Histidine 19 Residue Is Essential for Cell Internalization of Antifungal Peptide SmAPα1-21 Derived from the α-Core of the Silybum marianum Defensin DefSm2-D in Fusarium graminearum.

The synthetic peptide SmAPα1-21 (KLCEKPSKTWFGNCGNPRHCG) derived from DefSm2-D defensin α-core is active at micromolar concentrations against the phytopathogenic fungus Fusarium graminearum and has a multistep mechanism of action that includes alteration of the fungal cell wall and membrane permeabilization. Here, we continued the study of this peptide's mode of action and explored the correlation between the biological activity and its primary structure. Transmission electron microscopy was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. New peptides were designed by modifying the parent peptide SmAPα1-21 (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Membrane permeability and subcellular localization in conidia were studied by confocal laser scanning microscopy (CLSM). Reactive oxygen species (ROS) production was assessed by fluorescence spectroscopy and CLSM. SmAPα1-21 induced peroxisome biogenesis and oxidative stress through ROS production in F. graminearum and was internalized into the conidial cells' cytoplasm. SmAPH19R and SmAPH19A were active against F. graminearum with minimal inhibitory concentrations (MICs) of 38 and 100 µM for SmAPH19R and SmAPH19A, respectively. The replacement of His19 by Ala produced a decrease in the net charge with a significant increase in the MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. Like SmAPα1-21, SmAP2H19A and SmAP2H19R produced the permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, SmAPH19R and SmAPH19A were localized in the conidia cell wall. The replacement of His19 by Ala turned all the processes slower. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His19 residue in the internalization.

Mapping Resistance to Argentinean Fusarium (Graminearum) Head Blight Isolates in Wheat.

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum (Schwabe), is a destructive disease worldwide, reducing wheat yield and quality. To accelerate the improvement of scab tolerance in wheat, we assessed the International Triticeae Mapping Initiative mapping population (ITMI/MP) for Type I and II resistance against a wide population of Argentinean isolates of F. graminearum. We discovered a total of 27 additive QTLs on ten different (2A, 2D, 3B, 3D, 4B, 4D, 5A, 5B, 5D and 6D) wheat chromosomes for Type I and Type II resistances explaining a maximum of 15.99% variation. Another four and two QTLs for thousand kernel weight in control and for Type II resistance, respectively, involved five different chromosomes (1B, 2D, 6A, 6D and 7D). Furthermore, three, three and five QTLs for kernel weight per spike in control, for Type I resistance and for Type II resistance, correspondingly, involved ten chromosomes (2A, 2D, 3B, 4A, 5A, 5B, 6B, 7A, 7B, 7D). We were also able to detect five and two epistasis pairs of QTLs for Type I and Type II resistance, respectively, in addition to additive QTLs that evidenced that FHB resistance in wheat is controlled by a complex network of additive and epistasis QTLs.

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum.

Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved ß-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.