A mitogen-activated protein kinase kinase required for induction of cytokinesis and appressorium formation by host signals in the conidia of Colletotrichum gloeosporioides

Differentiation of fungal conidia of phytopathogens into the infection structure, appressorium, requires contact with a hard surface and host signals. The molecular signaling involved in the induction of this differentiation is poorly understood. We report the cloning of a mitogen-activated protein kinase kinase (MEK), CgMEK, from Colletotrichum gloeosporioides and its role in the induction of these developmental processes involved in pathogenesis. Disruption of CgMEK1 resulted in the loss of its ability to form appressoria in response to the host's signals and a loss of virulence. Results of confocal microscopic examination of germinating conidia of the gene-disrupted mutants were similar to those for wild-type conidia treated with an MEK inhibitor, suggesting that CgMEK1 is involved in two developmental processes in the differentiation into appressorium: (1) polarized cell division, with the preferential increase in F-actin in one of the daughter nuclei after nuclear division and the formation of septum; and (2) differentiation of the germ tube into an appressorium. CgMEK1 is required for the differentiation.     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.     

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

CtPMK1, a mitogen‐activated‐protein kinase gene,is required for conidiation,appressorium formation,and pathogenicity of Colletotrichum truncatum on soybean

Colletotrichum truncatum, the causal agent of soybean anthracnose, invades host plants by forming a specialised infection structure called an appressorium. Mitogen‐activated protein kinase (MAPK) genes have been shown to play vital roles in several phytopathogenic fungi in regulating various infection processes, including spore germination, melanised appressorium formation, appressorial penetration and subsequent invasive growth in host plants. In this study, we identified and characterised the first Fus3/Kss1‐related MAPK gene, CtPMK1, in Colletotrichum truncatum, which is related to PMK1 in Magnaporthe oryzae. Disruption of CtPMK1 in C. truncatum resulted in a mutant with slightly reduced mycelial growth (‐30%) and melanisation that is deficient in sporulation (‐99%), as observed in the CMK1 mutant of Colletotrichum lagenarium (a synonym of Colletotrichum orbiculare, which is now the accepted name for this taxon). In contrast to CMK1 of C. lagenarium, conidia from the Ctpmk1 mutant germinated normally on glass slides and onion epidermal surfaces. Our findings suggest that there are differences in the types of in vitro functions controlled by PMK1, even between closely related species. Furthermore, the Ctpmk1 mutant failed to form appressoria or hyphopodia, subsequently resulting in the complete loss of pathogenicity on host plants. Overall, the results indicate that the Fus3/Kss1‐related MAPK gene has a conserved role in infection structure formation and pathogenicity in phytopathogenic fungi.     

The mitogen-activated protein kinase kinase kinase BcOs4 is required for vegetative differentiation and pathogenicity in Botrytis cinerea

The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.     

The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.     

A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea

Insertional mutagenesis is an effective way to study the infection mechanism of fungal pathogens. In an attempt to identify the genes involved in appressorium formation from Magnaporthe grisea, we carried out Agrobacterium tumefaciens mediated transformation (ATMT) of the fungus. Analysis of the region flanking the T-DNA integration site in one of the appressorium mutants showed insertion in a gene coding a 78 amino acid protein (MGA1), showing no significant homology to any of the known proteins. The mutant mga1 caused neither foliar nor root infection. Complementation of the mutated gene with the full length wild type gene restored appressorium formation as well as rice infection demonstrating the involvement of this gene in pathogenicity of M. grisea. In an indirect immunolocalisation assay, the MGA1 expression was seen predominantly in germ tube and appressoria. The mutant was impaired in glycogen and lipid mobilization required for appressorium formation. The glycerol content in the mycelia of the mutant under hyperosmotic stress conditions was less as compared to wild type and was thus unable to tolerate the hyperosmotic stress induced by sorbitol. We hypothesize that MGA1 plays a crucial role in signal transduction leading to the metabolism of glycogen and lipids, which is a part of appressorium differentiation process.     

The transmembrane protein AaSho1 is essential for appressorium formation and secondary metabolism but dispensable for vegetative growth in pear fungal Alternaria alternata

The high-osmolarity glycerol response (HOG) pathway is pivotal in environmental stress response, differentiation and virulence of Alternaria alternata. The synthetic high osmolarity sensitive sensor Sho1 has been postulated to regulate the HOG pathway. To determine the regulatory role of transmembrane protein Sho1 on vegetative growth, secondary metabolism and infection structure formation, a gene (AaSho1) encoding Sho1 was cloned and characterized from A. alternata (JT-03). Sequence analysis showed that AaSho1 has all four characteristic transmembrane domains and the SH3 domain present in another Sho1 gene from several filamentous fungal. The quantitative RT-PCR analysis showed that fruit wax extract significantly up-regulated AaSho1 gene expression in vitro. Pharmacological experiments showed that A. alternata treated with nystatin, a specific AaSho1 inhibitor, had no significant effect on the morphology of A. alternata and the invasive growth in pear fruit. However, nystatin treatment significantly reduced spore germination rates on different wax-coated hydrophobic surfaces, with 58.00, 46.70 and 83.72% reduced for fruit wax, beeswax and paraffin coated. Meanwhile, the secondary metabolism altenuene (ALT), tentoxin (TEN) toxin, and melanin content were also affected by nystatin treatment. These findings suggest that AaSho1 is required for the infection structure differentiation and secondary metabolism of A. alternata in response to physiochemical signals on the host surfaces.     

An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis

The signal transduction pathways that control cytokinesis in plants are largely uncharacterized. Here, we provide genetic evidence that mitogen-activated protein kinase kinase kinases (MAPKKKs) play a role in the control of plant cell division. Using a reverse-genetic approach, we isolated plants carrying knockout alleles of the Arabidopsis MAPKKK genes ANP1, ANP2, and ANP3. The resulting single-mutant plants displayed no obvious abnormal phenotypes; two of the three double-mutant combinations displayed defects in cell division and growth; and the triple-mutant combination was not transmitted through either male or female gametes. The molecular and structural phenotypes displayed by the double mutants support a model in which the ANP family of MAPKKKs positively regulates cell division and growth and may negatively regulate stress responses.     

The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum

Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, Clpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.     

Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.     

The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation

DNA topoisomerase I (Topo1) contributes to vital biological functions, but its regulation is not clearly understood. The BTBD1 protein was recently cloned on the basis of its interaction with the core domain of Topo1 and is expressed particularly in skeletal muscle. To determine BTBD1 functions in this tissue, the in vitro model used was the C2C12 mouse muscle cell line, which expresses BTBD1 mainly after myotube differentiation. We studied the effects of a stably overexpressed BTBD1 protein truncated of the 108 N-terminal amino-acid residues and harbouring a C-terminal FLAG tag (Delta-BTBD1). The proliferation speed of Delta-BTBD1 C2C12 cells was significantly decreased and no myogenic differentiation was observed, although these cells maintained their capacity to enter adipocyte differentiation. These alterations could be related to Topo1 deregulation. This hypothesis is further supported by the decrease in nuclear Topo1 content in Delta-BTBTD1 proliferative C2C12 cells and the switch from the main peripheral nuclear localization of Topo1 to a mainly nuclear diffuse localization in Delta-BTBTD1 C2C12 cells. Finally, this study demonstrated that BTBD1 is essential for myogenic differentiation.     

Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1

Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Delta and pbs2Delta cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Delta cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.