Table 1.
Wild-type and mutant strains of Magnaporthe oryzae used in this study.
Figure 1.
MoSfl1 co-immunoprecipitates with Pmk1.
Western blots of total proteins (Total) and proteins eluted from the anti-FLAG M2 beads (Elution) of transformant GSF7 that expressed the MoSFL1-3×FLAG construct were detected with the anti-FLAG and anti-MAPK antibodies. Total proteins isolated from the wild-type strain (70-15) and detection with the anti-actin antibody was included as the control.
Figure 2.
Structural elements of MoSfl1 and complementation assays with the yeast sfl1 mutant.
A. The MoSfl1 protein contains a heat shock factor (HSF) domain (black box), a MAPK docking site (triangle), three putative MAP kinase phosphorylation sites (T231, S474, and T508), and two putative PKA phosphorylation sites (S211 and S554). The amino acid sequence of the MAPK docking site is conserved among Sfl1 and its orthologs from Magnaporthe oryzae, Aspergillus flavus, and Candida albicans. B. Expression of MoSFL1 suppressed the flocculation defect of the yeast sfl1 mutant. Cultures of Saccharomyces cerevisiae wild type strain BY4741 (SFL1) and transformants of Δsfl1 carrying pYES2 or pYES2-MoSFL1 were shaken at 30°C for 16 h and kept still for 15 min before being photographed.
Figure 3.
The MoSFL1 gene replacement vector and mutants.
A. The MoSFL1 genomic region and gene replacement construct. The upstream and downstream flanking sequences were amplified with primers 1F/2R and 3F/4R, respectively, and connected with the hph cassette by double joint PCR. B, BamHI. B. Southern blot of BamHI-digested DNA of Ku80, Mosfl1mutant GK102, and ectopic transformant E115 was hybridized with the downstream sequence of MoSFL1 amplified with primers 3F and 4R as the probe. Because of the BamHI site located in the middle of the MoSFL1 gene, the Mosfl1 deletion mutant had a 12.8-kb band instead of the wild-type 2.7-kb band.
Table 2.
Phenotypes of the Mosfl1 mutant in growth, development, and differentiation.
Figure 4.
Infection assays with the Mosfl1 mutant.
A. Seedlings of rice cultivar CO-39 were sprayed with conidia of Ku80, Mosfl1 mutant GK102, and complemented transformant C49. Typical leaves were photographed 7 days post inoculation (dpi). B. Detached barley leaves were drop inoculated with conidia from the same set of strains. The concentrations of conidium suspensions (conidia/ml) were marked on the left. Inoculation with 0.25% gelatin was the negative control.
Figure 5.
Penetration assays with the Mosfl1 mutant.
Rice leaf sheaths inoculated with conidia from Ku80 and Mosfl1 mutant GK102 were examined 48 hpi. The mutant was restricted in invasive growth compared with the wild type. A, appressorium; IH, invasive hyphae. Bar = 10 µm.
Figure 6.
Increased heat sensitivity in the Mosfl1 and pmk1 mutants.
A. Five-day-old CM cultures of Ku80, Mosfl1 mutant GK102, and complemented transformant C49. B. Seven-day-old CM cultures of the wild type Guy11, pmk1 mutant nn78, and cpkA mutant I-27. All of the cultures were incubated at 25°C (top) or 30°C (bottom). While the growth rates of Ku80 and Guy11 were not affected, the production of aerial hyphae was reduced in the Mosfl1 and pmk1 mutants when cultured at 30°C.
Figure 7.
qRT-PCR assay of MoHSP30, MoHSP98, BUF1, COS1, CON2,CON7, and HTF1 expression in the Ku80 and Mosfl1 mutant strains.
The relative expression levels of MoHSP30, MoHSP98, BUF1, COS1, CON2, CON7, and HTF1 were compared between the GK102 and Ku80 strains (arbitrarily set to 1) cultured at 30°C. Mean and standard error were calculated with data from three biological replicates.
Figure 8.
Expression of MoSFL1 in different developmental stages.
RNA samples used for qRT-PCR assays were isolated from conidia (CO), appressoria (AP), vegetative hyphae (VH), and infected plants (IP). The relative expression level of MoSFL1 was compared to that of the VH stage (arbitrarily set to 1). Mean and standard error were calculated with data from three biological replicates.