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.