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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.

Dong Y, Li Y, Zhao M, Jing M, Liu X, Liu M, Guo X, Zhang X, Chen Y, Liu Y, Liu Y, Ye W, Zhang H, Wang Y, Zheng X, Wang P, Zhang Z - PLoS Pathog. (2015)

Bottom Line: A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15.In addition, 134 candidate effectors with various segregation patterns were identified.Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China; Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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ppat.1004801.g008: 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.

Mentions: The Δiug6, Δiug9, and Δiug18 mutants were examined for phenotypes including conidiogenesis. Conidiation in 10-d-old cultures of the iug6 mutant was reduced dramatically, by ~17-fold, compared with WT (Fig 6B). The Δiug9 and Δiug18 mutants showed approximately 14% and 32% reduction in conidiation on SDC medium (Fig 6B). Microscopic observations showed that Δiug6, Δiug9, and Δiug18 mutants produced significantly fewer conidia than the WT strain (Fig 6A). To determine whether IUG6, IUG9, and IUG18 affect the expression of conidiation-related genes including MoCOM1, MoHOX2, MoCON7, MoCOS1, and MoSTUA [41,42,43,44,45], we measured and found that their expression was decreased (Fig 6C). To determine whether Δiug6, Δiug9, and Δiug18 have defects in pathogenicity, conidial suspensions (5 x 104 spores /ml) were sprayed onto 2-wk-old susceptible rice seedlings (CO-39 & LTH). Only small, necrotic-like dark brown spots were observed in Δiug6-infected rice leaves in comparison to controls (Fig 7A and 7B). When the lesions were excised, surface sterilized with 70% ethanol for 1 min, and incubated with light and humidity on 4% water agar for 2 days, as described in a previous study [46], no fungal growth or conidia occurred. The Δiug9 and Δiug18 mutants also resulted in a reduction in disease symptoms on rice 7 days after inoculation (Fig 8A). The mean lesion density per unit area of the mutants was significantly lower than that of WT (Fig 8B). Disease symptoms of three approximately 6 cm long rice blades from the same parts of plants infected by Δiug9 mutant or WT were also quantified using a ‘lesion-type’ scoring assay [9], which showed that lesion types 4 and 5 (severe, coalescing) were rarely produced by the Δiug9 mutant (Fig 8C). In addition, fungal DNA in rice was significantly lower in infection by Δiug9 and Δiug18 mutants than that by the WT as determined by M. oryzae 28S rDNA quantitation [47] (Fig 8D).


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.

Dong Y, Li Y, Zhao M, Jing M, Liu X, Liu M, Guo X, Zhang X, Chen Y, Liu Y, Liu Y, Ye W, Zhang H, Wang Y, Zheng X, Wang P, Zhang Z - PLoS Pathog. (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383609&req=5

ppat.1004801.g008: 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.
Mentions: The Δiug6, Δiug9, and Δiug18 mutants were examined for phenotypes including conidiogenesis. Conidiation in 10-d-old cultures of the iug6 mutant was reduced dramatically, by ~17-fold, compared with WT (Fig 6B). The Δiug9 and Δiug18 mutants showed approximately 14% and 32% reduction in conidiation on SDC medium (Fig 6B). Microscopic observations showed that Δiug6, Δiug9, and Δiug18 mutants produced significantly fewer conidia than the WT strain (Fig 6A). To determine whether IUG6, IUG9, and IUG18 affect the expression of conidiation-related genes including MoCOM1, MoHOX2, MoCON7, MoCOS1, and MoSTUA [41,42,43,44,45], we measured and found that their expression was decreased (Fig 6C). To determine whether Δiug6, Δiug9, and Δiug18 have defects in pathogenicity, conidial suspensions (5 x 104 spores /ml) were sprayed onto 2-wk-old susceptible rice seedlings (CO-39 & LTH). Only small, necrotic-like dark brown spots were observed in Δiug6-infected rice leaves in comparison to controls (Fig 7A and 7B). When the lesions were excised, surface sterilized with 70% ethanol for 1 min, and incubated with light and humidity on 4% water agar for 2 days, as described in a previous study [46], no fungal growth or conidia occurred. The Δiug9 and Δiug18 mutants also resulted in a reduction in disease symptoms on rice 7 days after inoculation (Fig 8A). The mean lesion density per unit area of the mutants was significantly lower than that of WT (Fig 8B). Disease symptoms of three approximately 6 cm long rice blades from the same parts of plants infected by Δiug9 mutant or WT were also quantified using a ‘lesion-type’ scoring assay [9], which showed that lesion types 4 and 5 (severe, coalescing) were rarely produced by the Δiug9 mutant (Fig 8C). In addition, fungal DNA in rice was significantly lower in infection by Δiug9 and Δiug18 mutants than that by the WT as determined by M. oryzae 28S rDNA quantitation [47] (Fig 8D).

Bottom Line: A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15.In addition, 134 candidate effectors with various segregation patterns were identified.Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China; Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China.

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.

No MeSH data available.


Related in: MedlinePlus