Table 1.
Wild-type and mutant strains of Magnaporthe oryzae used in this study.
Table 2.
Putative Mac1-interacting genes identified by affinity purification.
Figure 1.
Assays for the interaction of CAP1 with MAC1 and its function in yeast.
A. The domain structure of M. oryzae Cap1, S. cerevisiae Srv2, and human Cap1(hCap1). ACB, AC-binding domain; P1 and P2, proline-rich regions; AB; actin-binding domain. B. Yeast transformants expressing the CAP1 prey and MAC1CT bait constructs were assayed for growth on SD-Trp-Leu and SD-His plates and ß-galactosidase activities (LacZ). The Mst11-Mst50 and Pmk1-Mst50 interactions were the positive and negative controls. C. Co-IP assays. Western blots of total proteins and proteins eluted from anti-FLAG M2 beads from transformant CMT (CAP1-GFP and MAC1CT-3×FLAG) and transformant MCF (MAC1CT-3×FLAG) were detected with anti-FLAG or anti-GFP antibodies. D. Yeast cells (103 to 106 cells/ml) of BY4741, Δsrv2 mutant, and Δsrv2 -CAP1 or Δsrv2 -pYES2 transformants were assayed for growth on YPGal (galactose) plates with or without 5 mM H2O2 or 1 M NaCl.
Figure 2.
BiFC assays for the Cap1-Mac1 interaction.
Vegetative hyphae, conidia, and appressoria of transformant CMB14 expressing the CAP1-NYFP and MAC1CT-CYFP constructs were examined by DIC and epifluorescence microscopy. Bar = 10 µm.
Table 3.
Phenotypes of the cap1 mutant in growth, conidiation, and plant infection.
Figure 3.
Appressorium formation assays on hydrophobic surfaces.
A. Appressoria formed by Ku80, Δcap1 mutant HC83, and Δcap1/CAP1 transformant CH07 on the hydrophobic surface of GelBond membranes at 24 h. B. Appressoria formed by hyphal tips on hydrophobic surfaces after incubation for 48 h. Bar = 10 µm.
Figure 4.
Deletion of CAP1 suppressed the formation of appressoria on hydrophilic surfaces in the RAS2DA transformant.
Conidia of the RAS2DA and the cap1 RAS2DA transformants were incubated on the hydrophilic surface of GelBond membranes. After incubation for 24 h, melanized appressoria were formed by the RAS2DA transformant but not the cap1 RAS2DA transformant.
Figure 5.
CAP1 functions in the cAMP signaling pathway.
A. The intracellular cAMP level in Guy11, Δmac1 mutant, Ku80, Δcap1 mutant HC83, and Δcap1/CAP1 transformant CH07. Mean and standard deviation were calculated with results from three independent biological replicates. B. Appressorium formation assays with Ku80, HC83, and CH07 in the presence of 5 mM cAMP on the hydrophobic surface of GelBond membranes. C. Appressorium formation assays on the hydrophilic surface of GelBond membranes in the absence (upper panel) or presence (lower panel) of 5 mM cAMP. Bar = 10 µm.
Figure 6.
Infection and penetration assays with the Δcap1 mutant.
A. Leaves of two-week-old rice seedlings were sprayed or injected with conidial suspensions of Ku80, Δcap1 mutant, and complemented strain Δcap1/CAP1. Inoculation with 0.25% gelatin was used as the negative control. Typical leaves were photographed 7 dpi. B. Penetration assays with rice leaf sheaths. Invasive hyphae formed by Ku80, Δcap1 mutant, and complemented strain Δcap1/CAP1 in plant cells were examined 48 and 72 hpi. A, appressorium; IH, invasive hyphae. Bar = 10 µm.
Figure 7.
Subcellular localization of Cap1 and LifeAct in M. oryzae.
A. Germ tubes (1 h), developing appressoria (2 h), young appressoria (7 h), and mature appressoria (14 h) of the CAP1-GFP (left, Δcap1/CAP1-GFP) and LifeAct-GFP transformants were examined by DIC or epifluorescence microscopy. Bar = 5 µm. B. Invasive hyphae produced by the CAP1-GFP (left, Δcap1/CAP1-GFP) and LifeAct-GFP transformants in rice leaf sheath were examined 24 or 48 hpi. A, appressorium; IH, invasive hyphae. Bar = 10 µm.
Figure 8.
Cytochalasin A (CytA) treatment disrupted normal subcellular localization of Cap1 in vegetative hyphae (A) and during appressorium formation (B).
