Figure 1. The disease symptoms on the rice cultivars Nipponbare and Hui1586 infected by blast fungi.
(A-B) Hui1586 exhibited strong leaf blast resistance in a natural nursery at two locations, Jinggangshan, Jiangxi Province (A) and Yichang, Hubei Province, China (B). (C) Blast resistance of Hui1586 plants using spraying inoculation in a greenhouse. Representative leaves obtained 7-day postinoculation of blast strain Guy11.
Figure 2. Overview of transcriptome data and differentially expressed genes (DEGs) in the rice response to blast fungus. (A) Principal component analysis of the time-series transcriptome data in Nipponbare (Nip) and Hui1586 treated with the blast isolate Guy11 (M.o) or H2O. (B) Hierarchical clustering of the samples for the normalized FPKM (fragments per kilobase of transcript per million mapped reads) values of all detectable genes. (C) The numbers of up- and downregulated genes by M. oryzae in Nipponbare or Hui1586 compared with the mock treatment are shown. (D) Venn diagram of total DEGs in Nipponbare compared with Hui1586.
Figure 3. Analysis of differentially expressed genes (DEGs) between Nipponbare and Hui1586 in the mock treatment. (A) The numbers of DEGs between Nipponbare and Hui1586 at the indicated time points after H2O treatment. (B) Venn diagram of DEGs in (A). (C-D) KEGG pathway (C) and GO (D) enrichment analysis of 1331 DEGs in (B). The y-axis represents the negative log10-transformed Q -value (blue bars) and gene numbers (yellow dots). (E) Enrichment of the LRR and NLRgenes in the 1331 DEGs compared with their ratio in the rice genome.
Figure 4. Pathway enrichment of DEGs in the rice response to blast fungus. (A) Venn diagram of DEGs induced by M. oryzae in Nipponbare and Hui1586 compared with mock treatment within 48 hpi. Up- and downregulated DEGs are indicated by upward and downward pointing arrows, respectively. (B) Hierarchical clustering of all the DEGs at 12 hpi based on the log2 fold change in transcript levels in Nipponbare (Nip) and Hui1586 (Hui) with M. oryzae treatment relative to the mock treatment. (C) KEGG pathway and GO enrichment analysis of 3398 upregulated DEGs at 12 h in (A). The y-axis represents the negative log10-transformed Q -value (blue bars) and gene numbers (yellow dots). (D) The enrichment of ribosome- and rRNA-related genes in the 3398 DEGs in (C) compared with its ratio in the rice genome. (E) Hierarchical clustering of ribosome- and rRNA-related genes in the 3398 DEGs in (C) based on the log2 fold change as in (B).
Figure 5. Identification and pathway analysis of a “core” set of DEGs in Nipponbare and Hui1586. (A) Venn diagram of DEGs at the four time points in Nipponbare (Nip) and Hui1586. The numbers in the blue circle represent the overlapped DEGs between the two linked groups. The upward and downward arrows indicate up- and downregulated DEGs, respectively. (B) Hierarchical clustering of the 321 + 32 DEGs in (A) based on normalized FPKM values in Nipponbare and Hui1586. (C) KEGG pathway enrichment analysis of 321 DEGs in (A). The y-axis represents the negative log10-transformed Q -value (blue bars) and gene numbers (yellow dots).
Figure 6. The core genes are involved in biotic and abiotic stress responses. (A) The bar plot shows that among the 321 core genes, the number of genes that were differentially regulated in rice treated with Magnaporthe oryzae (GSE83219), Ustilaginoidea virens(GSE39049), Xanthomonas oryzae (GSE36272) or drought (GSE57950) in polished transcriptome data sets. (B) Hierarchical clustering of the 321 core genes based on the log2–fold change with various treatments as in (A). (C) Similarity comparation among the data sets as in (A) based on the log2–fold change of the 321 core genes.
Figure 7. Characterization of transcription factors andcis-regulatory motifs in differentially regulated genes in rice in response to M. oryzae. (A) Hierarchical clustering of the total 313 transcription factors (TFs) that were differentially regulated in both Nipponbare (Nip) and Hui1586 in response to M. oryzaeinfection. The rows of collapse represent the average value of genes in the indicated groups in each column/condition. (B) Enrichment of the TF families of the 205 TFs (Cluster I in (A)) compared to their ratio in each family in the rice genome. (C) Overrepresentation of known TF DNA binding motifs within the 500-bp promoter of the 321 core genes. Rows indicate motifs of corresponding TF families and are colored according to the P- value for enrichment. (D) Sequence logo depiction of the key overrepresented TF DNA binding motifs in the 500-bp promoter of the 321 core genes.
Figure 8. Phenotypic characterization of the rice perox4knockout lines. (A) Blast resistance of two independent perox4knockout lines compared with the parental Zhonghua11 (ZH11) plants. Three representative leaves collected at 3 d postinoculation of the blast isolate Guy11 are shown. (B) Lesion numbers per cm2 on the rice leaves (mean ± SD, n > 10 leaves) after inoculation with blast fungus as in (A). (C) Expression of defense marker genes (PR2, and PR5 ) in perox4 and ZH11 at the indicated times after infection of Guy11 measured by qRT-PCR. The y-axis represents the relative expression value (log2 – transformed, mean ± SD, n = 3) normalized toOsactin (Os03g0718100 ). *, Statistical significance (P < 0.01) determined by the Student’s t- test; ns, no significant difference. (D) Photograph of 10 seeds each for ZH11 and the perox4 mutant lines. Scale bars, 0.5 cm. (E) and (F) Grain width and thousand-gain weight of the ZH11 and perox4mutant. Error bars indicate the mean ± SD (n=3 biological replicates, each replicate contains 10 seeds for G and 1000 seeds for H), *, statistical significance (P < 0.01) determined by the Student’s t- test.
Figure 9. Disease resistance of the rice osmt1a/bknockout lines. (A) Blast resistance of two independent osmt1a/bknockout lines compared with the parental Nipponbare (Nip) plants. Three representative leaves collected at 3 d postinoculation of the blast isolate Guy11 are shown. (B) Lesion numbers per cm2 on rice leaves (mean ± SD, n > 10 leaves) after inoculation with blast fungus as in (A). (C) Expression of defense marker genes (PR5, and PAL ) in osmt1a/b and Nipponbare at the indicated times after infection of Guy11 measured by qRT-PCR. The y-axis represents the relative expression value (log2 – transformed, mean ± SD, n = 3) normalized to Osactin(Os03g0718100 ). *, Statistical significance (P < 0.01) determined by the Student’s t- test; ns, no significant difference.
Table 1. Inoculation experiment with different blast isolates showing that Hui1586 has broad spectrum resistance.