Global genome and transcriptome analyses of Magnaporthe oryzae epidemic isolate 98-06 uncover novel effectors and pathogenicity-related genes, revealing gene gain and lose dynamics in genome evolution

Abstract

Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions.

Fig 1. Global view of synteny alignments between isolates 98–06 and 70–15.

Scaffolds, genes, and secreted protein genes distribution in isolate 98–06 compared to 70–15. For each chromosome, the first line (i) represents the genomic scaffolds of 98–06 alignment with 70–15; the second line (ii) displays the chromosmes of 70–15; the third line (iii) and fourth line (iv) show all genes and secreted protein genes, respectively. The red small vertical lines in line (iii) and line (iv) show the isolate-specific genes in 98–06 compared to 70–15. Chr, chromosome.

Fig 2. Analysis of different signal pathways gene expression in rice.

Heat maps in boxes indicate the expression of individual genes, and the chart plots indicate the aggregate expression levels of the pathway genes. Green box, transcription factors. Red circle, genes for the nearest heat map.

Fig 3. Clustering analysis of gene expression patterns of pathogenicity-related genes and effectors through RNA-Seq.

(A) CAST assay shows 14 M. oryzae expression clusters of 64 reported pathogenicity genes and ten effectors of M. oryzae at different stages. The y-axis stands for the log2 average gene expression levels. The quantity of cluster member is marked at the right bottom of each pattern line. (B) Heat map showing expression levels of known pathogenicity genes and effectors of M. oryzae during compatible interactions. The color bar represents the log2 of (RPKM +1) value, ranging from blue (0.0) to red (10.0). Top, stage tree; left, gene tree. The pink rectangle indicates expression pattern of effectors. The blue rectangle indicates expression pattern relevant to pathogenicity.

Fig 4. Heat map of 645 small candidate effectors of 98–06.

645 candidate effectors were added to employ the hierarchical clustering, and divided into two groups (red rectangle and gray rectangle) according to the expression pattern of Avr-Pik and AvrPiz-t, providing 134 candidate effectors. The color bar represents the log2 of (RPKM +1) value, ranging from blue (0.0) to red (10.0).

Fig 5. Iug6, Iug9, Nup1, Nup2, and Nup3 suppress the cell death triggered by BAX.

(A) Agroinfiltration sites in N. benthamiana leaves expressing Iug6, Iug9, Nup1, Nup2, or Nup3 were challenged with A. tumefaciens expressing the BAX elicitin. The BAX-induced cell death was scored at 3 and 4 DAI. A. tumefaciens strain carrying pGR106-GFP was used as a negative control, and pGR106-BAX as a positive control. (B) Western blot analysis of GFP, Iug6, Iug9, Nup1, Nup2, Nup3, and Bax protein levels in plant tissues treated above. Proteins were extracted 60 h after the last infiltration. Equal amounts of protein lysate were loaded in each lane, as verified by Ponceau S staining. (C) Presence or absence polymorphisms of each candidate effectors are indicated by a colored or black tile across the 29 field isolates. Colored tile, presence; black tile, absence. Presence /absence patterns (top) and isolates (left) were hierarchically clustered.

Fig 6. Disruption of IUG genes lead to reduced conidiation.

(A) Development of conidia on conidiophores is affected by IUG genes deletion. Light microscopy was observed on strains grown on SDC medium for 7 days. Bars = 100 μm. (B) Statistical analysis of conidial production by wild type, Δiug6, Δiug9, Δiug18, and complemented strains. (C) Reduced expression patterns are found in five genes encoding conidiation-associated genes in all mutants. RNA was extracted from mycelia grown in liquid CM for 2 days. Error bars represent the standard deviation and asterisks represent significant differences (P<0.01).

Fig 7. IUG6 is involved in pathogenicity of M. oryzae.

