Regulation of cAMP and cAMP dependent protein kinase during conidial germination and appressorium formation inColletotrichum trifolii

cot-1 mutants belong to a class of Neurospora crassa colonial temperature-sensitive (cot) mutants that exhibit abnormal polar extension and branching patterns when grown at restrictive temperatures. cot-1 encodes a Ser/Thr protein kinase that is structurally related to the human myotonic dystrophy kinase which, when impaired, confers a disease that involves changes in cytoarchitecture and ion homeostasis. When grown under restrictive conditions, cot-1 cultures exhibited enhanced medium acidification rates, increased relative abundance of sodium, and increased intracellular glycerol content, indicating an ion homeostasis defect in a hyperbranching mutant. The application of ion transport blockers led to only mild suppression of the cot-1 phenotype. The presence of increased medium NaCl or sorbitol, H 2 O 2 , or ethanol levels significantly suppressed the cot-1 phenotype, restored ion homeostasis, and was accompanied by reduced levels of cyclic AMP-dependent protein kinase (PKA) activity. The cot-1 phenotype could also be partially suppressed by direct inhibition of PKA with KT-5720. A reduced availability of fermentable carbon sources also had a suppressive effect on the cot-1 phenotype. In contrast to the effect of extragenic ropy suppressors of cot-1, environmental stress-related suppression of cot-1 did not change COT1 polypeptide expression patterns in the mutant. We suggest that COT1 function is linked to environmental stress response signaling and that altering PKA activity bypasses the requirement for fully functional COT1.

Section: Discussionmentioning

confidence: 99%

Environmental Suppression of Neurospora crassa cot-1 Hyperbranching: a Link between COT1 Kinase and Stress Sensing

Gorovits

Yarden

2003

“…The wild-type strain of C. trifolii race 1 (7) was used throughout this study. C. trifolii cultures were routinely grown at 25°C on YPSS agar plates (28). For isolation of protoplasts, DNA, RNA, and proteins, mycelia were grown in stationary liquid YPSS for 3 to 7 days.…”

Section: Methodsmentioning

confidence: 99%

Colletotrichum trifolii TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation

Chen

Dickman

2002

Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus (TB3), which is a functional homolog of the Neurospora crassa COT1 kinase, has been isolated in our laboratory and appears to be associated with hyphal elongation and branching. In this report we show that light treatment rapidly induces TB3 expression and hyphal branching frequency. Western analysis showed TB3 localization in both the cytoplasm and nucleus, but not in membranes. Moreover, indirect immunofluorescence indicated that TB3 levels were most abundant in the nucleus. To further evaluate the subcellular distribution of TB3, a TB3::GFP fusion construct was inserted into C. trifolii. Results indicated that the cellular location of TB3 changed during fungal growth and development. Consistent with previous observations, TB3 was localized in both the cytoplasm and the nucleus but was preferentially localized in the nucleus during extended hyphal growth. The amino terminus of TB3 contains two relatively long polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts can activate transcription. These results suggest that TB3 may be positioned in a signaling cascade regulating proper hyphal growth and development by functioning as a transcription factor.A distinctive feature of filamentous fungi is their mode of growth and development via vegetative propagagules known as hyphae. Hyphae grow in a polarized manner and account, in part, for the foraging success of fungi either as saprophytes or pathogens. Coordinated control of hyphal growth and development in filamentous fungi is achieved by activating intracellular signaling cascades in response to environmental stimuli, including nutrient availability, light, and plant host. In addition to these environmental cues that influence hyphal growth, genetic factors have also been identified. In Neurospora crassa, the serine/threonine protein kinase, COT1, has been shown to be required for hyphal elongation (29). Mutations in cot-1 are temperature sensitive; colonial growth occurs at or above 32°C, while normal radial growth is observed at or below 25°C (17). Moreover, disruption of cot-1 results in severe growth defects as disruptants grow little, if at all. cot-1 expression is also photoinducible, and light illumination increases apical hyphal cell branching frequency (16). Immunoblotting with COT1 antibody detects the COT1 kinase in the cytoplasm, membrane, and nucleus (13).Colletotrichum trifolii is the causal agent of alfalfa anthracnose (1). Pathogenicity of C. trifolii depends on proper growth and appressorium differentiation, which are believed to be regulated through the activation of intracellular signal transduction pathways in response to host stimuli (9). However, our understanding of the genes and biochemical pathways which govern hyphal growth and morphogenesis are not entirely clear. Previously, we cloned and characterized a serine/threonine protein kinase gene, tb3 (3), a...

“…Spore germination is a key step in the colonization process of a new environment by filamentous fungi. Recent studies of C. trifolii have revealed that various signaling molecules, including calcium, cyclic AMP (cAMP), calmodulin, and protein kinases, are important players in regulating spore germination (14,46,50). Here we described the involvement of the small GTPase Cdc42 in this process; it will be of interest to examine the relationship between Cdc42 and these other signaling components.…”

mentioning

confidence: 90%

“…Previous studies of C. trifolii have shown that an increase in endogenous cAMP levels by using cAMP-elevating agents, such as 8-Br-cAMP, sodium fluoride, and 3-isobutyl-1-methylxanthine, also results in induction of appressorial differentiation on a noninductive surface, suggesting a possible linkage between CtCdc42 and the cAMP signaling pathway (50). Moreover, several signaling molecules in C. trifolii that are important for appressorium development have been isolated and characterized, including the catalytic subunit of PKA, calmodulin, and lipid-induced protein kinase (14,46,49,50). Molecular analysis of the connection of CtCdc42 with these molecules will improve the understanding of signaling pathways involved in appressorium development in C. trifolii.…”

mentioning

confidence: 98%

“…The mechanism(s) of appressorium development has been intensively studied in phytopathogenic fungi, including Magnaporthe grisea and other Colletotrichum spp. The results of these studies indicated that cAMP/PKA-and MAPK-mediated signaling pathways are operative during appressorium development (10,46,(48)(49)(50). Interestingly, disruption of MAGB, encoding an inhibitory group I subunit of a heterotrimeric G protein (G␣i), affects the appressorium formation and pathogenicity of M. grisea (26), suggesting the possibility that G protein may participate in the process of appressorium formation.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Cdc42 Is Required for Proper Growth and Development in the Fungal Pathogen Colletotrichum trifolii

Chen

Min

et al. 2006

Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.