Proteomic and Transcriptomic Analyses Provide Novel Insights into the Crucial Roles of Host-Induced Carbohydrate Metabolism Enzymes in Xanthomonas oryzae pv. oryzae Virulence and Rice-Xoo Interaction

Abstract

Background: Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a devastating rice disease. The Xoo-rice interaction, wherein wide ranging host- and pathogen-derived proteins and genes wage molecular arms race, is a research hotspot. Hence, the identification of novel rice-induced Xoo virulence factors and characterization of their roles affecting rice global gene expression profiles will provide an integrated and better understanding of Xoo-rice interactions from the molecular perspective.

Results: Using comparative proteomics and an in vitro interaction system, we revealed that 5 protein spots from Xoo exhibited significantly different expression patterns (|fold change| > 1.5) at 3, 6, 12 h after susceptible rice leaf extract (RLX) treatment. MALDI-TOF MS analysis and pathogenicity tests showed that 4 host-induced proteins, including phosphohexose mutase, inositol monophosphatase, arginase and septum site-determining protein, affected Xoo virulence. Among them, mutants of two host-induced carbohydrate metabolism enzyme-encoding genes, ΔxanA and Δimp, elicited enhanced defense responses and nearly abolished Xoo virulence in rice. To decipher rice differentially expressed genes (DEGs) associated with xanA and imp, transcriptomic responses of ΔxanA-treated and Δimp-treated susceptible rice were compared to those in rice treated with PXO99^A at 1 and 3 dpi. A total of 1521 and 227 DEGs were identified for PXO99^A vs Δimp at 1 and 3 dpi, while for PXO99^A vs ΔxanA, there were 131 and 106 DEGs, respectively. GO, KEGG and MapMan analyses revealed that the DEGs for PXO99^A vs Δimp were mainly involved in photosynthesis, signal transduction, transcription, oxidation-reduction, hydrogen peroxide catabolism, ion transport, phenylpropanoid biosynthesis and metabolism of carbohydrates, lipids, amino acids, secondary metabolites, hormones, and nucleotides, while the DEGs from PXO99^A vs ΔxanA were predominantly associated with photosynthesis, signal transduction, oxidation-reduction, phenylpropanoid biosynthesis, cytochrome P450 and metabolism of carbohydrates, lipids, amino acids, secondary metabolites and hormones. Although most pathways were associated with both the Δimp and ΔxanA treatments, the underlying genes were not the same.

Conclusion: Our study identified two novel host-induced virulence factors XanA and Imp in Xoo, and revealed their roles in global gene expression in susceptible rice. These results provide valuable insights into the molecular mechanisms of pathogen infection strategies and plant immunity.

Keywords: Carbohydrate metabolism enzyme; Differentially expressed genes; Host-induced proteins; Pathogenicity; Xoo-rice interaction.

Fig. 1

Identification of host-induced proteins of X. oryzae pv. oryzae PXO99^A in the in vitro assay system using comparative proteomic analysis. Representative 2-DE profiles of the total proteins of X. oryzae pv. oryzae at 3, 6, and 12 h after treatment with NB plus rice leaf extract (+RLX; left gel) and NB (−RLX; right gel). Protein spots that were significantly altered (|fold change| > 1.5) in +RLX groups compared to -RLX groups at all three time points are indicated by red arrows and circles. These protein spots were excised from silver-stained gels and identified via MALDI-TOF-MS. Detailed information regarding 5 successfully identified proteins was provided in Table 1. The experiments were repeated three times independently, with similar results

Fig. 2

Four of 5 host-induced proteins were involved in X. oryzae pv. oryzae PXO99^A virulence on susceptible rice plants. A Inoculation of PXO99^A, ΔxanA, Δimp, ΔrocF, ΔminD, Δbfr and their complemented strains onto the susceptible rice IR24 by the leaf clipping method. Representative disease symptoms were recorded 16 dpi (days post inoculation). B Lesion length caused by tested strains on leaves of susceptible rice IR24 at 16 dpi. Values are the means ± standard deviation (SD) from three independent experiments. Asterisks indicate significant differences compared with wild-type (t-test, **P < 0.01). PXO99^A is wild-type X. oryzae pv. oryzae; ΔxanA, the xanA deletion mutant; ΔxanA(xanA), the complemented strain of ΔxanA; Δimp, the imp deletion mutant; Δimp(imp), the complemented strain of Δimp; ΔrocF, the rocF deletion mutant; ΔrocF(rocF), the complemented strain of ΔrocF; ΔminD, the minD deletion mutant; ΔminD(minD), the complemented strain of ΔminD; Δbfr, the bfr deletion mutant; Δbfr(bfr), the complemented strain of Δbfr