(A) The IUG6 deletion leads to a significant reduction in pathogenicity on rice leaves. 4 ml of conidial suspension (5 x 10⁴ spores /ml) for each strain was sprayed on two-week-old rice seedlings (O. sativa cv CO-39) and 60 healthy rice plants were used in each independent experiment. Diseased leaves were harvested 7 days after inoculation. (B) Leaves of 4-week-old rice seedlings were injected with conidial suspensions of 98–06, Δiug6 and complemented strain. Diseased leaves were photographed 7 days after inoculation. (C) Rice leaf sheath penetration assay indicates severely confined growth of the Δiug6 mutant hyphae at 48 hpi compared to 98–06. 50 infection sites were examined for each experiment and experiments were repeated twice with similar results. Bars = 20 μm. AP: appressoria. (D) Infection hyphae were observed in the cells on the back side of barley leaves at 48 hpi. 50 infection sites were examined for each experiment and experiments were repeated twice with similar results. Bars = 20 μm.

Fig 8. IUG9 and IUG18 are involved in pathogenicity of M. oryzae.

(A) Disease symptoms were reduced on rice leaves inoculated with Δiug9 and Δiug18 mutants. Conidial suspension (5 x 10⁴ spores/ml) of the wild-type strain 98–06, mutants and complemented strains were inoculated on rice (cv. LTH), and incubated for 7 days. (B) Bar chart of mean lesion density of seedlings infected with isolate 98–06 and the Δiug18 mutant per unit area. Mean lesion density was significantly reduced in Δiug18 mutant infections. Error bars represent the standard deviation and asterisks represent significant differences (P<0.01). (C) Quantification of lesion types (0, no lesion; 1, pinhead-sized brown specks; 2, 1.5-mm brown spots; 3, 2–3-mm grey spots with brown margins; 4, many elliptical grey spots longer than 3 mm; 5, coalesced lesions infecting 50% or more of the leaf area) reveals no difference in lesion types 1–3; however, the Δiug9 mutant make rarely lesions of types 4 and 5. Lesions were photographed and measured or scored at 7 days post-inoculation (dpi) and experiments were repeated twice with similar results. (D) Severity of blast disease was evaluated by quantifying M. oryzae genomic 28S rDNA relative to rice genomic Rubq1 DNA (7 days post-inoculation). Mean values of three determinations with standard deviations are shown. The asterisks indicate a significant difference from the 98–06 (P < 0.01). (E) Percentage of difference infection hyphae type (I = no infection hyphae; II = only one infection hyphae; III = two or three branches of the infection hyphae; IV = more than three branches of infection hyphae), occupied by each strain in the reverse side cells of barley 32 h after inoculation. The total number of appressorium-mediated penetration and infection is indicated (top right corner, N = 100). (F) Typical infection sites of rice leaf sheath inoculated with 98–06 strain, Δiug18, Δiug9 mutants, and complemented strains, showing greater fungal proliferation and tissue invasion by the wild-type strain. Infectious growth was observed at 30 hpi. Bars = 50 μm.

Fig 9. Iug6 and Iug9 proteins accumulate at BICs in sheath epidermal cells.

(A) Cellular localization of Iug6:GFP in M. oryzae during biotrophic growth on epidermal rice cells at 27 hpi. Fluorescence was observed accumulating preferentially at BICs. Merged DIC and GFP images and GFP fluorescence alone are shown. BICs are indicated by arrows. Bars = 10 μm. (B) Secretion of Iug9:GFP at 27 hpi. Fluorescence was at BICs. Bars = 10 μm. (C) AvrPiz-t:GFP was observed preferential BIC localization at 30 hpi. Bars = 10 μm.

Fig 10. Over-expression of IUG6 or IUG9 in Guy11 suppresses defense-related genes in rice.

(A) qRT-PCR on IUG6 or IUG9 at different fungal developmental stages in Guy11 overexpression transformants in comparison with mycelium phase of isolate 98–06. RNA was extracted from mycelia and infectious stages (8, 24, and 48 hpi), respectively. (B) Expression of PR1a and Cht1 marker genes in the infected rice is suppressed in the presence of Iug6 or Iug9. RNA samples were collected from rice plants (O. sativa cv CO-39) 0, 24, and 48 h after inoculation with the Guy11, 98–06, or OE strains. The average threshold cycle (Ct) of triplicate reactions was normalized by the stable expressions ACTIN gene in O. sativa. Three independent biological experiments were performed and yielded similar results. Error bars in the figure represent the standard deviation.