- Email: [email protected]
Regulation of cAMP and cAMP dependent protein kinase during conidial germination and appressorium formation inColletotrichum trifolii
Physiological and Molecular Plant Pathology (1997) 50, 117–127
Regulation of cAMP and cAMP dependent protein kinase during conidial germination and appressorium formation in Colletotrichum trifolii Z. Y and M. B. D Department of Plant Pathology, Uniersity of Nebraska, Lincoln, NE 68583, U.S.A. (Accepted for publication December 1996)
Colletotrichum trifolii is the causal agent of alfalfa anthracnose. To investigate the involvement of cAMP}cAMP-dependent protein kinase pathway during conidial germination and appressorial differentiation, enzymatic levels of cAMP-dependent protein kinase were measured from conidia, germinating conidia, appressoria and mycelia. Highest levels of activity were found in germinating conidia, intermediate levels in conidia, and only basal levels of activity in appressoria and mycelia. Using a specific inhibitor of cAMP-dependent protein kinase, KT5720, both conidial germination and appressorial differentiation were impaired when conidia and germinating conidia were treated, respectively. Endogenous cAMP levels were also measured. Highest cAMP levels were found in conidia. Germinating conidia contained 70 % less cAMP and the lowest level of cAMP was found in mycelia. Using a pharmacological approach, including 8-Br-cAMP, sodium fluoride and 3-isobutyl-1-methylxanthine, all of which increase endogenous cAMP levels, we showed induction of appressorial differentiation on a non-inductive surface (1±5 % water agar). These data strongly suggest that cAMP}cAMP-dependent protein kinase pathway plays a key role during morphogenesis of C. trifolii. # 1997 Academic Press Limited
INTRODUCTION
Colletotrichum trifolii Bain & Essary causes anthracnose of alfalfa (Medicago satia L.). As with many fungal pathogens, the pathogenicity of C. trifolii depends on cellular morphogenesis, initiating with conidial attachment to host surfaces, the induction of conidial germination, followed by germ tube elongation, appressorial differentiation, and subsequent cuticular penetration [5 ]. This genetically programmed prepenetration process is essential for successful colonization [3 ], since interference with these events either via mutation [18 ] or chemical inhibitors [22 ] prevents disease development. Although this developmental sequence is both essential for infection and common to many fungal pathogens, the mechanism by which fungi sense the host surface and presumably respond to such environmental cues to trigger infection-related morphogenesis is not well understood. Fungi possess effective sensing mechanisms for recognizing topographical features and other signals that induce germ tube morphogenesis [11, 23 ]. A well studied Abbreviations used in text : PKA, cAMP dependent protein kinase. 0885–5765}97}02011711 $25.00}0}pp960077
# 1997 Academic Press Limited
118
Z. Yang and M. B. Dickman
example of infection structure development occurs in the bean rust fungus, Uromyces appendiculatus Unger. Uredospore germlings of this fungus form appressoria specifically over stomata where polarized growth ceases and differentiation begins [11 ]. As the germ tube grows across the surface of the leaf, it orients its direction of growth at right angles to the surface ridges of the leaf. Germ tubes thus grow in straight lines rather than randomly, maximizing the probability of finding a stomate. The surface feature which triggers differentiation is stomatal architecture. In an elegant series of experiments to demonstrate this, Wynn [23 ] used plaster replicas of the leaf. Subsequently Hoch and colleagues defined leaf conditions more precisely by producing silicon wafer templates by electron beam lithography and reactive ion etching. They showed that a precise window of ridge height was required to induce differentiation, which, not coincidentally, is similar to the height of a stomatal ridge [11 ]. Ridges outside this range did not induce differentiation. While appressorial development is required for fungal penetration and subsequent infection, conidial germination is required for appressorial formation. Thus, infection depends on conidial germination. While numerous studies have focused on sporulation and to a lesser extent appressorial formation, far less attention has been given to the germination process which initiates development [5 ]. Moreover, although a few genes expressed during the early stages of the infection process have been identified, their role in development and pathogenesis remains unclear [5, 6 ]. We have been studying the signalling process during prepenetration development in C. trifolii to elucidate the biochemical pathways responsible for the morphological switches. Previously we have shown the importance of calcium and calmodulin for conidial germination and appressorial development [22 ], and have characterized a glutamine rich serine–threonine protein kinase (TB3) that is maximally expressed during germination and is possibly involved in hyphal growth [2 ]. Additional potentially important signalling molecules which could mediate prepenetration events are the second messenger cAMP and its partner, cAMP-dependent protein kinase (PKA). cAMP is well characterized as a regulator of development in numerous eukaryotes [20 ]. cAMP is known to influence conidiation in Neurospora crassa and dimorphism in certain other fungi [17 ]. The addition of cAMP to bean rust germlings induced one round of mitosis and septum formation, processes normally associated with appressorial development [10 ]. Of particular significance, the recent work of Lee and Dean [12 ] confirmed the importance of the cAMP–PKA pathway in fungal development. cAMP has been shown to be a requisite for appressorial formation in Magnaporthe grisea (Krause and Webster). Subsequently, the gene encoding the M. grisea PKA catalytic subunit was cloned. Inactivation of PKA by gene replacement prevented appressorial formation [15 ]. In the corn smut pathogen Ustilago maydis Corda, cAMP and PKA appear to have key roles in regulating the dimorphic switch between budding and filamentous growth [8 ]. Thus, there is precedent for involvement of the cAMP–PKA pathway in phytopathogenic fungal developmental switching and development, although the molecular details are still sketchy. In this report, the role of cAMP and PKA in C. trifolii morphogenesis is assessed. In this study, we show that PKA enzyme activity as well as cAMP levels are developmentally regulated. In addition, interference with this pathway by specific inhibitors suggests the importance of cAMP and PKA for both germination and
cAMP and cAMP dependent protein kinase in C. trifolii
119
differentiation. Lastly, inappropriate regulation of this pathway by pharmacological chemicals induces abnormal appressorial development. MATERIALS AND METHODS
