Figure 1.
Identification of the T-DNA-tagged genes MoSOM1 and MoCDTF1.
(A) Colonies of the wild type strain Guy11 and three T-DNA insertional mutants. (B) Barley and rice leaf segments were inoculated with the mycelia from Guy11, YX-145, YX-1303 and YX-864, H2O was used as the control. (C) Position of the T-DNA insertion in YX-145, YX-1303 and YX-864 mutants. The nucleotide in the brackets was deleted by T-DNA integration. The arrows (▾) indicate the T-DNA (2.2 kb) insertion positions in MoSOM1, MoCDTF1 and MoMSB2 genes, respectively. The thick boxes represent the coding regions and the thin line joining these coding regions indicates the position of the introns.
Figure 2.
The Δmosom1, Δmocdtf1 and Δmosom1Δmocdtf1 mutants are nonpathogenic.
(A) Barley and rice segments inoculated with the mycelia from the wild-type strain Guy11, YX-145, SK27 (Δmosom1), ES16 (ectopic transformant), SC1 (Δmosom1+MoSOM1), CTK15 (Δmocdtf1), CTC1 (Δmocdtf1+MoCDTF1) and D-3 (Δmosom1Δmocdtf1). a = unwounded leaf and b = abraded leaf. (B) Root infection assays. The mutants SK27 and D-3 were non-pathogenic when inoculated onto rice roots, but the mutant CTK15 was still able to cause some disease symptom. Arrows indicate necrotic lesions. H2O was used as the control. Photographs were taken at 5 days after inoculation.
Figure 3.
MoSOM1 and MoCDTF1 are required for vegetative growth and pigmentation.
(A) Mycelium growth and pigmentation were significantly impaired in SK27 (Δmosom1), CTK15 (Δmocdtf1) and D-3 (Δmosom1Δmocdtf1) mutants. The mutants and the wild type strain (Guy11) were inoculated on CM medium and cultured at 25°C for 10 days (top). Reduced pigmentation was observed from the colony back side of the mutants (middle). Mycelium growth patterns of the strains in liquid CM medium at 25°C for 48 h (bottom). Scale bars = 5 mm. (B) Bar chart showing the colony diameters of Guy11, YX-145, SK27, ES16 (ectopic transformant), SC3 (Δmosom1+MoSOM1), YX-1303, CTK15, CTC1 (Δmocdtf1+MoCDTF1) and D-3. Error bars represent standard deviation. Asterisks indicate significant difference at P = 0.01.
Figure 4.
MoSOM1 and MoCDTF1 are essential for producing asexual/sexual spores and appressoria.
(A) Bar chart showing the conidial production. The YX-145, SK27 (Δmosom1), YX-1303, CTK15 (Δmocdtf1) and D-3 (Δmosom1Δmocdtf1) mutants were unable to produce any conidia, while the wild type strain Guy11, SC3 (Δmosom1+MoSOM1) and CTC1 (Δmocdtf1+MoCDTF1) formed numerous conidia on CM medium at 25°C for 10 days. Error bars represent standard deviation. (B) Microscopic observation of conidial development. Aerial hyphae were significantly reduced and conidiophores were not observed in the SK27, CTK15 and D-3 mutants. The wild-type strain Guy11 and the complementation strains (SC3 and CTC1) formed normal conidiophores and numerous conidia. All the tested strains grown on CM medium for 4 days were examined by light microscopy and photographed. Scale bar = 20 µm. (C) Fertility assay. Guy11×TH3 formed numerous perithecia on oatmeal medium after 3-week incubation in an inductive condition, while no perithecia was observed for the crosses of SK27×TH3, CTK15×TH3 and D-3×TH3. (D) Appressorium formation assay. Mycelium fragments of Guy11, SK27, CTK15, D-3, SC3 and CTC1 were placed on hydrophobic GelBond film surfaces to allow appressorium development, respectively. Appressorium formation was observed after 24 h incubation at 25°C in darkness. Numerous appressoria were produced by Guy11 and the complementation strains (SC3 and CTC1), however, no appressoria were observed for the inoculation of SK27, CTK15 and D-3. A = appressorium; H = hypha. Scale bar = 10 µm.
Figure 5.
Both Δmosom1 and Δmocdtf1 mutants show an easily wettable phenotype.
(A) Surface hydrophobicity of the wild type strain Guy11 and the mutants SK27 (Δmosom1) and CTK15 (Δmocdtf1) was assessed by placing a 10 µl drop water, 250 µg/ml tween20, 0.2% SDS+50 mM EDTA, and 0.2% gelatin on the 7-day-old cultures, respectively. Drops of water and 0.2% gelatin remained on the cultures of Guy11 and the old mycelium of the mutant CTK15, while the others were soaked into colonies. The photographs were taken after 12 h incubation. (B) Expression of the genes coding hydrophobins in the Δmosom1 (SK27) and Δmocdtf1 (CTK15) mutants measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Error bars represent the standard deviation. Asterisks indicate significant difference at P = 0.01.
Figure 6.
Intracellular localization of MoSom1-green fluorescent protein.
