A nontoxic polypeptide oligomer with a fungicide potency under agricultural conditions which is equal or greater than that of their chemical counterparts

Abstract

There are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural conditions. The reasons behind may be multiple where the sensitivity to the Sun UV radiation can come in first place. Here we describe a multifunctional glyco-oligomer with 210 kDa which is mainly composed by a 20 kDa polypeptide termed Blad that has been previously shown to be a stable intermediary product of β-conglutin catabolism. This oligomer accumulates exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Blad-oligomer reveals a plethora of biochemical properties, like lectin and catalytic activities, which are not unusual per si, but are remarkable when found to coexist in the same protein molecule. With this vast range of chemical characteristics, antifungal activity arises almost as a natural consequence. The biological significance and potential technological applications of Blad-oligomer as a plant fungicide to agriculture, its uniqueness stems from being of polypeptidic in nature, and with efficacies which are either equal or greater than the top fungicides currently in the market are addressed.

Fig 1. Resistance to inactivation of the lectin activity of Blad-oligomer.

Blad-oligomer was purified, incubated for 10 min at 0°C in the presence of water (control, lanes 1 and 5), 4 N HCl (lane 2), 8 N HCl (lane 3) and 12 N HCl (lane 4). The protein samples were subsequently analysed by SDS-PAGE and stained for total protein (50 μg per lane; A) or blotted onto a membrane and probed with anti-blad oligomer antibodies (15 μg per lane; B) or with affinity purified, anti-ubiquitin antibodies (50 μg per lane; C). Molecular masses of standards are indicated in kDa.

Fig 2. Detection of phosphoryl groups in Blad-oligomer.

Blad-oligomer, α, β, and γ-conglutins were purified from L. albus, subjected to SDS-PAGE, and stained for total protein (A) or analysed for the presence of phosphoryl group using the Pro-Q diamond phosphoprotein gel stain (B). Lanes 1, 2, 3, 4: Blad-oligomer, α, β, and γ-conglutins, respectively. Lanes M: molecular mass standards (kDa). The position of the positive (+) and negative (-) phosphorylated markers is shown in B.

Fig 3. Susceptibility to proteolysis of the Blad-oligomer.

Pure 210 kDa protein (lanes 1) was mixed with proteolytic enzymes [pronase in (A); trypsin in (B); proteinase K in (C); α-chymotrypsin in (D); subtilisin in (E)] and incubated at room temperature for 1 h (lanes 2), 2 h (lanes 3) or 2 h followed by addition of pure ribulose bisphosphate carboxylase (55 μg) and a further 1 h incubation (lanes 4). In lanes 5, pure ribulose bisphosphate carboxylase (55 μg) was incubated for 1 h with the corresponding proteolytic enzyme. Lanes 6 and 7 contain pure ribulose bisphosphate carboxylase (55 μg) or the corresponding protease (20 μg), respectively. Lanes M: molecular mass standards (kDa).

Fig 4. Purification of Blad-oligomer by chitin-affinity chromatography.

(A,B) Pure Blad-containing protein (1.2 absorbance units; A) or the total globulin fraction from 8-days germinated Lupinus cotyledons (10 absorbance units; B), respectively, were loaded into a chitin column previously equilibrated with 50 mM Tris-HCl buffer, pH 7.5. The column was washed and the bound proteins eluted with 0.05 N HCl (beginning of elution is marked with a vertical arrow). One mL fractions were collected. SDS-PAGE analysis of the polypeptide patterns of Blad-oligomer by the standard, extensive procedure or purified by the single step, chitin-affinity chromatography procedure are illustrated in the figure.

Fig 5. Percentage of inhibition of radial growth of several phytopathogenic fungi in an agar medium containing two different concentrations of Blad-oligomer (4.5 and 9.0 μM).

1: B. cinerea, 2: C. acutatum, 3: C. dematium, 4: C. gloeosporiodes, 5: C. graminicola, 6: E. turcicum, 7: F. oxysporum, 8: F. solani, 9: M. fijiensis, 10: M. phaseolina, 11: S. sclerotiorum (CBS 128069), 12: S. sclerotiorum (CEV—micelium), 13: S. sclerotiorum (CEV—sclerotia). Values and error bars represent the mean and standard deviation of triplicate measurements.