A self-balancing circuit centered on MoOsm1 kinase governs adaptive responses to host-derived ROS in Magnaporthe oryzae
- Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, China
- The Key Laboratory of Plant Immunity, Nanjing Agricultural University, China
Figures
Figure 1
Time-course images of ROS accumulation during rice sheath infection by M. oryzae.
(A) The conidial suspension of Guy11 (1 × 105 spores/ml) was inoculated in the excised rice sheath of 4-weeks-old rice seedlings LTH and K23. The invasive hyphae growth was observed at 20, 24, 36, …
Figure 2 with 1 supplement
MoOsm1 phosphorylation and nuclear translocation in response to ROS stress.
(A) Phosphorylation of MoOsm1 in Guy11 in infection of LTH. Total proteins were extracted from LTH leaves 0, 8, 20, 24, 48, and 72 hr. Eluted proteins bound to the anti-GFP beads were analyzed by …
Figure 2—figure supplement 1
Localization of MoOsm1 under H2O2 stress in the conidium and mycelium.
(A) Phosphorylation of MoOsm1 in Guy11 under oxidative stress. Immunoblots were performed with total proteins from hyphae of Guy11 treated with H2O2 for 0, 10, 30, and 60 min. MoOsm1 phosphorylation …
Figure 3 with 1 supplement
Phosphorylation-mediated monomerization of MoOsm1 results in its nuclear accumulation.
(A) Localization of MoOsm1, MoOsm1Y173D, and MoOsm1Y173A in conidia. MoOsm1-GFP, MoOsm1Y173D-GFP, MoOsm1Y173A-GFP, and H1-RFP were observed by confocal fluorescence microscopy. Bars = 5 μm. (B) …
Figure 3—figure supplement 1
Thr171 and Tyr173 are important phosphorylation sites in MoOsm1.
(A) MoOsm1 phosphopeptide (QDPQMTGYVSTR) in the strain, which treated with H2O2 was identified by mass spectrometer analysis and the phosphorylated sites were Thr171 and Tyr-173. (B) Co-IP assay …
Figure 4 with 1 supplement
MoOsm1 phosphorylates MoAtf1 in the nucleus.
(A) Co-IP assay. Western blot analysis of total proteins (T) extracted from MoOsm1-GFP/MoAtf1-FLAG, MoOsm1Y173A-GFP/MoAtf1-FLAG, MoOsm1Y173D-GFP/MoAtf1-FLAG, MoOsm1NES-GFP/MoAtf1-FLAG strains, …
Figure 4—figure supplement 1
Phosphorylation of MoAtf1 in vitro.
In vitro phosphorylation analysis by the fluorescence detection in the tube (FDIT) method. Purified proteins of MoOsm1-HIS, MoAtf1-GST, and MoAtf1S124A-GST were used for the protein kinase reaction …
Figure 5 with 3 supplements
MoAtf1 is one of the regulators of the oxidation regulatory pathway.
ChIP-Seq assays showed that MoAtf1 binds to the promoters of 16 oxidation regulation pathway genes. Output- and input-DNA was visualized in blue and pink. The red arrow indicates gene direction. The …
Figure 5—figure supplement 1
Putative MoAtf1-interacting proteins identified by affinity purification.
Figure 5—figure supplement 2
Phosphorylation of MoAtf1 on S124 blocks MoAtf1 and MoTup1 interaction.
(A) Y2H analysis of the interaction between MoAtf1 and MoTup1. The pGADT7 and pGBKT7 fused with specific genes were co-introduced into the yeast AH109 strain, and transformants were plated on …
Figure 5—figure supplement 3
Expression levels of the oxidation regulatory pathway genes in the ∆Moatf1 mutant.
(A) The relative expression levels of putative oxidation regulation pathway genes in ∆Moatf1 mutant were analyzed by qRT-PCR and normalized to that of ACTIN. Error bars represent the standard …
Figure 6 with 5 supplements
MoPtps are important in host-derived ROS scavenging by M. oryzae.
(A) and (B) Excised rice sheaths from 3-week-old rice seedlings were inoculated with conidial suspension (1 × 105 spores/ml). Infectious growth was observed at 24- and 36-hr post-inoculation (hpi). …
Figure 6—figure supplement 1
Southern blot analysis of MoPTP1 and MoPTP2 deletional mutants.