(A) MoSom1 was localized to the nucleus. MoSom1 C-terminal green fluorescent protein (GFP) fusion strategy was conducted to generate GFP expression transformants. One of the transformants, SC3, was used for MoSom1 localization assay. Conidia and mycelia of SC3 were stained by DAPI (4′-6-Diamidino-2-phenylinodle). The merged image of GFP and DAPI staining showed that MoSom1-GFP localized in the nucleus. (B) The patterns of MoSOM1 expression and nuclear division during appressorium development. Conidia of the strain SC3 was allowed to germinate on hydrophobic GelBond film surfaces. Photographs were taken at various time intervals. (C) Expression of MoSOM1 during invasive growth. The assay was performed by placing 30 µl conidial suspension of SC3 on onion epidermis. Photographs were taken at 48 h after incubation. Arrows indicate conidium, appressorium or invasive hypha inside cells. C = conidium; A = appressorium; IH = invasive hypha. BF = bright field. All of bars = 10 µm.
Figure 7.
Functional analysis of putative LisH domain and nuclear localization sequences of MoSom1.
(A) Sequence alignment of the LisH domain between Magnaporthe oryzae MoSom1 and other regulatory proteins. Identical residues are shaded in black and conserved residues are shaded in gray. Abbreviations correspond to species names. The fungal species, proteins and GenBank accession numbers are: Mo, M. oryzae MoSom1_XP_362263; Af, Aspergillus fumigatus putative Som1_XP_746706; An, A. nidulans putative OefA_AAW55626; Gz, Gibberella zeae putative Som1_XP_382826; Nc, Neurospora crassa putative Som1_AAF75278; Ss, Sclerotinia sclerotiorum putative Som1_XP_001598877; Ca, Candida albicans Flo8_AAQ03244; Sc, Saccharomyces cerevisiae Flo8_DAA07769. (B) Position of LisH domain and two predicted nuclear localization sequences (NLSs) in MoSom1 protein. (C) Functional analysis of domains. Like Δmosom1 mutants, deletion of LisH domain of MoSOM1 resulted in pleiotropic defects. The predicted NLS of PSKRVRL but not PKKK was required for MoSom1 localization to the nucleus and its function. The predicted NLS (PPKRKKP) was also crucial for the function and localization of MoCdtf1 protein. +, normal asexual or sexual sporulation; -, not any conidia or perithecia. The LisH domains were predicted at: http://smart.embl-heidelberg.de/; The NLSs were predicted at: http://psort.hgc.jp/form2.html.
Figure 8.
MoSOM1 can complement S. cerevisae flo8 defects in haploid invasive growth and diploid pseudohyphal development.
(A) MoSOM1 could complement S. cerevisiae Δflo8 in invasive growth. The strains were grown on YPD at 30°C for 3 days. WT, Haploid wild-type (MY1384); Δflo8, HLY850; Δflo8+pYES2, HLY850 carrying a pYES2 vector; Δflo8+MoSOM1, HLY850 expressing MoSOM1. (B) MoSOM1 could complement S. cerevisiae Δflo8 in diploid pseudohyphal development. All strains were grown on SLAD at 30°C for 4 days except HLY852 was grown on SLAD with 200 mg/L URA (SIGMA). WT, diploid wild-type (CGx68); Δflo8, HLY852; Δflo8+pYES2, HLY852 carrying a pYES2 vector; Δflo8+MoSOM1, HLY852 expressing MoSOM1.
Figure 9.
MoSom1 interacts with MoStu1, MoCdtf1 and CpkA in M. oryzae.
(A) Strong interaction between MoSom1 and MoStu1 (an APSES protein) and MoCdtf1 in yeast two-hybrid assays. The Leu+ and Trp+ yeast transformants were assayed for growth on SD-Trp-Leu-His-Ade medium at specified concentrations 1×105, 1×104, 1×103, 1×102 and 10 cells each 10 µl droplet. (B) Weak interaction between MoSom1 and CpkA in a yeast two-hybrid assay with the presence of 5 mM cAMP. Transformants were tested for growth on SD-Trp-Leu-His-Ade medium with or without 5 mM cAMP.
Figure 10.
Differential gene expression analysis on transcriptomes of the Δmosom1 mutant and Guy11 strains.
(A) qRT-PCR validated the SAGE results. qRT-PCR was carried out to confirm the SAGE results through random selection of ten genes that were down-regulated in the Δmosom1 mutant (SK27). The value of log2 ratio (Δmosom1/Guy11) is in bracket after gene ID number. The level of gene expression in Guy11 was taken as 1 and the relative expression in SK27 mutant was normalized based on 1. Error bars represent the standard deviation. (B) Numbers of altered genes expressing in Δmosom1 mutant. >2.0 or >1.5, genes whose expression were up-regulated as indicated by expression profiling and the log2 ratio (Δmosom1/Guy11) values were more than 2 or 1.5; <−2 or <−1.5, genes whose expression were down-regulated as indicated by expression profiling and the log2 ratio (Δmosom1/Guy11) values were less than −2 or −1.5.
Table 1.
SAGE analysis for several known pathogenicity-related genes.
Figure 11.
Model of the cAMP/PKA signaling pathway in M. oryzae.
In this model, the rice blast fungus responds to external physical cues from the rice leaf surface that are detected by receptors such as Pth11 and are transmitted via the heterotrimeric G-protein leading to Mac1 adenylate cyclase activation. This activates the cyclic AMP dependent protein kinase A, CpkA, which in turn acts upstream of MoSom1, a transcriptional regulator that acts through a set of transcription factors including MoStu1, MoCdtf1 and others to bring about infection-assocaited development.