Shared and distinct functions of two Gti1/Pac2 family proteins in growth, morphogenesis and pathogenicity of Magnaporthe oryzae
Summary Gti1/Pac2 are conserved family proteins that regulate morphogenic transition in yeasts such as Schizosaccharomyces pombe and Candida albicans, and they also control toxin production and pathogenicity in filamentous fungus Fusarium graminearum. To test the functions of Gti1/Pac2 paralogues Mo...
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| Published in | Environmental microbiology Vol. 16; no. 3; pp. 788 - 801 |
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| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Oxford
Blackwell Publishing Ltd
01.03.2014
Blackwell Wiley Subscription Services, Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Summary
Gti1/Pac2 are conserved family proteins that regulate morphogenic transition in yeasts such as Schizosaccharomyces pombe and Candida albicans, and they also control toxin production and pathogenicity in filamentous fungus Fusarium graminearum. To test the functions of Gti1/Pac2 paralogues MoGti1 and MoPac2 in the rice blast fungus Magnaporthe oryzae, we generated respective ΔMogti1 and ΔMopac2 mutant strains. We found that MoGti1 and MoPac2 exhibit shared and distinct roles in hyphal growth, conidiation, sexual reproduction, stress responses, surface hydrophobility, invasive hyphal growth and pathogenicity. Consistent with the putative conserved function of MoGti1, we showed that MoGti1‐GFP is localized to the nucleus, whereas MoPac2‐GFP is mainly found in the cytoplasm. In addition, we provided evidence that the nuclear localization of MoGti1 could be subject to regulation by MoPmk1 mitogen‐activated protein kinase. Moreover, we found that the reduced pathogenicity in the ΔMopac2 mutant corresponds with an increased expression of plant defence genes, including PR1a, AOS2, LOX1, PAD4, and CHT1. Taken together, our studies provide a comprehensive analysis of two similar but distinct Gti1/Pac2 family proteins in M. oryzae, which underlines the important yet conserved functions of these family proteins in plant pathogenic fungi. |
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| Bibliography: | istex:E673577F615FC4FC5D862DE1E3B057DD86EFE245 Fig. S1. Expression analysis of MoGTI1 and MoPAC2 in the ΔMoap1 mutant. Transcriptional levels of MoGTI1 and MoPAC2 in wild-type, ΔMogti1 and ΔMopac2 strains before and after exposure to H2O2 were measured by qRT-PCR.Fig. S2. MoGti1 and MoPac2 are two members of the fungal Gti1/Pac2 domain family of M. oryzae and their transcription profiles at different stages of fungal development. A. Bioinformatics search of the amino acid sequence revealed both of proteins containing the typical Gti1/Pac2 family domain (white box), MoGti1 contains three phosphorylation sites (P), an NLS and a potential leucine-rich NES. B. The phase-specific expression of MoGTI1 and MoPAC2 was quantified by qRT-PCR, with the synthesis of cDNA from each sample including infectious growth, vegetative growth and conidia. hpi: hours post-inoculation.Fig. S3. Targeted gene replacement and complementation. A and B. A 1.4 kb fragment of the MoGTI1 and a 1.42 kb fragment of the MoPAC2 coding region were replaced by a 1.4 kb fragment containing the hygromycin B resistance cassette to create the ΔMogti1 and ΔMopac2 alleles respectively. C and D. Southern hybridization analysis was used to validate the deletion of MoGTI1 and MoPAC2 genes and the addition of a single copy integration of the HPH gene. E. Semiquantitative RT-PCR was carried out to confirm the deletion and reintroduction of MoGTI1 and MoPAC2 genes.Fig. S4. The ΔMogti1 mutants have defects in conidial morphology and germ tube differentiation. A. Conidia of the wild type and mutants were observed under a light microscope. B. Conidia were stained with 1 μg ml−1 calcofluor white (CFW) for 5 min in dark and observed under an epifluorescence microscope. C. Appressorium formation was allowed in inductive condition. Conidia from each strain were incubated on hydrophobic surfaces for 24 h and photographed.Fig. S5. Conidial