Strains and culture conditions Colletotrichum trifolii race 1 (ATCC 66954) was used throughout these studies [4 ]. The fungus was stored as mycelia on dry filters at ®20 °C and propagated in YPSS liquid medium [21 ]. Mycelia were collected by filtration after 4–5 days’ growth in liquid medium at room temperature with shaking (125 rpm). To obtain germinated conidia and appressoria, washed conidia were suspended (10' conidia ml−") in water and the suspension poured into glass Petri dishes (5 ml per Petri dish) at ambient temperature. After " 80 % of the conidia germinated (2–3 h), a rubber policeman was used to scrub the surface of the Petri dish to resuspend germinating conidia, and these germinating conidia were collected by centrifugation at 5 000 rpm at 4 °C for 10 min. The pellet was immediately frozen in liquid nitrogen and stored at ®80 °C. Appressoria were collected by inducing germinated conidia to differentiate on a hard surface [22 ] and collected as described above. PKA actiity assay Tissue from different developmental stages (conidia, germinating conidia, appressoria and mycelia) was ground with acid-washed glass beads (70–150 microns, Sigma) in liquid nitrogen with protein extraction buffer (5 m EDTA, 50 m Tris, pH 7±5) containing protease inhibitors aprotinin (25 µg ml−") and PMSF (1 m). After incubating on ice for 20 min, the mixture was centrifuged at 12 000 rpm in a JA-20 rotor (Beckman) at 4 °C for 20 min. The supernatant was collected and used in a PKA activity assay. For all assays, the final protein concentration was 5 µg µl−" as determined by the Bradford method [1 ]. To assay for PKA activity, protein extracts were added to tubes containing 50 m Tris, pH 7±5, 10 m MgCl , 0±25 mg ml−" BSA, # 100 µ [γ®$#P] ATP and 50 µ kemptide (model substrate) and incubated at 30 °C for 5 min. Twenty microlitres from each treatment were spotted onto a phosphocellulose disc, acid-washed three times and rinsed with water three times. Discs were then counted in a scintillation counter. PKA specific protein kinase activation was determined with a pseudosubstrate inhibitor peptide, a 17 amino acid peptide sequence (20, Gibco). The relative PKA activity was calculated as the ratio between the sample and mycelia values in liquid scintillation counting, using mycelia values as the baseline. cAMP assay The fresh weight of conidia, germinated conidia and mycelia was measured after freezing in liquid nitrogen. The frozen tissue was ground into powder with a mortar and pestle and incubated with 6 % trichloroacetic acid (TCA) on ice for 20 min. Approximately 10-fold excess of 6 % TCA was added, based on the volume of the ground tissue. After centrifugation at 4 °C for 10 min, the supernatants were collected and extracted five times with water-saturated ether. After extraction, the samples were lyophilized and stored at ®20 °C. cAMP content was determined according to the
120
Z. Yang and M. B. Dickman
manufacturer’s instructions (Amersham cAMP ["#&l] assay system). Briefly, the assay is based on competition between unlabelled cAMP and a fixed amount of "#&l-labelled cAMP for a limited number of binding sites on a cAMP specific antibody. In the presence of known amounts of antibody and radioactive ligand used, the quantity of labelled ligand bound by antibody is inversely proportional to the concentration of added non-radioactive cAMP. Thus the concentration of cellular (unlabelled) cAMP can be determined from a standard curve. Effect of KT5720 on conidial germination and appressorial differentiation Conidia were collected from liquid culture 1–2 days after sporulation. Conidia were concentrated by centrifugation (5 000 rpm for 10 min at 4 °C). The pellet was washed twice in double distilled H O and resuspended (5¬10&®10' conidia ml−") in double # distilled H O or double distilled H O with KT5720 (Calbiochem), a specific inhibitor # # of PKA. 1 m KT5720 was dissolved in DMSO and stored in the dark (4 °C) by covering with aluminium foil. Germination tests were performed by placing 100 µl of the conidial suspension on glass slides and incubating in the dark. Final concentrations of KT5720 used in these tests were based on Gadbois et al. [7 ]. Percentage germination of conidia was measured by counting under a light microscope. For each test, a minimum of 100 conidia were counted in three replicates. To examine the effect of KT5720 on appressorial formation, conidia were resuspended in YPSS media and placed onto glass slides. After germ tubes were observed under the microscope, the media was washed off and replaced with either water or water containing 10 m of KT5720. Appressoria were counted 2 h after this treatment and analysed as described above. Induction of appressorial deelopment on non-inductie surfaces Conidia were collected and resuspended as previously described. Six hundred microlitres of conidia suspension were spread over 1±5 % water agar plates. After " 80 % of the conidia had germinated, the water was removed and replaced with 600 µl of solutions containing 8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX), sodium fluoride (NaF) or forskolin at concentrations indicated in the text. All of these chemicals were purchased from Sigma (St. Louis, MO, U.S.A.). NaF and 8-Br-cAMP were dissolved in ddH O to a final concentration of 100 m for 8-Br-cAMP and 1 for # NaF. Forskolin and IBMX were dissolved in DMSO at 250 m as stock solutions. The percentage of appressorial differentiation was counted following these treatments using a light microscope. RESULTS
Deelopmental profile of PKA actiity To examine whether PKA is involved in C. trifolii development, enzymatic activity of PKA was determined in fungal tissue from different developmental stages. Activities were measured by phosphorylation of kemptide, a specific model substrate of PKA. As shown in Fig. 1, PKA activity was highest in germinating conidia, intermediate in ungerminated conidia, and low in vegetative mycelia and mature appressoria. KT5720, a specific PKA inhibitor (Ki ¯ 56 n) yielded similar levels of enzyme
cAMP and cAMP dependent protein kinase in C. trifolii
121
inhibition to the commercial inhibitor peptide (Ki ¯ 2±3 n) (Ki for both inhibitors were obtained from CalBiochem catalogue), strongly suggesting this assay specifically reflects PKA enzymatic activity.
Relative PKA activity X-fold over mycelia and appressoria
15
10
5
0
1
2
3
4
F. 1. Relative PKA activity in different developmental stages of C. trifolii. 1 : conidia ; 2 : germinating conidia ; 3 : appressoria ; 4 : mycelia. Results are the average from three independent assays. The values on vertical line are arbitrary units, determined as the ratio of counts (CPM) from conidia, germinating conidia with respect to the counts from mycelia. Mycelia and appressoria samples served as the baseline.
Effect of PKA inhibition in conidial germination and appressorial differentiation Since maximal levels of PKA activity occurred during conidial germination and since PKA has been shown to be important for appressorial differentiation in M. grisea [12, 15 ], we examined the role of PKA during these processes using KT5720. 10 µ KT5720 effectively prevented conidial germination (Table 1). Since KT5720 is unstable in the light (report from manufacturer), following inhibition, the conidia were incubated for a period in the light. Conidial germination resumed to near control levels, indicating that PKA inhibition by this compound is reversible and not lethal (unpublished data). When C. trifolii conidia in water attached to a hard surface and germinated, appressoria rapidly formed (1–2 h). However, when conidia were suspended in rich media such as YPSS, appressoria did not develop after conidial germination [20 and unpublished data]. To assess the role of PKA in appressorial formation, conidia were germinated in YPSS media on glass slides. Following germ tube formation, the media was replaced with water or water containing 10 µ KT5720. As shown in Table 2, as with conidial germination, appressorial differentiation was significantly reduced when PKA was inhibited, although not to the extent observed during conidial germination.
122
Z. Yang and M. B. Dickman
A
B
C
D
F. 2. Effect of KT5720 on C. trifolii conidial differentiation. (A) Conidia in water alone ; (B) conidia treated in water containing 10 µ of KT5720 ; (C) Conidia suspended in YPSS media and replaced with water ; (D) conidia suspended in YPSS media and replaced with water containing 10 µ KT5720. All the experiments were done on glass slides. The pictures were taken from the same experiments as described in Tables 1 and 2 in an identical time span.
