Subcellular localization of Cap1 and LifeAct in <i>M. oryzae</i>.

<p><b>A</b>. Germ tubes (1 h), developing appressoria (2 h), young appressoria (7 h), and mature appressoria (14 h) of the <i>CAP1</i>-GFP (left, Δ<i>cap1/CAP1</i>-GFP) and LifeAct-GFP transformants were examined by DIC or epifluorescence microscopy. Bar = 5 µm. <b>B</b>. Invasive hyphae produced by the <i>CAP1</i>-GFP (left, Δ<i>cap1/CAP1</i>-GFP) and LifeAct-GFP transformants in rice leaf sheath were examined 24 or 48 hpi. A, appressorium; IH, invasive hyphae. Bar = 10 µm.</p

Infection and penetration assays with the Δ<i>cap1</i> mutant.

<p><b>A</b>. Leaves of two-week-old rice seedlings were sprayed or injected with conidial suspensions of Ku80, Δ<i>cap1</i> mutant, and complemented strain Δ<i>cap1/CAP1</i>. Inoculation with 0.25% gelatin was used as the negative control. Typical leaves were photographed 7 dpi. <b>B</b>. Penetration assays with rice leaf sheaths. Invasive hyphae formed by Ku80, Δ<i>cap1</i> mutant, and complemented strain Δ<i>cap1/CAP1</i> in plant cells were examined 48 and 72 hpi. A, appressorium; IH, invasive hyphae. Bar = 10 µm.</p

Putative Mac1-interacting genes identified by affinity purification.

<p>Putative Mac1-interacting genes identified by affinity purification.</p

A hypothetical model of the function of Cap1.

<p>In <i>M. oryzae</i>, 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 <i>Δcap1</i> mutant. Reduced intracellular cAMP may somehow affect the proper activation of Pmk1, which can explain the formation of subapical swollen bodies in the <i>Δcap1</i> 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 <i>CAP1</i> 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 Cap1^ΔAB protein may be hyperactive in activating Mac1 via Ras2. Therefore, exogenous cAMP may overstimulate Ras signaling in the <i>CAP1</i>^ΔAB-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.</p

Phenotypes of the <i>cap1</i> mutant in growth, conidiation, and plant infection.

a<p>Growth rate and conidiation were measured on complete medium (CM). Mean and standard deviation were calculated with results from three replicates.</p>b<p>Appressorium formation is expressed as the percentage of germ tubes producing appressorium.</p>c<p>Lesion formation was examined on infected rice leaves 7 days post-inoculation.</p><p>Means and SD values were calculated from at least three independent experiments.</p>d<p>Appressorium penetration was assayed on onion epidermis at 48 h after inoculation.</p>*<p>The same letter indicated there was no significant difference. Different letters were used to mark statistically significant differences (P = 0.05).</p

Cytochalasin A (CytA) treatment disrupted normal subcellular localization of Cap1 in vegetative hyphae (A) and during appressorium formation (B).

<p><b>A.</b> Hyphae of the <i>CAP1</i>-GFP transformant treated with (<b>c</b> and <b>d</b>) or without (<b>a</b> and <b>b</b>) CytA were examined by DIC and fluorescence microscopy. Panels <b>a</b> and <b>c</b> were GFP images. Panels <b>b</b> and <b>d</b> were composites of GFP and DIC images. <b>B.</b> Conidia harvested from the <i>CAP1</i>-RFP transformant were incubated on hydrophobic surfaces for 16 h and examined by DIC and fluorescence microscopy. Panels <b>a</b> and <b>b</b> were non-treatment controls. Panels <b>c</b> and <b>d</b> were samples treated with CytA for 15 min. before examination. Panels <b>b</b> and <b>d</b> were composites of GFP and DIC images. Bar = 5 µm.</p

Melanized conidium compartments were appressorium-like structures.

<p><b>A.</b> Conidia of a transformant of the Δ<i>cap1</i>/<i>CAP1</i>^ΔAB mutant expressing the <i>GAS2</i>-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>B.</b> DAPI staining of melanized conidium compartments formed by the Δ<i>cap1</i>/<i>CAP1</i>^ΔAB transformant. <b>C.</b> Conidia from the Ku80, Δ<i>cap1</i>/<i>CAP1</i>^ΔAB, and Δ<i>pmk1</i>/<i>CAP1</i>^ΔAB strains HC1-39 were assayed for appressorium formation in the presence of 5 mM cAMP on hydrophilic surfaces. Bar = 10 µm.</p

BiFC assays for the Cap1-Mac1 interaction.

<p>Vegetative hyphae, conidia, and appressoria of transformant CMB14 expressing the <i>CAP1</i>-NYFP and <i>MAC1</i>^CT-CYFP constructs were examined by DIC and epifluorescence microscopy. Bar = 10 µm.</p

Appressorium formation assays on hydrophobic surfaces.

<p><b>A.</b> Appressoria formed by Ku80, Δ<i>cap1</i> mutant HC83, and Δ<i>cap1/CAP1</i> transformant CH07 on the hydrophobic surface of GelBond membranes at 24 h. <b>B.</b> Appressoria formed by hyphal tips on hydrophobic surfaces after incubation for 48 h. Bar = 10 µm.</p

Functions of different domains in the localization of Cap1.

<p>Appressoria (16 h, <b>A</b>) and vegetative hyphae (<b>B</b>) of transformants of the Δ<i>cap1</i> mutant HC83 expressing the <i>CAP1</i>^ΔAB-, <i>CAP1</i>^ΔP2-, <i>CAP1</i>^ΔAC-, or <i>CAP1</i>^ΔP1-GFP construct were examined under DIC or epifluorescence microscopy. Bar = 10 µm.</p