Molecular Analysis of MgO Nanoparticle-Induced Immunity against Fusarium Wilt in Tomato

Abstract

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a devastating soilborne disease in tomatoes. Magnesium oxide nanoparticles (MgO NPs) induce strong immunity against Fusarium wilt in tomatoes. However, the mechanisms underlying this immunity remain poorly understood. Comparative transcriptome analysis and microscopy of tomato roots were performed to determine the mechanism of MgO NP-induced immunity against FOL. Eight transcriptomes were prepared from tomato roots treated under eight different conditions. Differentially expressed genes were compared among the transcriptomes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that in tomato roots pretreated with MgO NPs, Rcr3 encoding apoplastic protease and RbohD encoding NADPH oxidase were upregulated when challenge-inoculated with FOL. The gene encoding glycine-rich protein 4 (SlGRP4) was chosen for further analysis. SlGRP4 was rapidly transcribed in roots pretreated with MgO NPs and inoculated with FOL. Immunomicroscopy analysis showed that SlGRP4 accumulated in the cell walls of epidermal and vascular vessel cells of roots pretreated with MgO NPs, but upon FOL inoculation, SlGRP4 further accumulated in the cell walls of cortical tissues within 48 h. The results provide new insights into the probable mechanisms of MgO NP-induced tomato immunity against Fusarium wilt.

Keywords: Fusarium oxysporum; Solanum lycopersicum; apoplast; glycine-rich protein; nanoparticles; plant defense; transcriptome.

Figure 1

Seven comparative groups (①~⑦) of DEGs.

Figure 2

Number of DEGs in seven comparative groups. (A) Number of upregulated and downregulated genes. (B) Venn diagrams showing the number of DEGs in different three sample comparisons.

Figure 3

KEGG enrichment pathway: MAPK signaling pathway-plant. The red and blue boxes represent genes that are upregulated and downregulated, respectively. The yellow boxes represent genes that are both upregulated and downregulated. The red frames indicate MAPKKK, MAKK, and MAPK genes, from left to right. The blue frames indicate pathways with reduced expression. The red arrows indicate the upregulated OXI1 gene. The green arrows show the upregulated RbohD gene. Black solid arrows indicate stimulation/activation. Black dashed arrows indicate indirect effect.

Figure 3

KEGG enrichment pathway: MAPK signaling pathway-plant. The red and blue boxes represent genes that are upregulated and downregulated, respectively. The yellow boxes represent genes that are both upregulated and downregulated. The red frames indicate MAPKKK, MAKK, and MAPK genes, from left to right. The blue frames indicate pathways with reduced expression. The red arrows indicate the upregulated OXI1 gene. The green arrows show the upregulated RbohD gene. Black solid arrows indicate stimulation/activation. Black dashed arrows indicate indirect effect.

Figure 4

KEGG enrichment pathway: Plant-pathogen interaction. The red and blue boxes represent genes that are upregulated and downregulated, respectively. The yellow boxes represent genes that are both upregulated and downregulated. The red arrows show the upregulated Rcr3 gene. The green arrow shows the upregulated cyclic nucleotide gated channels (CNGCs) gene. Black solid arrows indicate stimulation/activation. Black dashed arrows indicate indirect effect.

Figure 4

KEGG enrichment pathway: Plant-pathogen interaction. The red and blue boxes represent genes that are upregulated and downregulated, respectively. The yellow boxes represent genes that are both upregulated and downregulated. The red arrows show the upregulated Rcr3 gene. The green arrow shows the upregulated cyclic nucleotide gated channels (CNGCs) gene. Black solid arrows indicate stimulation/activation. Black dashed arrows indicate indirect effect.

Figure 4

KEGG enrichment pathway: Plant-pathogen interaction. The red and blue boxes represent genes that are upregulated and downregulated, respectively. The yellow boxes represent genes that are both upregulated and downregulated. The red arrows show the upregulated Rcr3 gene. The green arrow shows the upregulated cyclic nucleotide gated channels (CNGCs) gene. Black solid arrows indicate stimulation/activation. Black dashed arrows indicate indirect effect.

Figure 5

qRT-PCR analysis of DEGs in the seven comparative groups. 1, “H₂O1h vs. MgO1h”; 2, “H₂O7d vs. MgO7d”; 3, “H₂O7d vs. H₂O7d + FOL1h”; 4, “MgO7d vs. MgO7d + FOL1h”; 5, “H₂O7d vs. H₂O7d + FOL21d”; 6, “MgO7d vs. MgO7d + FOL21d”.

Figure 6

Heatmap analysis to detect genes that show remarkable upregulation (asterisk) when MgO-pretreated tomato plants were inoculated with Fusarium oxysporum f. sp. lycopersici compared to those before FOL inoculation.

Figure 7

Time course of SlGRP4 expression in tomato roots pretreated with MgO NPs and challenge-inoculated with Fusarium oxysporum f. sp. lycopersici.

Figure 8

Immunomicroscopy of SlGRP4 protein (A–D) and detection of reactive oxygen species (ROS) (E–H) in tomato root tissues. (A) Control (no MgO treatment, no FOL inoculation), (B) tomato root 2 days after inoculation with FOL without MgO pretreatment, (C) tomato root treated with MgO NPs for 7 days (pretreatment only), (D) tomato root 2 days after inoculation with FOL with Mg NPs pretreatment, (E) control (no MgO treatment, no FOL inoculation), (F) tomato root 1 h after inoculation with FOL without MgO pretreatment, (G) tomato root treated with MgO NPs for 7 days (pretreatment only), and (H) tomato root 1 h after inoculation with FOL with Mg NPs pretreatment. Yellow arrows represent immuno-stained SlGRP4 proteins. Red arrows represent ROS (brown precipitate) detected by diaminobenzidine (DAB) staining. Scale bars represent 100 μm.

Figure 9

Microscopy images of tomato stems (10 cm above ground) and main roots 21 days after inoculation with Fusarium oxysporum f. sp. lycopersici. Cross sections of each tissue were stained with lactophenol cotton blue. White arrows point to the mycelial masses of F. oxysporum f. sp. lycopersici. Asterisk indicates the dark brown discoloration of vascular bundles. Scale bars represent 100 μm.