T 1 Effect of KT5720 on conidial germination in C. trifolii*
Treatment HO # 1 µ KT5720 10 µ KT5720
Germinated conidia† (%³..) 92±7³3±2 84±6³8±0 2±3³1±5
*The data were based on at least six independent tests. †100 conidia first encountered under the microscope (not germinated and germinated) were counted from each slide with three replicates per test. Germination occurred and readings were taken 2–3 h after conidia were placed onto glass slides.
cAMP leels in C. trifolii during deelopment Endogenous levels of cAMP were measured for C. trifolii in conidia, germinating conidia and mycelia. As shown in Table 3, the highest levels of cAMP were found in conidia, with nearly 70 % reduction of cAMP level in germinating conidia for 2–3 h. Mycelia had negligible levels of cAMP. The lack of correlation between cAMP levels
cAMP and cAMP dependent protein kinase in C. trifolii
123
T 2 Effect of KT5720 on appressorial development in C. trifolii*
Treatment HO # 10 µ KT5720
Percentage of appressoria† (%³..) 78±4³0±1 14±2³3±3
*Conidia of C. trifolii germinated in YPSS medium on glass slides. After germination, YPSS medium was replaced with either water or water containing 10 µ KT5720. †Appressoria were counted 2 h after treatment. 100 germinated conidia (with or without appressoria) first encountered under the microscope were counted from each slide with three replicates per test. The result was based on at least six independent tests.
T 3 cAMP leels of C. trifolii in conidia, germinating conidia and mycelia
Tissue Conidia Germinating conidia Mycelia Appressoria
cAMP concentration* (pmols mg−" fresh weight) 0±37³0±09 0±13³0±05 0±06³0±01 ND
*The data are the averages of 3 independent assays. cAMP content was determined as described in Materials and Methods. ND ¯ not determined.
and PKA enzymatic activity during these stages might reflect breakdown of cAMP during tissue collection. It has been reported that cAMP levels in fungi can change within 2 min during extraction [17 ]. Alternatively, cAMP levels might be high in conidia, and rapidly induce germination via induction of PKA when conditions for germination are appropriate. Effect of IBMX, 8-Br-cAMP, NaF and Forskolin on appressorial differentiation In order to examine the role of the cAMP-PKA pathway in C. trifolii morphogenesis further, we determined the effects of established regulators of cAMP. IBMX is an effective inhibitor of cyclic nucleotide phosphodiesterase, which degrades cAMP to AMP. Forskolin has been commonly used as an activator of adenylate cyclase, which utilizes ATP to form cAMP. NaF is also an activator of adenylate cyclase while 8-BrcAMP is a stable analogue of cAMP. These chemicals are widely used in studying cAMP functions in fungi and many other organisms [10, 12, 17 ] as they increase the endogenous levels of cAMP, either by promoting its synthesis or inhibiting degradation. Previously, we demonstrated that appressorium induction of C. trifolii requires germ tube contact with a hard surface, and that hydrophobic surfaces stimulate higher frequencies of development than hydrophilic surfaces [22 ]. Since our studies indicated
124
Z. Yang and M. B. Dickman
that C. trifolii conidia germinate but do not differentiate into appressoria on relatively soft surfaces such as 1±5 % water agar, the non-inductive surface of 1±5 % water agar was used to determine the effects of these various chemicals on C. trifolii conidial differentiation. As shown in Table 4 and Fig. 3, raising cAMP levels in conidia by treatment with NaF, 8-Br-cAMP or by IBMX resulted in appressorial differentiation on this non-inductive surface, although the frequencies were significantly lower than found on inductive surfaces. Forskolin was ineffective in appressorial induction (unpublished data). When these treatments were performed in 1±5 % YPSS agar, no induction of appressoria could be observed, even at higher concentrations of these chemicals (unpublished data). Thus limiting nutrients are also required in addition to elevated cAMP levels to cause abnormal differentiation. Similar observations have been reported for Colletotrichum lindemuthianum Lams. [14 ]. A
B
F. 3. Induction of C. trifolii appressorial differentiation on 1±5 % water agar by 8-Br-cAMP. (A) Conidia spread on 1±5 % water agar ; (B) Conidia spread on 1±5 % water agar containing 5 m 8-Br-cAMP. This result is from the same experiments as described in Table 3. After evaluation, Petri dishes were stored at 4 °C for 5–6 h prior to photography.
cAMP and cAMP dependent protein kinase in C. trifolii
125
T 4 Effect of 8-Br-cAMP, IBMX, and NaF on C. trifolii conidial differentiation on non-inductie surfaces*
Treatment HO # 5 m 10 m 5 m 10 m 5 m
IBMX IBMX NaF NaF 8-Br-cAMP
Appressoria† (%³..) 1±0³0±8 12±8³5±6 14±2³4±8 10±0³4±7 13±7³4±6 20±1³3±9
*1±5 % water agar was used as non-inductive surface. †The percentage of differentiated appressoria was determined as follows : for each test, 100 germinated conidia (having long germ tubes with or without appressoria) were counted microscopically from each Petri dish. Each treatment was performed as described in Materials and Methods. The data were based on six independent experiments with three replicates per test.
DISCUSSION
Invasion of alfalfa by C. trifolii requires penetration of leaves or stems through a highly coordinated series of morphological events [5 ]. When conidia of C. trifolii land on the host surface, attachment triggers conidial germination. Appressorial differentiation follows germ tube formation and adherence to the host cuticle, but only in the presence of a hard surface and limiting nutrients. The appropriate timing of these switches is essential for host penetration and subsequent colonization. Interference with this process at any point prevents disease development in intact host tissue. This genetically programmed series of morphological changes is likely to be triggered by signalling pathways. To understand the events and key processes surrounding conidial germination and leading to appressorial differentiation, we examined the involvement of the cAMP–PKA pathway. Several lines of evidence support the involvement of this pathway in fungal morphogenesis. For example, the addition of exogenous cAMP or inhibitors of phosphodiesterase can induce synchronous zoospore germination in Blastocladiella emersonii [9 ]. In addition, PKA was shown to be developmentally regulated [13 ]. PKA activity was low during vegetative growth, increased during sporulation and was maximum in spores. During germination, total PKA activity decreased. Biochemical evidence for the role of cAMP in morphogenesis has been described for several fungi [17 ]. In N. crassa, mutants defective in cAMP conidiated poorly [17 ]. In Mucor rouxii, cAMP levels drop during emergence of hyphal germ tubes and remain low during hyphal growth [16, 19 ]. In contrast, in certain dimorphic fungi, intracellular cAMP levels increase during germ tube formation and hyphal growth [see 8 ]. In U. maydis, a dimorphic basidiomycete, low cAMP levels promote hyphal extension in preference to budding growth. Defects in adenylate cyclase or mutations in the regulatory subunit of PKA, which prevent PKA activation, lead to filamentous growth of U. maydis [8 ]. Similarly, a mutation in the cot-1 gene in N. crassa results in extensive mycelial branching and reduced hyphal extension [25 ]. The COT-1 sequence resembles the catalytic domain of PKA, although direct induction of this gene
126
Z. Yang and M. B. Dickman
