Induction of Ca2+-Calmodulin Signaling by Hard-Surface Contact Primes Colletotrichum gloeosporioides Conidia To Germinate and Form Appressoria

ABSTRACT Hard-surface contact primes the conidia of Colletotrichum gloeosporioides to respond to plant surface waxes and a fruit-ripening hormone, ethylene, to germinate and form the appressoria required for infection of the host. Our efforts to elucidate the molecular events in the early phase of the hard-surface contact found that EGTA (5 mM) and U73122 (16 nM), an inhibitor of phospholipase C, inhibited (50%) germination and appressorium formation. Measurements of calmodulin (CaM) transcripts with a CaM cDNA we cloned from C. gloeosporioides showed that CaM was induced by hard-surface contact maximally at 2 h and then declined; ethephon enhanced this induction. The CaM antagonist, compound 48/80, completely inhibited conidial germination and appressorium formation at a concentration of 3 μM, implying that CaM is involved in this process. A putative CaM kinase (CaMK) cDNA of C. gloeosporioides was cloned with transcripts from hard-surface-treated conidia. A selective inhibitor of CaMK, KN93 (20 μM), inhibited (50%) germination and appressorium formation, blocked melanization, and caused the formation of abnormal appressoria. Scytalone, an intermediate in melanin synthesis, reversed the inhibition of melanization but did not restore appressorium formation. The phosphorylation of 18- and 43-kDa proteins induced by hard-surface contact and ethephon was inhibited by the treatment with KN93. These results strongly suggest that hard-surface contact induces Ca2+-calmodulin signaling that primes the conidia to respond to host signals by germination and differentiation into appressoria.

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A Mitogen-Activated Protein Kinase Kinase Required for Induction of Cytokinesis and Appressorium Formation by Host Signals in the Conidia of Colletotrichum gloeosporioides

The Plant Cell ◽

10.2307/3871133 ◽

2000 ◽

Vol 12 (8) ◽

pp. 1331 ◽

Cited By ~ 1

Author(s):

Yeon-Ki Kim ◽

Tomonori Kawano ◽

Daoxin Li ◽

Pappachan E. Kolattukudy

Keyword(s):

Protein Kinase ◽

Colletotrichum Gloeosporioides ◽

Mitogen Activated Protein Kinase ◽

Appressorium Formation ◽

Mitogen Activated Protein ◽

Host Signals ◽

Protein Kinase Kinase

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A Mitogen-Activated Protein Kinase Kinase Required for Induction of Cytokinesis and Appressorium Formation by Host Signals in the Conidia of Colletotrichum gloeosporioides

The Plant Cell ◽

10.1105/tpc.12.8.1331 ◽

2000 ◽

Vol 12 (8) ◽

pp. 1331-1343 ◽

Cited By ~ 46

Author(s):

Yeon-Ki Kim ◽

Tomonori Kawano ◽

Daoxin Li ◽

Pappachan E. Kolattukudy

Keyword(s):

Protein Kinase ◽

Colletotrichum Gloeosporioides ◽

Mitogen Activated Protein Kinase ◽

Appressorium Formation ◽

Mitogen Activated Protein ◽

Host Signals ◽

Protein Kinase Kinase

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Early Expression of the Calmodulin Gene, Which Precedes Appressorium Formation in Magnaporthe grisea, Is Inhibited by Self-Inhibitors and Requires Surface Attachment

Journal of Bacteriology ◽

10.1128/jb.181.11.3571-3577.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3571-3577 ◽

Cited By ~ 55

Author(s):

Zhi-Mei Liu ◽

Pappachan E. Kolattukudy

Keyword(s):

Magnaporthe Grisea ◽

Fluorescent Protein ◽

Early Gene ◽

Plant Surface ◽

Appressorium Formation ◽

Gfp Expression ◽

Surface Attachment ◽

Calmodulin Gene ◽

Surface Waxes ◽

Green Fluorescent

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 andColletotrichum 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 ofM. 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.

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Two Novel Genes Induced by Hard-Surface Contact ofColletotrichum gloeosporioides Conidia

Journal of Bacteriology ◽

10.1128/jb.182.17.4688-4695.2000 ◽

2000 ◽

Vol 182 (17) ◽

pp. 4688-4695 ◽

Cited By ~ 20

Author(s):

Yeon-Ki Kim ◽

Zhi-Mei Liu ◽

Daoxin Li ◽

Pappachan E. Kolattukudy

Keyword(s):

Leucine Zipper ◽

Transmembrane Domain ◽

Early Stage ◽

Coiled Coil ◽

Phytopathogenic Fungi ◽

Heptad Repeat ◽

Hard Surface ◽

Molecular Events ◽

Surface Wax ◽

Surface Contact

ABSTRACT Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen, Colletotrichum gloeosporioides, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode Colletotrichum hard-surface induced proteins are designated chip genes. In this study, we report the characterization of CHIP2 and CHIP3 genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. TheCHIP2 product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The CHIP3 product would be a nine-transmembrane-domain-containing protein. RNA blots showed that CHIP2 and CHIP3 are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressorium-forming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting thatC. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.

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