Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by self-inhibitors and requires surface attachment

Abstract

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in Colletotrichum gloeosporioides and Colletotrichum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.

FIG. 1

Time course of development of GFP fluorescence by conidia at a low density. Conidia from the cam(p)::EGFP::Hph transformant were placed on a polystyrene surface at a low density (10⁴/ml) for a 4-h period. Then the GFP fluorescence images were collected by confocal microscopy as described in Materials and Methods. The GFP fluorescence of six conidia observed at 2 h in the presence of 0.3 mM cycloheximide (CH) is shown at the right.

FIG. 2

Levels of GFP fluorescence of M. grisea conidia affected by conidial density, conidial surface lipids as self-inhibitors, and plant surface wax. Conidia were placed on a polystyrene surface for 2 h at a high density (10⁵/ml) (A), at a high density (10⁵/ml) with plant surface wax (0.2 μg/μl) (B), without wax (C), at a low density (10⁴/ml) (D), at a low density (10⁴/ml) with conidial surface lipids (0.2 μg/μl) (E), and at a low density (10⁴/ml) with conidial surface lipids (0.2 μg/μl) plus plant surface wax (0.3 μg/μl) (F). The fluorescence images were collected by confocal microscopy as described in Materials and Methods.

FIG. 3

Levels of GFP fluorescence of M. grisea conidia (left) and percentages of appressorium formation (right) at a low conidial density and at a high density with or without plant surface wax. Conidia were subjected to hard-surface treatment for 2 h at a low density (10⁴/ml) (A), at a high density (10⁵/ml) (B) and at a high density (10⁵/ml) with cabbage leaf surface wax (0.2 μg/μl) (C) and to no treatment (D).

FIG. 4

Northern blot showing reversal of inhibition of cam gene expression caused by a high conidial density and plant surface wax. The number of hours on a polystyrene surface without (lanes with H prefix) or with (lanes with W prefix) cabbage leaf surface wax (0.25 μg/μl) are indicated (conidial density, ∼10⁵/ml). Lane 0 contains nontreated conidia as a control. Total RNA (10 μg/lane) was loaded, and the ethidium bromide staining of 28S and 18S rRNAs was the same for all lanes. The probe was a ³²P-labeled, 450-bp cDNA. Experimental details are noted in the text.

FIG. 5

(Left) Inhibition of development of GFP fluorescence in M. grisea conidia at a low conidial density by conidial surface lipids and reversal of this inhibition by plant surface wax. (Right) Percentages of appressorium formation under the same conditions as in the left panel. Shown are results with a low conidial density (10⁴/ml) (A), a low conidial density (10⁴/ml) with conidial surface lipids (0.2 μg/μl) (B), a low conidial density (10⁴/ml) with conidial surface lipids (0.2 μg/μl) plus plant surface wax (0.3 μg/μl) (C), and untreated conidia (D).

FIG. 6

(A) Effects of ConA on conidial attachment (⧫), fluorescence increase (●), and appressorium formation (□). (B) Correlation between fluorescence increase and percentages of appressorium formation. Conidia at 10⁵/ml were placed on a polystyrene surface in 100 μl of phosphate buffer with plant surface wax (0.25 μg/μl) in the presence of different concentrations of ConA. Two sets of duplicate samples were prepared for each concentration of ConA. Two samples were left overnight for observation of appressorium formation. After 2 h, the unattached conidia of the other two samples were removed by pipetting and the volume was adjusted to 200 μl. Conidial density was determined under a microscope with a hemacytometer. The removed conidia in 100 μl were recovered by centrifugation, resuspended in 10 μl of fixer, and placed on a polystyrene surface for confocal microscopic analysis along with the attached conidia. Values are averages of results from three experiments.