by cAMP has not yet been demonstrated [25 ]. Dean’s group has shown that cAMP regulates appressorial development in M. grisea and that disruption of the PKA catalytic subunit gene prevented the formation of appressoria [12, 15 ]. Thus there is convincing evidence that cAMP and PKA can regulate morphogenesis in fungi. We have shown that the cAMP-PKA pathway is likely to be operative during the prepenetration developmental sequences of C. trifolii. cAMP levels were determined in three distinct fungal developmental stages. Levels were highest in conidia and reduced as germ tubes formed and mycelial growth occurred. These patterns are similar to those found in M. rouxii and U. maydis. As cAMP has basically one known biochemical activity, that is binding to PKA [20 ], we examined PKA activity during these stages. Relative activity was highest in germinating conidia and lowest in mycelia and appressoria. The lack of a direct correlation between cAMP levels and PKA enzyme activity might reflect the fact that cAMP triggers PKA activity via binding to the regulatory subunit of PKA. cAMP binding to the regulatory subunit results in its release from the catalytic subunit and this release results in PKA induction. PKA activity would therefore be highest during germination. We also used a pharmacological approach to address the functional relevance of the cAMP-PKA pathway. KT5720, a highly specific inhibitor of PKA, almost completely prevented conidial germination at 10 µ. When conidia were germinated on an inductive surface and then treated with KT5720, appressorial differentiation was also effectively impaired. On hard surfaces (e.g. glass slides) which induce germination and differentiation, raising endogenous cAMP levels either by increasing synthesis with adenylate cyclase activators or by decreasing degradation with inhibitors of phosphodiesterase, we found increases in both the rate of conidial germination and the level of appressorial differentiation (unpublished). On a non-inductive surface (1±5 % water agar), where appressoria are rarely if ever observed, the addition of 8-Br-cAMP, IBMX and NaF all induced appressoria differentiation. Similar results have been reported in the rice blast pathogen M. grisea [12 ]. When water agar is replaced by nutrient media, appressoria do not differentiate, even in the presence of the aforementioned effectors. Thus, starvation or nutrient limitation is also necessary for differentiation ; increasing cAMP levels alone is not sufficient for differentiation. Forskolin, an activator of adenylate cyclase had no effect on C. trifolii as has been reported for the ascomycete M. grisea, but differing from the reports with the basidiomycete, U. phaseoli, possibly reflecting taxonomic differences [10, 12 ]. Alternatively, forskolin may be poorly taken up or inactivated in C. trifolii (and M. grisea). While the evidence suggests the importance of the cAMP-PKA pathway in these prepenetration events, these data are limited. For example, PKA inhibitors prevent appressorial differentiation, but PKA enzyme levels in mature appressoria were low. This may reflect the unreliability of this biochemical assay. Also, C. trifolii spores do not germinate synchronously, and thus appressoria were only collected after maturity at which point PKA may be unnecessary. Alternatively, since germination and appressorial differentiation are tightly coupled, once triggered under inductive conditions, PKA levels induced during germination may be sufficient to complete the process. To address this problem more directly, we have isolated the regulatory and catalytic subunit genes of PKA in C. trifolii (unpublished data). Characterization and functional analyses of these genes are in progress.
cAMP and cAMP dependent protein kinase in C. trifolii
127
This work was supported by the United States–Israel Binational Agricultural Research and development Fund (BARD) and by the DOE}NSF}USDA Program on Collaborative Research in Plant Biology grant 92-37310-7821. REFERENCES 1. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 : 249–254. 2. Buhr TL, Oved S, Truesdell GM, Huang C, Yarden O, Dickman MB. 1996. A kinase-encoding gene from Colletotrichum trifolii complements a colonial growth mutant of Neurospora crassa. Molecular and General Genetics 251 : 565–572. 3. Churchill A, Baker C, O’Neill N, Elgin J. 1988. Development of Colletotrichum trifolii races 1 and 2 on alfalfa clones resistant and susceptible to anthracnose. Canadian Journal of Botany 66 : 75–81. 4. Dickman M. 1988. Whole cell transformation of the fungal pathogen Colletotrichum trifolii. Current Genetics 14 : 241–246. 5. Dickman MB, Buhr TL, Warwar V, Truesdell GM, Huang CX. 1995. Molecular signals during early stages of alfalfa anthracnose. Canadian Journal of Botany 73 : 1169–1177. 6. Dickman MB. 1995. The molecular biology of fungal-plant interactions. In : Leslie JF, Fredrisksen RA, eds. Disease Analysis through Genetics and Biotechnology. Ames, Iowa : Iowa State University Press, 149–195. 7. Gadbois D, Grissman H, Tobey R, Bradbury M. 1992. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proceedings of National Academy of Sciences of the United States 89 : 8626–8630. 8. Gold S, Duncan G, Barret K, Kronstad J. 1994. cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes and Deelopment 8 : 2805–2816. 9. Gomes SL, Mennucci L, Maia JCC. 1980. Induction of Blastocladiella emersonii germination by 3«,5«monophosphate. Cell Differentiation 9 : 169–179. 10. Hoch HC, Staples RC. 1984. Evidence that cAMP initiates nuclear division and infection structure formation in the bean rust fungus, Uromyces phaseoli. Experimental Mycology 8 : 37–46. 11. Hoch HC, Staples RC, Whitehead B, Comeau J, Wolf ED. 1987. Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235 : 1659–1662. 12. Lee YH, Dean R. 1993. cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea. The Plant Cell 5 : 693–700. 13. Marques M, Juliani M, Maia J, Gomes S. 1989. Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii. European Journal of Biochemistry 178 : 803–810. 14. Mercer PC, Wood RKS, Greenwood AD. 1971. Initial infection of Phaseolus ulgaris by Colletotrichum lindemuthianum. In : Preece TF, Dickinson CH, eds. Ecology of Leaf Surface Micro-organisms. London : Academic Press, 381–389. 15. Mitchell T, Dean R. 1995. The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. The Plant Cell 7 : 1869–1878. 16. Orlowski M. 1991. Mucor dimorphism. Microbiology Reiew 55 : 234–258. 17. Pall ML. 1981. Adenosine 3,5-phosphate in fungi. Microbiology Reiew 45 : 462–480. 18. Perpetua NS, Kubo Y, Okuno T, Furusawa I. 1994. Restoration of pathogenicity of a penetrationdeficient mutant of Colletotrichum lagenarium by DNA complementation. Current Genetics 25 : 41–46. 19. Pewerchin MA, Van Laere AJ. 1984. Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores. Journal of Bacteriology 158 : 575–579. 20. Taylor SS, Buechler JA, Yonemoto W. 1990. cAMP-dependent protein kinase : framework for a diverse family of regulatory enzymes. Annual Reiew of Biochemistry 59 : 971–1005. 21. Tuit J. 1968. Plant Pathological Methods. Minneapolis : Burgess Publishing. 22. Warwar V, Dickman MB. 1996. Effects of calcium and calmodulin on spore germination and appressorium development in Colletotrichum trifolii. Applied and Enironmental Microbiology 62 : 74–79. 23. Wynn WK. 1976. Appressorium formation over stomates by the bean rust fungus : response to a surface contact stimulus. Phytopathology 66 : 136–146. 24. Wynn WK. 1981. Tropic and taxic responses of pathogens to plants. Annual Reiew of Phytopathology 19 : 237–255. 25. Yarden O, Plamann M, Ebbole DJ, Yanofsky C. 1992. Cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. The EMBO Journal 11 : 2159–2166.