Fig. 3

Detection and assessment of cellular defense responses in rice IR24 at 1 and 3 d after infection with PXO99^A, ΔxanA or Δimp. A DAB staining and visualization of hydrogen peroxide in IR24 rice. 5-week-old rice leaves were detached and infiltrated with DAB staining solution (1 mg/mL, pH 3.8) used for oxidative burst detection after inoculation with H₂O, PXO99^A, ΔxanA and Δimp via 1-ml needleless syringes. PXO99^A-inoculated leaves were used as controls. Orange-brown deposits were visualized after the leaves were cleared in absolute ethanol. Representative photos were taken at 1 and 3 dpi. B Quantification of H₂O₂ levels in rice leaves after inoculation with H₂O, PXO99^A, ΔxanA and Δimp via 1-ml needleless syringes. Values are the means and standard deviation of three independent experiments, each of which comprised two replicates. Asterisks indicate significant differences compared with controls (t test, ** indicates P < 0.01)

Fig. 4

Histogram presentation of Gene Ontology (GO) and Clusters of Orthologous Groups (COGs) classifications of DEGs from four pairwise comparisons: PXO99^A vs ΔxanA (1d), PXO99^A vs ΔxanA (3d), PXO99^A vs Δimp (1d) and PXO99^A vs Δimp (3d). A GO annotations and classifications of the DEGs. All DEGs from each pairwise comparison were classified into three main GO categories (biological process, cellular component and molecular function) and 28 dominant subcategories were presented. The x-axis shows the names of GO terms and categories. The y-axis indicates the number of DEGs in each category. B COG annotations and classifications of the DEGs. All DEGs from each pairwise comparison were assigned to 21 categories in the COG classification. The y-axis shows the description of the 21 functional categories, and the x-axis indicates the number of DEGs in each category. Any one DEG may be categorized into different GO and COG classes. Detailed information is shown in Table S2 and Table S3

Fig. 5

KEGG pathway enrichment analysis of DEGs from comparison groups PXO99^A vs ΔxanA (1d), PXO99^A vs ΔxanA (3d), PXO99^A vs Δimp (1d) and PXO99^A vs Δimp (3d). Histogram of the top 33 significantly enriched pathways with the highest representation of the DEGs. The names of the KEGG pathways are listed along the x-axis. The y-axis indicates the number of enriched genes in different comparison groups. Asterisks indicate significant enrichment (*P < 0.05). The detailed information is shown in Table S4

Fig. 6

MapMan analysis and comparison of the metabolic changes in IR24 rice at 1 and 3 d after infection with the mutants ΔxanA and Δimp relative to those with PXO99^A infection. In each comparison group, the corresponding DEGs with |log2 (fold change)| ≥ 1 were imported into MapMan software. The gray circles indicate that no differentially expressed genes were matched in this process. The red and blue squares attached to each metabolic pathway represent up- and downregulated genes, respectively. The color intensity represents the gene expression level (log2 ratio mutant/PXO99^A), as indicated by the color scale. Detailed information is shown in Table S6

Fig. 7

MapMan visualization and comparison of the DEGs associated with the biotic stress pathway among IR24 rice infected by PXO99^A, ΔxanA or Δimp at 1 and 3 dpi. In each comparison group, the corresponding DEGs with |log2 (fold change)| ≥ 1 were imported into MapMan software. The gray circles indicate missing data. The DEGs successfully matched to the biotic stress pathway are represented by colored squares, where the red squares and blue squares indicate up- and downregulated genes, respectively. The color intensity represents the gene expression level (log2 ratio mutant/PXO99^A), as indicated by the color scale. Detailed information is presented in Table S7

Fig. 8

The qRT-PCR validation of 9 randomly selected DEGs from RNA-Seq data. The genes eEF1a and actin were used as internal standard. PXO99^A_1d and PXO99^A_3d were as control groups at 1dpi and 3dpi respectively. Gene expression level in the control group was set to 1.0. The data were expressed as the mean fold change (means ± SD, n = 3) relative to the corresponding control group. The values were significantly different to the control when the relative expression level (fold change) ≥ 2 or ≤ 0.5