Figure 1.
The MSB2 and SHO1 orthologs in M. oryzae.
A. qRT-PCR assay of MoMSB2 and MoSHO1 expression in the wild-type, pmk1, and mst12 mutant strains. The relative expression level of MoMSB2 and MoSHO1 in the mutants was compared to that of the wild-type strain (arbitrarily set to 1). Mean and standard error were calculated with data from three biological replicates. B. Schematic drawing of domains identified in MoMsb2 and MoSho1. SP, signal peptide; STR, serine/threonine rich region; HMH, Hkr1-Msb2 homology domain; TM, transmembrane domain; CT, cytoplasmic tail; SH3, and Src homology 3 domain. C. Colonies of XK1-25 (WT) and sho1 mutant transformed with pYES2 (GL1) or pMoSHO1 (GL2). Photos were taken after incubation for two days on YPGal plates with or without 1.5 M sorbitol.
Table 1.
Wild-type and mutant strains of Magnaporthe oryzae used in this study.
Table 2.
Phenotype characterization of the mutants generated in this study.
Figure 2.
Appressorium formation and plant infection assays.
A. Conidia from wild-type strains 70-15 and Ku80, Momsb2 mutant M6, Mosho1 mutant S72, Mosho1 Momsb1 mutant MS88, and an ectopic transformant Ect16 were incubated on hydrophobic surfaces for 24 h. Bar = 20 µm. B. Rice leaves sprayed with conidia from the same set of strains. Typical leaves were photographed 7 dpi.
Figure 3.
Appressorium formation assays with intact rice leaves.
A. Rice leaves inoculated with conidia from strains Ku80, M6 (Momsb2), and MS88 (Mosho1 Momsb2). Melanized appressoria were observed 24 hpi. B. Appressoria formed by the same set of strains on rice leaf surface examined under SEM. The mutants produced appressoria at the tip of long germ tubes. Bar = 10 µm. Arrows marked appressoria.
Figure 4.
Assays for the effects of different treatments on appressorium formation.
Conidia from strains Ku80, M6 (Momsb2), S72 (Mosho1), and MS88 (Mosho1 Momsb2) mutants were incubated on: A) the hydrophilic surface in the presence of 10 µM 1,16-hexadecanediol; B) de-waxed leaves, and C) glass surface coated with the rice leaf wax extract. Representative germlings were photographed after 24 h incubation. Arrows marked appressoria. Bar = 20 µm.
Figure 5.
Appressorium formation induced by different waxes or wax components.
Conidia from strains Ku80 (WT), M6 (Momsb2), Mosho1 (S72), and MS88 (Momsb2 Mosho1) were place over microscope glass slides coated with bee waxes (A), paraffin waxes (B), C28 primary alcohol 1-Octacosanol (C), and C31 alkane hentricacontane (D). Bar = 10 µm.
Table 3.
Appressorium formation on glass slides coated with different waxes.
Figure 6.
Appressorium penetration assays with onion and rice epidermal cells.
A. Onion epidermal cells inoculated with Ku80, M6 (Momsb2), S72 (Mosho1), and MS88 (Momsb2 Mosho1) were examined 48 hpi. B. Epidermal cells of rice leaf sheaths were inoculated with the same set of strains and examined 48 hpi. A, appressorium; C, conidium; GT, germ tube; IH, infectious hypha. Bar = 10 µm.
Figure 7.
Assays for MAP kinase phosphorylation.
Western blots were conducted with proteins isolated from vegetative hyphae of the wild-type (70-15) and the Momsb2 (M6), Mosho1 (S72), and Mosho1 Momsb2 (MS88) mutant strains. A. The anti-MAPK and anti-TpEY antibodies detected the expression and phosphorylation levels of Pmk1 (42-kD) and Mps1 (46-kD), respectively. B. The 41-kD Osm1 band was detected with both an anti-P38 MAPK antibody and the anti-TpGY antibody.
Figure 8.
Effects of expressing the dominant active allele of MST7 on appressorium formation.
Conidia from transformants of the wild-type strain 70-15 (WDA2) and the Momsb2 mutant M6 (WDA12) expressing the MST7DA allele were incubated on hydrophobic (upper) or hydrophilic (lower) surface of Gelbond membranes for 24 h. Bar = 10 µm.
Figure 9.
Expression and subcellular localization of MoMsb2-eGFP.
A. Conidia, germ tubes, and young appressoria of the MoMSB2-eGFP (CM6) and MoMSB2ΔSP-eGFP (DSSM) transformants were examined by DIC or epifluoresence microscopy. B. In mature appressoria (24 h) of transformant CM6, GFP signals localized to small vacuole-like structures (marked with arrows). In transformant DSSM, appressorium formation was not observed and GFP signals localized in the cytoplasm. Bar = 10 µm. The same fields were examined under DIC (left) and epifluoresence microscopy (right).
Figure 10.
Functional characterization of different domains of the MoMSB2 gene.
A. Schematic drawing of different mutant alleles of MoMSB2. The numbers in the bracket indicate the amino acid residues deleted in each allele. B. Appressorium formation assays with transformants D5STR, D3STR, DSTR, DHMH, and DCT that expressed the MoMSB2Δ5STR-, MoMSB2Δ3STR-, MoMSB2ΔSTR-, MoMSB2ΔHMH-, and MoMSB2ΔCT-eGFP alleles. Bar = 10 µm. C. Rice leaves inoculated with conidia from the same set of strains. Like the original Momsb2 mutant, transformants DSTR and DHMH were significantly reduced in appressorium formation on hydrophobic surfaces and virulence.