Regulation of cAMP and cAMP dependent protein kinase during conidial germination and appressorium formation in Colletotrichum trifolii Z. Y and M. B. D Department of Plant Pathology, Uniersity of Nebraska, Lincoln, NE 68583, U.S.A. (Accepted for publication December 1996)
Colletotrichum trifolii is the causal agent of alfalfa anthracnose. To investigate the involvement of cAMP}cAMP-dependent protein kinase pathway during conidial germination and appressorial differentiation, enzymatic levels of cAMP-dependent protein kinase were measured from conidia, germinating conidia, appressoria and mycelia. Highest levels of activity were found in germinating conidia, intermediate levels in conidia, and only basal levels of activity in appressoria and mycelia. Using a specific inhibitor of cAMP-dependent protein kinase, KT5720, both conidial germination and appressorial differentiation were impaired when conidia and germinating conidia were treated, respectively. Endogenous cAMP levels were also measured. Highest cAMP levels were found in conidia. Germinating conidia contained 70 % less cAMP and the lowest level of cAMP was found in mycelia. Using a pharmacological approach, including 8-Br-cAMP, sodium fluoride and 3-isobutyl-1-methylxanthine, all of which increase endogenous cAMP levels, we showed induction of appressorial differentiation on a non-inductive surface (1±5 % water agar). These data strongly suggest that cAMP}cAMP-dependent protein kinase pathway plays a key role during morphogenesis of C. trifolii. # 1997 Academic Press Limited
INTRODUCTION
Colletotrichum trifolii Bain & Essary causes anthracnose of alfalfa (Medicago satia L.). As with many fungal pathogens, the pathogenicity of C. trifolii depends on cellular morphogenesis, initiating with conidial attachment to host surfaces, the induction of conidial germination, followed by germ tube elongation, appressorial differentiation, and subsequent cuticular penetration [5 ]. This genetically programmed prepenetration process is essential for successful colonization [3 ], since interference with these events either via mutation [18 ] or chemical inhibitors [22 ] prevents disease development. Although this developmental sequence is both essential for infection and common to many fungal pathogens, the mechanism by which fungi sense the host surface and presumably respond to such environmental cues to trigger infection-related morphogenesis is not well understood. Fungi possess effective sensing mechanisms for recognizing topographical features and other signals that induce germ tube morphogenesis [11, 23 ]. A well studied Abbreviations used in text : PKA, cAMP dependent protein kinase. 0885–5765}97}02011711 $25.00}0}pp960077
# 1997 Academic Press Limited
118
Z. Yang and M. B. Dickman
example of infection structure development occurs in the bean rust fungus, Uromyces appendiculatus Unger. Uredospore germlings of this fungus form appressoria specifically over stomata where polarized growth ceases and differentiation begins [11 ]. As the germ tube grows across the surface of the leaf, it orients its direction of growth at right angles to the surface ridges of the leaf. Germ tubes thus grow in straight lines rather than randomly, maximizing the probability of finding a stomate. The surface feature which triggers differentiation is stomatal architecture. In an elegant series of experiments to demonstrate this, Wynn [23 ] used plaster replicas of the leaf. Subsequently Hoch and colleagues defined leaf conditions more precisely by producing silicon wafer templates by electron beam lithography and reactive ion etching. They showed that a precise window of ridge height was required to induce differentiation, which, not coincidentally, is similar to the height of a stomatal ridge [11 ]. Ridges outside this range did not induce differentiation. While appressorial development is required for fungal penetration and subsequent infection, conidial germination is required for appressorial formation. Thus, infection depends on conidial germination. While numerous studies have focused on sporulation and to a lesser extent appressorial formation, far less attention has been given to the germination process which initiates development [5 ]. Moreover, although a few genes expressed during the early stages of the infection process have been identified, their role in development and pathogenesis remains unclear [5, 6 ]. We have been studying the signalling process during prepenetration development in C. trifolii to elucidate the biochemical pathways responsible for the morphological switches. Previously we have shown the importance of calcium and calmodulin for conidial germination and appressorial development [22 ], and have characterized a glutamine rich serine–threonine protein kinase (TB3) that is maximally expressed during germination and is possibly involved in hyphal growth [2 ]. Additional potentially important signalling molecules which could mediate prepenetration events are the second messenger cAMP and its partner, cAMP-dependent protein kinase (PKA). cAMP is well characterized as a regulator of development in numerous eukaryotes [20 ]. cAMP is known to influence conidiation in Neurospora crassa and dimorphism in certain other fungi [17 ]. The addition of cAMP to bean rust germlings induced one round of mitosis and septum formation, processes normally associated with appressorial development [10 ]. Of particular significance, the recent work of Lee and Dean [12 ] confirmed the importance of the cAMP–PKA pathway in fungal development. cAMP has been shown to be a requisite for appressorial formation in Magnaporthe grisea (Krause and Webster). Subsequently, the gene encoding the M. grisea PKA catalytic subunit was cloned. Inactivation of PKA by gene replacement prevented appressorial formation [15 ]. In the corn smut pathogen Ustilago maydis Corda, cAMP and PKA appear to have key roles in regulating the dimorphic switch between budding and filamentous growth [8 ]. Thus, there is precedent for involvement of the cAMP–PKA pathway in phytopathogenic fungal developmental switching and development, although the molecular details are still sketchy. In this report, the role of cAMP and PKA in C. trifolii morphogenesis is assessed. In this study, we show that PKA enzyme activity as well as cAMP levels are developmentally regulated. In addition, interference with this pathway by specific inhibitors suggests the importance of cAMP and PKA for both germination and
cAMP and cAMP dependent protein kinase in C. trifolii
119
differentiation. Lastly, inappropriate regulation of this pathway by pharmacological chemicals induces abnormal appressorial development. MATERIALS AND METHODS
