cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.     

The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.     

Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea.     

Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus.

We have investigated the role of the mitogen-activated protein kinase (MAPK) pathway in the differentiation of CD4+ and CD8+ T cells by looking specifically at the effects of inhibitors of MAPK-activating enzyme, MAPK/extracellular signal-related kinase (ERK) kinase (MEK), during the positive selection step from double-positive to single-positive (SP) thymocytes. Using a variety of transgenic/knockout mouse strain combinations that fail to differentiate individual lineages of SP thymocytes together with genetically engineered F(ab')2 reagents that induce maturation preferentially to either the CD4 or CD8 subpopulations, we show that induction of CD4 differentiation cells is highly sensitive to levels of MEK inhibition that have no effect on CD8 maturation. In addition, the presence of MEK inhibitor is able to modify signals that normally induce CD4 differentiation to instead promote CD8 differentiation. Finally, we show that continuous culture in the presence of inhibitor interferes with TCR up-regulation in SP thymocytes, suggesting that MAPK signaling may be involved in final maturation steps for both lineages. These data indicate that there is discrimination in the biochemical pathways that are necessary to specify CD4 and CD8 lineage commitment and can reconcile previously conflicting reports on the influence of MAPK activation in commitment and maturation of thymocytes.     

A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae

Insertional mutagenesis of Magnaporthe oryzae led to the identification of MCK1, a pathogenicity gene predicted to encode mitogen-activated protein kinase kinase kinase (MAPKKK) homologous to BCK1 in Saccharomyces cerevisiae. Targeted disruption of MCK1 resulted in the fungus undergoing autolysis and showing hypersensitivity to cell-wall-degrading enzyme. The mck1 produced significantly reduced numbers of conidia and developed appressoria in a slightly retarded manner compared with the wild type. Appressorium of the mck1 mutant was unable to penetrate into plant tissues, thereby rendering the mutant nonpathogenic. Cytorrhysis assay and monitoring of lipid mobilization suggested that the appressorial wall was altered, presumably affecting the level of turgor pressure within appressorium. Furthermore, the mck1 mutant failed to grow inside plant tissue. Complementation of the mutated gene restored its ability to cause disease symptoms, demonstrating that MCK1 is required for fungal pathogenicity. Taken together, our results suggest that MCK1 is an MAPKKK involved in maintaining cell wall integrity of M. oryzae, and that remodeling of the cell wall in response to host environments is essential for fungal pathogenesis.     

Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea

A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.     

MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Deltapmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a DeltacpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Deltamac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MAPK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.     

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

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.     

In vitro differentiation of haustorial mother cells of the wheat stem rust fungus,Puccinia graminis f. sp. tritici,triggered by the synergistic action of chemical and physical signals

Biotrophic plant pathogenic fungi often develop a sophisticated series of infection structures for non-destructive host tissue penetration. In vitro, early infection structures of rust fungi-germ tube, appressorium, substomatal vesicle, infection hyphae-can easily be induced, but in vitro differentiation rates of late infection structures-haustorial mother cells (hmc), haustoria-are low at best. Under appropriate conditions (humid atmosphere), a combination of physical (mild heat shock) and chemical signals (trans-2-hexen-1-ol) induced the in vitro differentiation of hmc in the wheat stem rust fungus, Puccinia graminis f. sp. tritici. Around two thirds of the in vitro differentiated germlings developed up to three hmc which were cytologically identical to hmc formed in planta. Efficient in vitro differentiation of hmc will allow us to analyse in molecular detail the processes involved in the induction and differentiation of this critically important developmental stage of the economically important plant pathogenic rust fungi.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.     

DYRK1A enhances the mitogen-activated protein kinase cascade in PC12 cells by forming a complex with Ras, B-Raf, and MEK1

Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) is the human homologue of the Drosophila mnb (minibrain) gene. In Drosophila, mnb is involved in postembryonic neurogenesis. In human, DYRK1A maps within the Down syndrome critical region of chromosome 21 and is overexpressed in Down syndrome embryonic brain. Despite its potential involvement in the neurobiological alterations observed in Down syndrome patients, the biological functions of the serine/threonine kinase DYRK1A have not been identified yet. Here, we report that DYRK1A overexpression potentiates nerve growth factor (NGF)-mediated PC12 neuronal differentiation by up-regulating the Ras/MAP kinase signaling pathway independently of its kinase activity. Furthermore, we show that DYRK1A prolongs the kinetics of ERK activation by interacting with Ras, B-Raf, and MEK1 to facilitate the formation of a Ras/B-Raf/MEK1 multiprotein complex. These data indicate that DYRK1A may play a critical role in Ras-dependent transducing signals that are required for promoting or maintaining neuronal differentiation and suggest that overexpression of DYRK1A may contribute to the neurological abnormalities observed in Down syndrome patients.     

Uncoupling Raf1 from MEK1/2 impairs only a subset of cellular responses to Raf activation

The Raf family of serine/threonine protein kinases is intimately involved in the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf effector proteins. However, accumulating evidence suggests that there are additional Raf substrates and that subsets of Raf-induced regulatory events are mediated independently of Raf activation of MEK1/2. To examine the possibility that there is bifurcation at the level of Raf in activation of MEK1/2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association uncouples Raf from activation of ERK1/2, induction of serum-response element-dependent gene expression, and induction of growth and morphological transformation. However, activation of NF-kappaB-dependent gene expression and induction of neurite differentiation were unimpaired. In addition, Raf-dependent activation of p90 ribosomal S6 kinase was only slightly impaired. These results support the hypothesis that Raf kinases utilize multiple downstream effectors to regulate distinct cellular activities.     

RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility

The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.     

Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.