morphology and appressorium formation were normal in the ΔMopac2 mutant. A. Conidia of the wild-type and ΔMopac2 mutant strains were observed under a light microscope. B. Appressorium formation was allowed in inductive condition. Conidia from each strain were incubated on hydrophobic surfaces for 24 h and photographed.Fig. S6. ΔMogti1 and ΔMopac2 stress assessment. A. The ΔMogti1 mutant is hypersensitive to H2O2. The wild-type strain Guy11, ΔMogti1 and ΔMopac2 strain were inoculated on CM with or without 2.5 or 5 mM H2O2, and cultured at 28°C for 7 days. B. The growth inhibition rate is estimated relative to the growth rate of each untreated control [inhibition rate = (the diameter of untreated strain - the diameter of treated strain)/(the diameter of untreated strain × 100%)]. Three repeats were performed and similar results obtained. Error bars represent the standard deviations and lowercase represent significant differences (P < 0.05). C. The ΔMogti1 mutant is more sensitive to hyperosmotic stress than Guy11. Colonies of the wild type, ΔMogti1 and ΔMopac2 grown on CM plates with or without 0.7 M NaCl, 1.2 M KCl or 1 M sorbitol, and cultured at 28°C for 7 days. D. The growth inhibition rate is estimated similar to above (B).Fig. S7. ΔMogti1 and ΔMopac2 pathogenicity test. A. Pathogenicity test of ΔMogti1 and ΔMopac2 mutants on barley leaves. Four millilitres of conidia suspension (5 × 104 spores ml−1) of each strain were sprayed on 7-day-old barley seedlings. Diseased leaves were photographed at 5 days after inoculation. B. Pathogenicity test of the mutant strain by conidia injection. Conidia, which were resuspended to 5 × 104 conidia ml−1 in 0.2% gelatin, were used for inoculation. Photographs were taken at 5 days after inoculation. C. Assays for fungal growth on surface-sterilized rice leaves inoculated by spray in Guy11 and ΔMopac2 mutant. Abundant conidia were observed on leaves inoculated with Guy11, but no fungal growth was observed on tiny lesion in the ΔMopac2 mutant. Arrows point to conidia.Fig. S8. Expression analysis of MoMPG1. Transcriptional levels of MoMPG1 in wild-type, ΔMogti1, ΔMopac2 and overexpressing transformant strains were measured by qRT-PCR.Fig. S9. Overexpression MoMPG1 in the mutants restores the surface hydrophobicity but not pathogenicity defect. A. Twenty microlitres of water or detergent solution of different concentration (0.02% SDS and 5 mM EDTA) were placed on the colony surfaces of wild-type, the mutants and MoMPG1 overexpression strains and photographed after 5 min. B. Four millilitres of conidia suspension (5 × 104 spores ml−1) of each strain were sprayed on 2-week-old rice seedlings. Diseased leaves were photographed at 7 days after inoculation.Fig. S10. Expression and localization of MoPAC2-GFP fusion gene construct and co-IP assay for the interaction of MoPmk1 with MoGti1. A. Conidium of ΔMopac2/MoPAC2-GFP transformant examined under an epifluorescence microscope. GFP signals were observed in the cytoplasm. B. Western blot analysis with total proteins (Total) isolated from transformants co-expressing the MoPMK1-3 × FLAG and MoGTI1-GFP constructs and proteins eluted from the anti-FLAG M2 beads (Elution). The presence of MoPmk1 and MoGti1 was detected with an anti-FLAG and anti-GFP antibody respectively.Fig. S11. Expression analysis of genes involved in conidiation in the ΔMogti1 and ΔMopac2 mutants. RNA isolated from vegetative hyphae of CM cultures was used for qRT-PCR analysis. Transcript levels of genes were indicated from three independent experients.Table S1. Primers used in this study.Table S2. Catagorization of MoGti1 regulated proteins with known function.Table S3. Catagorization of MoPac2-regulated proteins with known function. Fundamental Research Funds for the Central Universities - No. KYZ201105 ArticleID:EMI12204 NIH/NIAID - No. AI054958; No. AI074001 ark:/67375/WNG-4D30NSBH-P National Basic Research Program of China - No. 2012CB114000 Natural Science Foundation of China - No. 31271998 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 |
| ISSN: | 1462-2912 1462-2920 1462-2920 |
| DOI: | 10.1111/1462-2920.12204 |