Southern blot analysis of the gene knockout mutant with gene-specific probe (probe one for MoPTP1 and probe three for MoPTP2) and hygromycin phosphotransferase (HPH) probe (probe 2). The scheme of Mo…
Figure 6—figure supplement 2
MoPtps are not involved in mycelial growth and conidiation.
(A) Comparison of ∆Moptps and wild-type strain in colony morphology. Wild type, the ∆Moptps mutant and complementation strains were cultured at 28°C in darkness for 7 days. (B) Statistical analyses …
Figure 6—figure supplement 3
MoPtp1 and MoPtp2 are involved in dephosphorylation of MoOsm1.
(A) Pathogenicity assay. Conidial suspensions were sprayed onto two-week-old rice seedlings (CO-39). Diseased leaves were photographed after 7 days of inoculation. (B) Quantification of lesion type. …
Figure 6—figure supplement 4
Hyperphosphorylation on MoOsm1 Tyr173 causes virulence defect in M. oryzae.
(A) MoOsm1 phosphopeptide (QDPQMTGYVSTR) in the ΔMoptp2/MoOSM1-GFP strain was identified by mass spectrometer analysis and the phosphorylated sites were Tyr-173. (B) The pathogenicity assay on rice …
Figure 6—figure supplement 5
Appressorium formation in the ∆Moptps mutants.
Appressorium formation rates at different time points were calculated and statistically analyzed. The percentage at a given time was recorded by observing at least 200 conidia for each strain and …
Figure 7 with 2 supplements
MoPtps-mediated dephosphorylation of MoOsm1 leads to its nuclear exporting and monomerization.
(A) Pathogenicity assay. Conidial suspensions were sprayed onto two-week-old rice seedlings (CO-39). Diseased leaves were photographed after 7 days of inoculation. (B) Dephosphorylation of MoOsm1 …
Figure 7—figure supplement 1
A comparison of MoPtp1 and MoPtp2 functional domains.
A model of MoPtp1, MoPtp2, and mutants constructs. Black bars indicate the conserved phosphatase-domain (PTPc) in MoPtp1 and MoPtp2 that were mutanted in ∆Moptp1/MoPTP1∆ptpc and ∆Moptp2/MoPTP2∆ptpc. …
Figure 7—figure supplement 2
MoOsm1 phosphorylation levels in ∆Moptp1/2 mutants under oxidative stress.
Immunoblots were performed with total proteins from Guy11, ΔMoptp1, and ΔMoptp2 mutant strains without or with 5 mM H2O2 for 10, 30, and 60 min. The MoOsm1 phosphorylation level was detected using …
Figure 8 with 1 supplement
MoAtf1 binds to the promoter regions of MoPTP1 and MoPTP2.
(A) Expression analysis of MoPTP1 in the ∆Moatf1 mutant and wild-type strains. The expression of MoPTP1 was analyzed by qRT-PCR and normalized to that of actin gene (MGG_03982). MY represents …
Figure 8—figure supplement 1
The ChIP assay identifies MoPTP1 and MoPTP2 as putative targets of MoAtf1.
(A) The cis-element of MoAtf1 was predicted by MEME. The putative sequence of the cis-element obtained by analyzing the promoters of MoAtf1 binding genes with the MEME program. (B) Schematic …
Figure 9
MoAtf1 phosphorylation controls the transcription of MoPTP1 and MoPTP2 in response to oxidative stress.
(A) and (B) MoPTP1 and MoPTP2 expression analysis in Guy11, ∆Moosm1 mutant, and ∆Moosm1/MoAtf1S124D strains treated with H2O2 for 15, 30, and 60 min. Three independent biological experiments were …
Figure 10
A proposed model depicting MoOsm1/MoAtf1/MoTup1/MoPtp1/2 mediated ROS signaling and responses to host immunity.
Rice generates immunity, including ROS burst during its interaction with M. oryzae. Once host perception, M. oryzae induces MoOsm1 phosphorylation that disintegrates MoOsm1 dimerization leading to …
Additional files
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Supplementary file 1
574 putative binding proteins of MoAtf1 by CHIP assay.
Genomic location and Annotation ID of MoAtf1 binding genes.
- https://cdn.elifesciences.org/articles/61605/elife-61605-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61605/elife-61605-transrepform-v1.docx
