; Onc: Onconase carrier; P: (P)GKY20 peptide.</p

Asp-Pro cleavage efficiency comparison of ONC-DCless-H6-(P)GKY20 and ONC-DCless-H6-(PM)GKY20 fusion proteins.

<p>Samples were cleaved in 0.1 M acetic acid adjusted at pH 2.0 with HCl (60°C, 24 h) and analyzed on 20% SDS-PAGE. Lane M: marker (14.3 kDa). Lane 1: ONC-DCless-H6-(PM)GKY20 purified fusion protein. Lanes 2, 3: ONC-DCless-H6-(PM)GKY20 (lane 2) and ONC-DCless-H6-(P)GKY20 (lane 3) hydrolyzed proteins. Onc-P: Onconase/Peptide fusion protein; Onc: Onconase carrier; P: (P)GKY20 peptide.</p

Schematic representation of proteins with enzymatic activity enriched in <i>P. plurivora</i> secretome.

<p>The most represented categories for the oxidoreductases, transferases and hydrolases are also reported.</p

Protein expression of first generation mutants.

<p>(A) SDS-PAGE analysis of insoluble (lanes 1, 3) and soluble (lanes 2, 4) fractions after cell lysis of ONC(YY)-(P)GKY20 and ONC(EYEY)-(P)GKY20 fusion proteins, respectively. (B) SDS-PAGE analysis of soluble (lane 5) and insoluble (lane 6) fractions after cell lysis of ONC(EYEY)-H6-(P)GKY20 fusion protein. Lane M: marker (14.3 kDa).</p