In silico characterization, physiochemical analysis, and antifungal evaluation of the Zea mays PR-1 protein

Abstract

Pathogenesis-related protein 1 (PR-1) is a critical component of plant defense mechanisms, exhibiting significant antifungal activity. This study focused on the PR-1 protein from Zea mays and five other plant species (Triticum aestivum, Hordeum vulgare, Oryza sativa, Avena sativa, and Secale cereale) to explore their physicochemical, structural, and functional characteristics. The PR-1 gene from Z. mays was isolated and expressed in Escherichia coli BL21-DE3 cells, enabling structural characterization. Recombinant expression in E. coli enabled structural characterization and functional studies of the PR-1 protein, laying a foundation for future experiments. Sequence analysis revealed high similarity (65-90%) among PR-1 proteins across species, although physicochemical properties varied based on plant origin. Structural analysis showed a conserved beta-barrel structure surrounded by alpha-helices in all PR-1 proteins. In addition to in silico and structural characterization, antifungal bioassays were performed using the purified recombinant PR-1 protein against Fusarium oxysporum, a major fungal phytopathogen. These assays demonstrated notable antifungal activity, supporting the predicted defense role of PR-1 and laying experimental groundwork for its potential use in developing fungi-resistant transgenic crops.

Keywords: Zea mays; Pathogen resistance; Pathogenesis-related protein; Structural analysis.

Fig. 1

Amino acid analysis of PR-1 protein in different species of family Gramineae.

Fig. 2

Sequence alignment and phylogenetic tree of PR-1 proteins under study using UniProt blast tool.

Fig. 3

(a) PCR amplified product of PR-1. (b) Isolation of supercoiled cloning vector pTZ57R/T. (c) Digested plasmid of vector pTZ57R/T (d) PCR positive clones of expression vector pET15b-PR-1.

Fig. 4

Chromatogram available for the DNA sequencing of the PR-1 gene, and this sequencing was conducted using SnapGene software.

Fig. 5

The sequence homology of the Z. mays PR-1 gene was examined in relation to the reported genomic clone gene “(Sequence ID: AC116033.3)”s.

Fig. 6

Sequences generating noteworthy alignments with the sequenced of PR-1 gene from Z. mays were recognized.

Fig. 7

(A) Expression of PR-1 in E. coli. Fusion proteins were induced by adding IPTG and incubating the culture at 37°C for 4 h. Total protein isolated from cell culture before and after induction and solubilized and refolded protein (shown in M: Low molecular Protein marker, (Lane1) Uninduced sample. (Lane 2) IPTG induced (Lane 3) Solubilized inclusion, (Lane 4) Refolded protein from inclusions. (B) Purification of E. coli expressed PR-1 protein from affinity column (His-tag) chromatography. (shown in M: Low molecular Protein marker, (Lane1) folded PR1. (Lane 2) Column wash (Lane 3–7 Imidazole eluted fraction. (C) Protein dot blot of PR-1 expressed in and purified from re-folded bacterial inclusions (NC) Negative E. coli protein. (1) IPTG Induced (2) Solubilized inclusion (3) Folded protein (Lane 4–6) Imidazole eluted fractions. (D) The 3D modeled structure of the plant PR-1 protein in Z. mays reveals the presence of paired cysteines.

Fig. 8

Kyte and Doolittle mean hydrophobicity profile computed for the transmembrane regions of PR-1 protein O82086 (primary and secondary helices). Helical wheel representation of predicted helix of O82086 (Zea mays) PR-1 protein. Hydrophobic residues (V, L, I) are represented as blue squares and violet letters (A, G, P, Y), polar residues (E, Q, S, T) as red diamonds.

Fig. 9

Secondary structural features of maize PR-1 protein (O82086) predicted by SOPMA using default parameter.

Fig. 10

3D structure of PR-1 protein (082,086) was performed by homology model server Swiss model

Fig. 11

The graph shows the predicted local similarity to the target (y-axis) plotted against the protein residue number (x-axis) and protein size distribution of a set of structures. The scatter plot shows the normalized OMEAN4 score versus protein size in residues. The color of the points indicates the IZ-score, with blue points indicating IZ-score > 21, green issue displaying IZ-score > 2, and red points indicating IZ-score < 1.

Fig. 12

Z. mays PR-1 protein exhibited dose dependent antifungal activity against Fusarium oxysporum using disk diffusion. The image indicates distinct areas of inhibition surrounding disks that have been treated with Z. mays PR-1 protein that have been purified, indicating a direct relationship goes between the degree of fungal growth inhibition and the concentration of the protein. 1: 40 µg (17 mm zone), 2: 30 µg (15 mm zone), and 3: 20 µg (13 mm zone) present decreasing inhibitory effects with progressively smaller amounts of protein. The negative control (protein buffer) does not exhibit an inhibition zone around 4, which affirms the specific antifungal activity of the PR-1 protein.