A. Hyphae of the CAP1-GFP transformant treated with (c and d) or without (a and b) CytA were examined by DIC and fluorescence microscopy. Panels a and c were GFP images. Panels b and d were composites of GFP and DIC images. B. Conidia harvested from the CAP1-RFP transformant were incubated on hydrophobic surfaces for 16 h and examined by DIC and fluorescence microscopy. Panels a and b were non-treatment controls. Panels c and d were samples treated with CytA for 15 min. before examination. Panels b and d were composites of GFP and DIC images. Bar = 5 µm.
Figure 9.
Assays for co-localization of LifeAct and Cap1.
Vegetative hyphae, conidia, and appressoria (16 h) of transformant MC20 expressing the LifeAct-GFP and CAP1-mCherry constructs were examined under DIC and epifluorescence microscopy with GFP and mCherry-specific filters. The bottom panel was generated by merging the GFP and mCherry images.
Figure 10.
Functional analysis of the AB and ACB domains of CAP1.
A. Growth and colony morphology of wild type Ku80, Δcap1/CAP1ΔAB transformant, Δcap1/CAP1ΔACB transformant, and Δcap1 mutant HC83. B. Rice leaves sprayed with conidia from Ku80, Δcap1/CAP1ΔAB transformant, and Δcap1/CAP1ΔACB transformant.
Figure 11.
Functions of different domains in the localization of Cap1.
Appressoria (16 h, A) and vegetative hyphae (B) of transformants of the Δcap1 mutant HC83 expressing the CAP1ΔAB-, CAP1ΔP2-, CAP1ΔAC-, or CAP1ΔP1-GFP construct were examined under DIC or epifluorescence microscopy. Bar = 10 µm.
Figure 12.
Functional analysis of P2 domain of CAP1.
A. Appressorium formation assays with Ku80 and transformant XY244 (Δcap1/CAP1ΔP2) on hydrophobic surfaces by 24 h. B. Rice leaves sprayed with conidia of Ku80 and XY244. Transformant XY244 was significantly reduced in virulence.
Figure 13.
Appressorium formation assay with the CAP1ΔAB transformant.
Conidia from Ku80 and the CAP1ΔAB transformant were incubated for 24 h on the hydrophobic (A) or hydrophilic (B) surface of GelBond membranes. C. The presence of 5 mM cAMP stimulated appressorium formation in the CAP1ΔAB transformant on hydrophobic and hydrophilic surfaces. Bar = 10 µm.
Figure 14.
Melanized conidium compartments were appressorium-like structures.
A. Conidia of a transformant of the Δcap1/CAP1ΔAB mutant expressing the GAS2-GFP fusion construct were incubated on the hydrophilic surface of GelBond membranes in the presence of 5 mM cAMP. GFP signals were observed in the melanized conidium compartments and appressoria at 24 h. B. DAPI staining of melanized conidium compartments formed by the Δcap1/CAP1ΔAB transformant. C. Conidia from the Ku80, Δcap1/CAP1ΔAB, and Δpmk1/CAP1ΔAB strains HC1-39 were assayed for appressorium formation in the presence of 5 mM cAMP on hydrophilic surfaces. Bar = 10 µm.
Figure 15.
A hypothetical model of the function of Cap1.
In M. oryzae, cAMP signaling is the secondary messenger for surface sensing to initiate appressorium formation. Although their exact relationship is not clear, surface sensing signals must be transduced from the cAMP signaling to the Pmk1 MAP kinase pathway, which regulates appressorium development and plant penetration. Ras2 functions upstream both Pmk1 and cAMP signaling. Cap1 directly interacts with Mac1 and plays a role in the activation of Mac1, which may function downstream of Ras or MagB. In cells lacking Cap1, Mac1 cannot be fully activated, which leads to the reduced intracellular cAMP level and reduced appressorium formation. Exogenous cAMP partially suppresses the phenotype of Δcap1 mutant. Reduced intracellular cAMP may somehow affect the proper activation of Pmk1, which can explain the formation of subapical swollen bodies in the Δcap1 mutant. Cap1 may also interact with actin and is involved in cytoskeleton reorganization during appressorium morphogenesis. In addition, it is likely that Cap1 is involved in the feedback inhibition of Mac1 and Ras2 signaling by the activated Pmk1. Deletion of CAP1 may affect this process and result in the formation of branched germ tubes. The AB domain may play a critical role in the feedback inhibition by directly interact with Pmk1 or by being phosphorylated by Pmk1. Meanwhile, the Cap1ΔAB protein may be hyperactive in activating Mac1 via Ras2. Therefore, exogenous cAMP may overstimulate Ras signaling in the CAP1ΔAB-GFP transformant and result in the inappropriate activation of the Pmk1 pathway, which may be responsible for the formation of melanized conidium compartments and appressoria in the absence of visible germ tubes.