Omics-Based Mechanistic Insight Into the Role of Bioengineered Nanoparticles for Biotic Stress Amelioration by Modulating Plant Metabolic Pathways

Abstract

Bioengineered silver nanoparticles can emerge as a facile approach to combat plant pathogen, reducing the use of pesticides in an eco-friendly manner. The plants' response during tripartite interaction of plant, pathogen, and nanoparticles remains largely unknown. This study demonstrated the use of bioengineered silver nanoparticles in combating black spot disease caused by necrotrophic fungus Alternaria brassicicola in Arabidopsis thaliana via foliar spray. The particles reduced disease severity by 70-80% at 5 μg/ml without showing phytotoxicity. It elicited plant immunity by a significant reduction in reactive oxygen species (ROS), decreases in stress enzymes by 0.6-19.8-fold, and emergence of autophagy. Comparative plant proteomics revealed 599 proteins expressed during the interaction, where 117 differential proteins were identified. Among different categories, proteins involved in bioenergy and metabolism were most abundant (44%), followed by proteins involved in plant defense (20%). Metabolic profiling by gas chromatography-mass spectroscopy yielded 39 metabolite derivatives in non-polar fraction and 25 in the polar fraction of plant extracts. It was observed that proteins involved in protein biogenesis and early plant defense were overexpressed to produce abundant antimicrobial metabolites and minimize ROS production. Bioengineered silver nanoparticles performed dual functions to combat pathogen attack by killing plant pathogen and eliciting immunity by altering plant defense proteome and metabolome.

Keywords: metabolomics; nanoparticles; pesticide; plant pathogen; proteomics.

FIGURE 1

Photograph showing effect of silver nanoparticles in reducing disease severity after (A) 48 h (day 2) post infection (B) Day 5 post infection (C) reduction in necrosis of leaves (D) reduction in number of lesions formed per leaf (E) Assessment of disease parameters in terms of (a) number of lesions (b) number of spores (c) leaf area covered with lesion (d) chlorophyll content (e) fresh and dry weight in silver nanoparticles pre-treated plants as compared to other treatments. Cont-Control, SNP-Biogenic silver nanoparticles lone, AB-A. brassicicola infected plants, AB + SNP-A. brassicicola infected, treated with SNP, FW-fresh weight, DW-Dry weight. Values are the means ± SD of three replicates. Means sharing different alphabets “a”, “b” differ significantly from each other at p ≤ 0.05.

FIGURE 2

Scanning electron micrographs of adaxial surface of A. thaliana leaf. (A) Stomata of (a) control, (b) SNP, (c) AB alone, and (d) AB + SNP. S represents stomata and H represents hyphae of fungal pathogen. (Ba) Enlarged image of damaged stomata in AB alone and (b) enlarged image of normal stomata in AB + SNP treatment. (Ca,c) Image showing healthy mycelia and spore network on plant surface in AB alone. (b,d) Absence of mycelia network and damaged spores in AB + SNP. Cont, control; SNP, biogenic silver nanoparticles alone; AB, A. brassicicola-infected plants; AB + SNP, A. brassicicola-infected, treated with SNP.

FIGURE 3

Stress enzyme activities across all the treatments in terms of (A) SOD, (B) catalase, (C) GoPX, (D) PAL, and (E) PPO. Cont, control; SNP, biogenic silver nanoparticles alone; AB, A. brassicicola-infected plants; AB + SNP, A. brassicicola-infected, treated with SNP. Values are the means ± SD of three replicates. Means sharing different letters “a” and “b” differ significantly from each other at p ≤ 0.05.

FIGURE 4

Transmission electron micrographs of cell organelles of A. thaliana leaves across all treatments. (A) Sample image of A. thaliana leaf. (B) Autophagosomal structures in AB + SNP treatments. (a) Single membrane vesicles encapsulating several organelles (encircled with red) near chloroplast. (b) Membrane-bound autophagosomal structure near chloroplast. Arrow represents single-membrane autophagosomal structure, chloroplast is represented by (C). (C) Chloroplast of (a) control, (b) SNP, (c) AB alone, and (d) AB + SNP. Encircled image of AB represents damage in chloroplast. (D) Cell wall of (a) control, (b) SNP, (c) AB alone, and (d) AB + SNP. Arrow represents cell wall (CW). Cont, control; SNP, biogenic silver nanoparticles alone; AB, A. brassicicola-infected plants; AB + SNP, A. brassicicola-infected, treated with SNP. Yellow arrows represents SNPs.

FIGURE 5

(A) Clustering of differentially expressed proteins. Each protein is represented by a single row of colored boxes. Induction to suppression ranges from red to green. (B) Functional cataloging of differentially expressed proteins. The identified proteins were assigned a putative function using protein function databases and functionally grouped in five different categories as represented in the pie chart. Cont, control; SNP, biogenic silver nanoparticles alone; AB, A. brassicicola- infected plants; AB + SNP, A. brassicicola-infected, treated with SNP.

FIGURE 6

Clusterogram of differentially expressed proteins showing 11 clusters based on their expression profiles. (A) The SOTA cluster trees and (B) expression profiles of 11 different clusters. Pie chart represents respective contribution of proteins of different functional groups. Cont, control; SNP, biogenic silver nanoparticles alone; AB, A. brassicicola-infected plants; AB + SNP, A. brassicicola-infected, treated with SNP.

FIGURE 7

Total Ag content in leaves of A. thaliana. Cont, control; SNP, biogenic silver nanoparticles alone; AB, A. brassicicola-infected plants; AB + SNP, A. brassicicola-infected, treated with SNP. Values are the means ± SD of three replicates. Means sharing different letters “a” and “b” differ significantly from each other at p ≤ 0.05.

SCHEME 1

Molecular mechanism involved in amelioration of biotic stress by SNP during tripartite interaction of plant–pathogen–nanoparticles. Black spheres represent SNP, red squares represent proteins and metabolites upregulated, and green squares represent proteins and metabolites downregulated. Red arrows shows the differentially expressed proteins involved in different pathways. Black arrows demonstrate hypothesized mechanism linking differentially expressed proteome and metabolome. ROS, reactive oxygen species; HSP, heat shock proteins; GST, glutathione S transferase; MDHAR, monodehydroascorbatereductase.