Strains and culture conditions Colletotrichum trifolii race 1 (ATCC 66954) was used throughout these studies [4 ]. The fungus was stored as mycelia on dry filters at ®20 °C and propagated in YPSS liquid medium [21 ]. Mycelia were collected by filtration after 4–5 days’ growth in liquid medium at room temperature with shaking (125 rpm). To obtain germinated conidia and appressoria, washed conidia were suspended (10' conidia ml−") in water and the suspension poured into glass Petri dishes (5 ml per Petri dish) at ambient temperature. After " 80 % of the conidia germinated (2–3 h), a rubber policeman was used to scrub the surface of the Petri dish to resuspend germinating conidia, and these germinating conidia were collected by centrifugation at 5 000 rpm at 4 °C for 10 min. The pellet was immediately frozen in liquid nitrogen and stored at ®80 °C. Appressoria were collected by inducing germinated conidia to differentiate on a hard surface [22 ] and collected as described above. PKA actiity assay Tissue from different developmental stages (conidia, germinating conidia, appressoria and mycelia) was ground with acid-washed glass beads (70–150 microns, Sigma) in liquid nitrogen with protein extraction buffer (5 m EDTA, 50 m Tris, pH 7±5) containing protease inhibitors aprotinin (25 µg ml−") and PMSF (1 m). After incubating on ice for 20 min, the mixture was centrifuged at 12 000 rpm in a JA-20 rotor (Beckman) at 4 °C for 20 min. The supernatant was collected and used in a PKA activity assay. For all assays, the final protein concentration was 5 µg µl−" as determined by the Bradford method [1 ]. To assay for PKA activity, protein extracts were added to tubes containing 50 m Tris, pH 7±5, 10 m MgCl , 0±25 mg ml−" BSA, # 100 µ [γ®$#P] ATP and 50 µ kemptide (model substrate) and incubated at 30 °C for 5 min. Twenty microlitres from each treatment were spotted onto a phosphocellulose disc, acid-washed three times and rinsed with water three times. Discs were then counted in a scintillation counter. PKA specific protein kinase activation was determined with a pseudosubstrate inhibitor peptide, a 17 amino acid peptide sequence (20, Gibco). The relative PKA activity was calculated as the ratio between the sample and mycelia values in liquid scintillation counting, using mycelia values as the baseline. cAMP assay The fresh weight of conidia, germinated conidia and mycelia was measured after freezing in liquid nitrogen. The frozen tissue was ground into powder with a mortar and pestle and incubated with 6 % trichloroacetic acid (TCA) on ice for 20 min. Approximately 10-fold excess of 6 % TCA was added, based on the volume of the ground tissue. After centrifugation at 4 °C for 10 min, the supernatants were collected and extracted five times with water-saturated ether. After extraction, the samples were lyophilized and stored at ®20 °C. cAMP content was determined according to the
120
Z. Yang and M. B. Dickman
manufacturer’s instructions (Amersham cAMP ["#&l] assay system). Briefly, the assay is based on competition between unlabelled cAMP and a fixed amount of "#&l-labelled cAMP for a limited number of binding sites on a cAMP specific antibody. In the presence of known amounts of antibody and radioactive ligand used, the quantity of labelled ligand bound by antibody is inversely proportional to the concentration of added non-radioactive cAMP. Thus the concentration of cellular (unlabelled) cAMP can be determined from a standard curve. Effect of KT5720 on conidial germination and appressorial differentiation Conidia were collected from liquid culture 1–2 days after sporulation. Conidia were concentrated by centrifugation (5 000 rpm for 10 min at 4 °C). The pellet was washed twice in double distilled H O and resuspended (5¬10&®10' conidia ml−") in double # distilled H O or double distilled H O with KT5720 (Calbiochem), a specific inhibitor # # of PKA. 1 m KT5720 was dissolved in DMSO and stored in the dark (4 °C) by covering with aluminium foil. Germination tests were performed by placing 100 µl of the conidial suspension on glass slides and incubating in the dark. Final concentrations of KT5720 used in these tests were based on Gadbois et al. [7 ]. Percentage germination of conidia was measured by counting under a light microscope. For each test, a minimum of 100 conidia were counted in three replicates. To examine the effect of KT5720 on appressorial formation, conidia were resuspended in YPSS media and placed onto glass slides. After germ tubes were observed under the microscope, the media was washed off and replaced with either water or water containing 10 m of KT5720. Appressoria were counted 2 h after this treatment and analysed as described above. Induction of appressorial deelopment on non-inductie surfaces Conidia were collected and resuspended as previously described. Six hundred microlitres of conidia suspension were spread over 1±5 % water agar plates. After " 80 % of the conidia had germinated, the water was removed and replaced with 600 µl of solutions containing 8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX), sodium fluoride (NaF) or forskolin at concentrations indicated in the text. All of these chemicals were purchased from Sigma (St. Louis, MO, U.S.A.). NaF and 8-Br-cAMP were dissolved in ddH O to a final concentration of 100 m for 8-Br-cAMP and 1 for # NaF. Forskolin and IBMX were dissolved in DMSO at 250 m as stock solutions. The percentage of appressorial differentiation was counted following these treatments using a light microscope. RESULTS
Deelopmental profile of PKA actiity To examine whether PKA is involved in C. trifolii development, enzymatic activity of PKA was determined in fungal tissue from different developmental stages. Activities were measured by phosphorylation of kemptide, a specific model substrate of PKA. As shown in Fig. 1, PKA activity was highest in germinating conidia, intermediate in ungerminated conidia, and low in vegetative mycelia and mature appressoria. KT5720, a specific PKA inhibitor (Ki ¯ 56 n) yielded similar levels of enzyme
cAMP and cAMP dependent protein kinase in C. trifolii
121
inhibition to the commercial inhibitor peptide (Ki ¯ 2±3 n) (Ki for both inhibitors were obtained from CalBiochem catalogue), strongly suggesting this assay specifically reflects PKA enzymatic activity.
Relative PKA activity X-fold over mycelia and appressoria
15
10
5
0
1
2
3
4
F. 1. Relative PKA activity in different developmental stages of C. trifolii. 1 : conidia ; 2 : germinating conidia ; 3 : appressoria ; 4 : mycelia. Results are the average from three independent assays. The values on vertical line are arbitrary units, determined as the ratio of counts (CPM) from conidia, germinating conidia with respect to the counts from mycelia. Mycelia and appressoria samples served as the baseline.
Effect of PKA inhibition in conidial germination and appressorial differentiation Since maximal levels of PKA activity occurred during conidial germination and since PKA has been shown to be important for appressorial differentiation in M. grisea [12, 15 ], we examined the role of PKA during these processes using KT5720. 10 µ KT5720 effectively prevented conidial germination (Table 1). Since KT5720 is unstable in the light (report from manufacturer), following inhibition, the conidia were incubated for a period in the light. Conidial germination resumed to near control levels, indicating that PKA inhibition by this compound is reversible and not lethal (unpublished data). When C. trifolii conidia in water attached to a hard surface and germinated, appressoria rapidly formed (1–2 h). However, when conidia were suspended in rich media such as YPSS, appressoria did not develop after conidial germination [20 and unpublished data]. To assess the role of PKA in appressorial formation, conidia were germinated in YPSS media on glass slides. Following germ tube formation, the media was replaced with water or water containing 10 µ KT5720. As shown in Table 2, as with conidial germination, appressorial differentiation was significantly reduced when PKA was inhibited, although not to the extent observed during conidial germination.
122
Z. Yang and M. B. Dickman
A
B
C
D
F. 2. Effect of KT5720 on C. trifolii conidial differentiation. (A) Conidia in water alone ; (B) conidia treated in water containing 10 µ of KT5720 ; (C) Conidia suspended in YPSS media and replaced with water ; (D) conidia suspended in YPSS media and replaced with water containing 10 µ KT5720. All the experiments were done on glass slides. The pictures were taken from the same experiments as described in Tables 1 and 2 in an identical time span.
