Global expression profiling of transcription factor genes provides new insights into pathogenicity and stress responses in the rice blast fungus

Abstract

Because most efforts to understand the molecular mechanisms underpinning fungal pathogenicity have focused on studying the function and role of individual genes, relatively little is known about how transcriptional machineries globally regulate and coordinate the expression of a large group of genes involved in pathogenesis. Using quantitative real-time PCR, we analyzed the expression patterns of 206 transcription factor (TF) genes in the rice blast fungus Magnaporthe oryzae under 32 conditions, including multiple infection-related developmental stages and various abiotic stresses. The resulting data, which are publicly available via an online platform, provided new insights into how these TFs are regulated and potentially work together to control cellular responses to a diverse array of stimuli. High degrees of differential TF expression were observed under the conditions tested. More than 50% of the 206 TF genes were up-regulated during conidiation and/or in conidia. Mutations in ten conidiation-specific TF genes caused defects in conidiation. Expression patterns in planta were similar to those under oxidative stress conditions. Mutants of in planta inducible genes not only exhibited sensitive to oxidative stress but also failed to infect rice. These experimental validations clearly demonstrated the value of TF expression patterns in predicting the function of individual TF genes. The regulatory network of TF genes revealed by this study provides a solid foundation for elucidating how M. oryzae regulates its pathogenesis, development, and stress responses.

Figure 1. Identification and classification of 495 M. oryzae TF genes.

These TF genes were identified based on a domain search using InterProScan (www.ebi.ac.kr/interpro) and belong to 44 families. The TF families are indicated by circles. Domains are noted by small colored rectangles. The TF genes at the overlapping regions between families indicate those possessing more than one TF motif. Two green-colored letters designate the zinc-coordinating DNA-binding motif and helix-turn-helix motif. The blue-colored number (in the circle or at the left side of the circle) indicates the number of genes in each family.

Figure 2. Expression profiles of TF genes.

(A) Heat map showing expression patterns of 206 TF genes under 31 different conditions. The color for each gene indicates its expression level relative to its mean across all of the experiments. Red indicates up-regulation; black, no differential expression; green, down-regulation. Top, condition tree; left, gene tree. The pink bar at the top indicates five infection-related conditions. The yellow rectangles indicate distinct expression patterns in each group. (B) Percentages of up-regulated, not differentially expressed, and down-regulated TF genes under each condition.

Figure 3. Venn diagrams showing up-regulated TF genes at infection-related developmental stages and under in vitro oxidative stress conditions.

(A) The number of genes induced during conidiation (112), germination (51), and appressorium formation (52) is indicated. These TF genes are grouped into the following seven categories: (1) conidiation-associated up-regulation; (2) germination-associated up-regulation; (3) appressorium-associated up-regulation; (4) up-regulation during conidiation, germination, and appressorium formation; (5) up-regulation during conidiation and germination; (6) up-regulation during conidition and appressorium formation; (7) up-regulation during germination and appressorium formation. TF genes in groups 1 to 4 are listed in Table S5. (B) Venn diagram showing the number of genes up-regulated TF genes at 78 hpi and 150 hpi, and in response to two sources of oxidative stress, 1 mM H₂O₂ and methyl viologen. (C) PCA of the expression data from 206 TF genes in the following conditions: conidiation [Con], conidial germination [Ger], appressorium formation [App], infection (78 hpi and 150 hpi), 1 mM H₂O₂, 5 mM H₂O₂, and 10 mM methyl viologen [MV].

Figure 4. Functional analysis of selected TF genes.

Phenotypes of T-DNA insertion mutants in four genes up-regulated at 72 hpi (MGG_06279.6, MGG_04951.6, MGG_04521.6, and MGG_06434.6). (A) The T-DNA insertion sites for each mutated gene, and mutant phenotypes, including sensitivity to H₂O₂, infectious growth in rice sheath and disease symptoms in 3 week old rice seedling, are shown. (B) Quantitative RT-PCR analysis of transcripts from the four TF genes in the corresponding mutant.

Figure 5. Phenotype analyses of ΔMoaps1, ΔMoaps2, and complemented mutants.

(A) Expression patterns of MoAPS1 and MoAPS2 under five conditions: Con, conidiation; Ger, conidial germination; App, appressorium formation; and 78 hpi and 150 hpi. (B) Conidial production, conidial germination, and appressorium formation (left to right). The asterisk denotes a significant difference (at P<0.05). (C) Vegetative growth on CM agar. (D) Infectious growth in rice sheaths.

Figure 6. Expression profiles of 57 conidiation-specific TF genes in six TF gene deletion mutants.

The mutants included ΔMoaps1, ΔMoaps2, ΔMohox2, ΔMohox4, ΔMoleu3, and ΔMonit4. Up-regulated genes in the mutants (more than 2 fold) are indicated by red bars, and down-regulated genes (less than 0.5 fold) are noted by blue bars. The genes that did not show differential expression in the six mutants are marked in blue.

Figure 7. A model for the regulatory network controlling the expression of conidiation-specific TF genes.

Solid diamonds indicate the genes deleted in ΔMohox2, ΔMohox4, ΔMoaps1, ΔMoaps2, ΔMoleu3, and ΔMonit4. Spheres correspond to up-regulated (red line) or down-regulated (blue line) TF genes in one or more of these mutants. Different colors of the sphere indicate different TF families: Green (Zn₂Cys₆); black (C₂H₂); violet (Homobox); orange (APSES); red (GATA); blue (bHLH); olive (Myb); violet (Forkhead). A detailed description of these genes is shown in Tables S5 and S6.