T 1 Effect of KT5720 on conidial germination in C. trifolii*
Treatment HO # 1 µ KT5720 10 µ KT5720
Germinated conidia† (%³..) 92±7³3±2 84±6³8±0 2±3³1±5
*The data were based on at least six independent tests. †100 conidia first encountered under the microscope (not germinated and germinated) were counted from each slide with three replicates per test. Germination occurred and readings were taken 2–3 h after conidia were placed onto glass slides.
cAMP leels in C. trifolii during deelopment Endogenous levels of cAMP were measured for C. trifolii in conidia, germinating conidia and mycelia. As shown in Table 3, the highest levels of cAMP were found in conidia, with nearly 70 % reduction of cAMP level in germinating conidia for 2–3 h. Mycelia had negligible levels of cAMP. The lack of correlation between cAMP levels
cAMP and cAMP dependent protein kinase in C. trifolii
123
T 2 Effect of KT5720 on appressorial development in C. trifolii*
Treatment HO # 10 µ KT5720
Percentage of appressoria† (%³..) 78±4³0±1 14±2³3±3
*Conidia of C. trifolii germinated in YPSS medium on glass slides. After germination, YPSS medium was replaced with either water or water containing 10 µ KT5720. †Appressoria were counted 2 h after treatment. 100 germinated conidia (with or without appressoria) first encountered under the microscope were counted from each slide with three replicates per test. The result was based on at least six independent tests.
T 3 cAMP leels of C. trifolii in conidia, germinating conidia and mycelia
Tissue Conidia Germinating conidia Mycelia Appressoria
cAMP concentration* (pmols mg−" fresh weight) 0±37³0±09 0±13³0±05 0±06³0±01 ND
*The data are the averages of 3 independent assays. cAMP content was determined as described in Materials and Methods. ND ¯ not determined.
and PKA enzymatic activity during these stages might reflect breakdown of cAMP during tissue collection. It has been reported that cAMP levels in fungi can change within 2 min during extraction [17 ]. Alternatively, cAMP levels might be high in conidia, and rapidly induce germination via induction of PKA when conditions for germination are appropriate. Effect of IBMX, 8-Br-cAMP, NaF and Forskolin on appressorial differentiation In order to examine the role of the cAMP-PKA pathway in C. trifolii morphogenesis further, we determined the effects of established regulators of cAMP. IBMX is an effective inhibitor of cyclic nucleotide phosphodiesterase, which degrades cAMP to AMP. Forskolin has been commonly used as an activator of adenylate cyclase, which utilizes ATP to form cAMP. NaF is also an activator of adenylate cyclase while 8-BrcAMP is a stable analogue of cAMP. These chemicals are widely used in studying cAMP functions in fungi and many other organisms [10, 12, 17 ] as they increase the endogenous levels of cAMP, either by promoting its synthesis or inhibiting degradation. Previously, we demonstrated that appressorium induction of C. trifolii requires germ tube contact with a hard surface, and that hydrophobic surfaces stimulate higher frequencies of development than hydrophilic surfaces [22 ]. Since our studies indicated
124
Z. Yang and M. B. Dickman
that C. trifolii conidia germinate but do not differentiate into appressoria on relatively soft surfaces such as 1±5 % water agar, the non-inductive surface of 1±5 % water agar was used to determine the effects of these various chemicals on C. trifolii conidial differentiation. As shown in Table 4 and Fig. 3, raising cAMP levels in conidia by treatment with NaF, 8-Br-cAMP or by IBMX resulted in appressorial differentiation on this non-inductive surface, although the frequencies were significantly lower than found on inductive surfaces. Forskolin was ineffective in appressorial induction (unpublished data). When these treatments were performed in 1±5 % YPSS agar, no induction of appressoria could be observed, even at higher concentrations of these chemicals (unpublished data). Thus limiting nutrients are also required in addition to elevated cAMP levels to cause abnormal differentiation. Similar observations have been reported for Colletotrichum lindemuthianum Lams. [14 ]. A
B
F. 3. Induction of C. trifolii appressorial differentiation on 1±5 % water agar by 8-Br-cAMP. (A) Conidia spread on 1±5 % water agar ; (B) Conidia spread on 1±5 % water agar containing 5 m 8-Br-cAMP. This result is from the same experiments as described in Table 3. After evaluation, Petri dishes were stored at 4 °C for 5–6 h prior to photography.
cAMP and cAMP dependent protein kinase in C. trifolii
125
T 4 Effect of 8-Br-cAMP, IBMX, and NaF on C. trifolii conidial differentiation on non-inductie surfaces*
Treatment HO # 5 m 10 m 5 m 10 m 5 m
IBMX IBMX NaF NaF 8-Br-cAMP
Appressoria† (%³..) 1±0³0±8 12±8³5±6 14±2³4±8 10±0³4±7 13±7³4±6 20±1³3±9
*1±5 % water agar was used as non-inductive surface. †The percentage of differentiated appressoria was determined as follows : for each test, 100 germinated conidia (having long germ tubes with or without appressoria) were counted microscopically from each Petri dish. Each treatment was performed as described in Materials and Methods. The data were based on six independent experiments with three replicates per test.
DISCUSSION
Invasion of alfalfa by C. trifolii requires penetration of leaves or stems through a highly coordinated series of morphological events [5 ]. When conidia of C. trifolii land on the host surface, attachment triggers conidial germination. Appressorial differentiation follows germ tube formation and adherence to the host cuticle, but only in the presence of a hard surface and limiting nutrients. The appropriate timing of these switches is essential for host penetration and subsequent colonization. Interference with this process at any point prevents disease development in intact host tissue. This genetically programmed series of morphological changes is likely to be triggered by signalling pathways. To understand the events and key processes surrounding conidial germination and leading to appressorial differentiation, we examined the involvement of the cAMP–PKA pathway. Several lines of evidence support the involvement of this pathway in fungal morphogenesis. For example, the addition of exogenous cAMP or inhibitors of phosphodiesterase can induce synchronous zoospore germination in Blastocladiella emersonii [9 ]. In addition, PKA was shown to be developmentally regulated [13 ]. PKA activity was low during vegetative growth, increased during sporulation and was maximum in spores. During germination, total PKA activity decreased. Biochemical evidence for the role of cAMP in morphogenesis has been described for several fungi [17 ]. In N. crassa, mutants defective in cAMP conidiated poorly [17 ]. In Mucor rouxii, cAMP levels drop during emergence of hyphal germ tubes and remain low during hyphal growth [16, 19 ]. In contrast, in certain dimorphic fungi, intracellular cAMP levels increase during germ tube formation and hyphal growth [see 8 ]. In U. maydis, a dimorphic basidiomycete, low cAMP levels promote hyphal extension in preference to budding growth. Defects in adenylate cyclase or mutations in the regulatory subunit of PKA, which prevent PKA activation, lead to filamentous growth of U. maydis [8 ]. Similarly, a mutation in the cot-1 gene in N. crassa results in extensive mycelial branching and reduced hyphal extension [25 ]. The COT-1 sequence resembles the catalytic domain of PKA, although direct induction of this gene
126
Z. Yang and M. B. Dickman
by cAMP has not yet been demonstrated [25 ]. Dean’s group has shown that cAMP regulates appressorial development in M. grisea and that disruption of the PKA catalytic subunit gene prevented the formation of appressoria [12, 15 ]. Thus there is convincing evidence that cAMP and PKA can regulate morphogenesis in fungi. We have shown that the cAMP-PKA pathway is likely to be operative during the prepenetration developmental sequences of C. trifolii. cAMP levels were determined in three distinct fungal developmental stages. Levels were highest in conidia and reduced as germ tubes formed and mycelial growth occurred. These patterns are similar to those found in M. rouxii and U. maydis. As cAMP has basically one known biochemical activity, that is binding to PKA [20 ], we examined PKA activity during these stages. Relative activity was highest in germinating conidia and lowest in mycelia and appressoria. The lack of a direct correlation between cAMP levels and PKA enzyme activity might reflect the fact that cAMP triggers PKA activity via binding to the regulatory subunit of PKA. cAMP binding to the regulatory subunit results in its release from the catalytic subunit and this release results in PKA induction. PKA activity would therefore be highest during germination. We also used a pharmacological approach to address the functional relevance of the cAMP-PKA pathway. KT5720, a highly specific inhibitor of PKA, almost completely prevented conidial germination at 10 µ. When conidia were germinated on an inductive surface and then treated with KT5720, appressorial differentiation was also effectively impaired. On hard surfaces (e.g. glass slides) which induce germination and differentiation, raising endogenous cAMP levels either by increasing synthesis with adenylate cyclase activators or by decreasing degradation with inhibitors of phosphodiesterase, we found increases in both the rate of conidial germination and the level of appressorial differentiation (unpublished). On a non-inductive surface (1±5 % water agar), where appressoria are rarely if ever observed, the addition of 8-Br-cAMP, IBMX and NaF all induced appressoria differentiation. Similar results have been reported in the rice blast pathogen M. grisea [12 ]. When water agar is replaced by nutrient media, appressoria do not differentiate, even in the presence of the aforementioned effectors. Thus, starvation or nutrient limitation is also necessary for differentiation ; increasing cAMP levels alone is not sufficient for differentiation. Forskolin, an activator of adenylate cyclase had no effect on C. trifolii as has been reported for the ascomycete M. grisea, but differing from the reports with the basidiomycete, U. phaseoli, possibly reflecting taxonomic differences [10, 12 ]. Alternatively, forskolin may be poorly taken up or inactivated in C. trifolii (and M. grisea). While the evidence suggests the importance of the cAMP-PKA pathway in these prepenetration events, these data are limited. For example, PKA inhibitors prevent appressorial differentiation, but PKA enzyme levels in mature appressoria were low. This may reflect the unreliability of this biochemical assay. Also, C. trifolii spores do not germinate synchronously, and thus appressoria were only collected after maturity at which point PKA may be unnecessary. Alternatively, since germination and appressorial differentiation are tightly coupled, once triggered under inductive conditions, PKA levels induced during germination may be sufficient to complete the process. To address this problem more directly, we have isolated the regulatory and catalytic subunit genes of PKA in C. trifolii (unpublished data). Characterization and functional analyses of these genes are in progress.
cAMP and cAMP dependent protein kinase in C. trifolii
127
This work was supported by the United States–Israel Binational Agricultural Research and development Fund (BARD) and by the DOE}NSF}USDA Program on Collaborative Research in Plant Biology grant 92-37310-7821. REFERENCES 1. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 : 249–254. 2. Buhr TL, Oved S, Truesdell GM, Huang C, Yarden O, Dickman MB. 1996. A kinase-encoding gene from Colletotrichum trifolii complements a colonial growth mutant of Neurospora crassa. Molecular and General Genetics 251 : 565–572. 3. Churchill A, Baker C, O’Neill N, Elgin J. 1988. Development of Colletotrichum trifolii races 1 and 2 on alfalfa clones resistant and susceptible to anthracnose. Canadian Journal of Botany 66 : 75–81. 4. Dickman M. 1988. Whole cell transformation of the fungal pathogen Colletotrichum trifolii. Current Genetics 14 : 241–246. 5. Dickman MB, Buhr TL, Warwar V, Truesdell GM, Huang CX. 1995. Molecular signals during early stages of alfalfa anthracnose. Canadian Journal of Botany 73 : 1169–1177. 6. Dickman MB. 1995. The molecular biology of fungal-plant interactions. In : Leslie JF, Fredrisksen RA, eds. Disease Analysis through Genetics and Biotechnology. Ames, Iowa : Iowa State University Press, 149–195. 7. Gadbois D, Grissman H, Tobey R, Bradbury M. 1992. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proceedings of National Academy of Sciences of the United States 89 : 8626–8630. 8. Gold S, Duncan G, Barret K, Kronstad J. 1994. cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes and Deelopment 8 : 2805–2816. 9. Gomes SL, Mennucci L, Maia JCC. 1980. Induction of Blastocladiella emersonii germination by 3«,5«monophosphate. Cell Differentiation 9 : 169–179. 10. Hoch HC, Staples RC. 1984. Evidence that cAMP initiates nuclear division and infection structure formation in the bean rust fungus, Uromyces phaseoli. Experimental Mycology 8 : 37–46. 11. Hoch HC, Staples RC, Whitehead B, Comeau J, Wolf ED. 1987. Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235 : 1659–1662. 12. Lee YH, Dean R. 1993. cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea. The Plant Cell 5 : 693–700. 13. Marques M, Juliani M, Maia J, Gomes S. 1989. Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii. European Journal of Biochemistry 178 : 803–810. 14. Mercer PC, Wood RKS, Greenwood AD. 1971. Initial infection of Phaseolus ulgaris by Colletotrichum lindemuthianum. In : Preece TF, Dickinson CH, eds. Ecology of Leaf Surface Micro-organisms. London : Academic Press, 381–389. 15. Mitchell T, Dean R. 1995. The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. The Plant Cell 7 : 1869–1878. 16. Orlowski M. 1991. Mucor dimorphism. Microbiology Reiew 55 : 234–258. 17. Pall ML. 1981. Adenosine 3,5-phosphate in fungi. Microbiology Reiew 45 : 462–480. 18. Perpetua NS, Kubo Y, Okuno T, Furusawa I. 1994. Restoration of pathogenicity of a penetrationdeficient mutant of Colletotrichum lagenarium by DNA complementation. Current Genetics 25 : 41–46. 19. Pewerchin MA, Van Laere AJ. 1984. Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores. Journal of Bacteriology 158 : 575–579. 20. Taylor SS, Buechler JA, Yonemoto W. 1990. cAMP-dependent protein kinase : framework for a diverse family of regulatory enzymes. Annual Reiew of Biochemistry 59 : 971–1005. 21. Tuit J. 1968. Plant Pathological Methods. Minneapolis : Burgess Publishing. 22. Warwar V, Dickman MB. 1996. Effects of calcium and calmodulin on spore germination and appressorium development in Colletotrichum trifolii. Applied and Enironmental Microbiology 62 : 74–79. 23. Wynn WK. 1976. Appressorium formation over stomates by the bean rust fungus : response to a surface contact stimulus. Phytopathology 66 : 136–146. 24. Wynn WK. 1981. Tropic and taxic responses of pathogens to plants. Annual Reiew of Phytopathology 19 : 237–255. 25. Yarden O, Plamann M, Ebbole DJ, Yanofsky C. 1992. Cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. The EMBO Journal 11 : 2159–2166.