Nitric oxide NO is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC.
Correspondence and Footnotes. Several mammalian AC isoforms have been cloned These isoforms are involved critically in mammalian signal transduction pathways where they regulate gene transcription, metabolism, and ion channel activity 4,5.
ACs are induced or inhibited by a system of three components associated with the plasma membrane: G protein-coupled receptors, stimulatory and inhibitory heterotrimeric G proteins, and the AC catalytic entity itself 6. Furthermore, forskolin binds to the AC catalytic core 9,10 by joining the two domains of the core using a combination of hydrophobic and hydrogen-bonding interactions that are distributed equally between the two domains It has been shown that all mammalian AC isoforms are activated by both forskolin and the GTP-bound a subunit of the stimulatory G protein G s The diverse activities of NO include blood vessel relaxation and modulation of immune responses, pathogen killing, and inhibition of platelet aggregation and adhesion.
Moreover, NO controls the activity of enzymes and ion channels, and serves as a neuromodulator and neurotransmitter in the central and peripheral nervous systems There is also evidence that NO may be a physiological intracellular regulator of mitochondrial respiration and gene activity, and can interact with oxygen-derived radicals to produce other highly reactive substances 15, In addition, NO is involved in the development of tolerance to and withdrawal from morphine 17, Overproduction of NO in mammalian systems may contribute to cell damage or cell death Although the actions of the enzymes AC and NOS in promoting two signal transduction pathways have been already characterized, little information is available about the relation between these two pathways.
It has been well established that NO activates the second messenger guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers such as AC.
It has been previously demonstrated that the inhibition of the endogenous neuroblastoma cell line AC by NO is related to isoform type VI 21 , but little information is available on the effects of NO on other mammalian AC isotypes, as in AC genes transfected to mammalian cultured cell lines. Transient transfection of COS-7 cells was performed as described 22, Twenty-four hours later, the cells from each cm plate were trypsinized and recultured on a well plate, and after an additional 24 h, cells were assayed for cAMP content as a measure of AC activity as described below.
The assay was performed in triplicate as described previously 22,25, Briefly, cells cultured on well plates were incubated for 2 h with 0. This medium was replaced with 0. After 10 min of incubation at room temperature, the medium was removed and the reaction terminated by the addition of perchloric acid containing 0. For this purpose, the COS-7 cell system, which readily allows transient transfection of the desired AC isozymes, was employed.
In the concentration range between 0. Figure 1. One hundred percent represents the control AC activity observed in stimulated transfected COS-7 cells. Similar results were obtained with the forskolin-stimulated AC VI isozyme. Furthermore, in all the experiments performed, the basal accumulation of cAMP, a measure of cAMP generated in the absence of hormonal stimulation, was unaffected by treatment with SNP data not shown. The results also suggest that the inhibitory effect of SNP is independent of forskolin binding in these isoforms, resulting in a differential effect of SNP on the various AC isotypes.
It is also interesting to mention that the inhibitory effect of SNP was on the AC I and AC VI isoforms, which are not closely related to each other either in terms of enzymatic stimulation or of genetic homology. The complete coding sequence of AC type I is known, and there is some information concerning functional domains of the enzyme. An examination of the amino acid sequence of AC type I reveals several sequences which exhibit the general properties of calmodulin-binding domains.
It has been reported that a synthetic peptide, corresponding to amino acids from AC I, binds with high affinity and inhibits calmodulin stimulation of the enzyme, suggesting that this sequence may correspond to the calmodulin-binding domain of the enzyme Interestingly, this sequence contains two cysteine residues, uncommon for calmodulin-binding domains.
One possible explanation for this inhibition could be the oxidation of sulfhydryl groups of neighboring cysteines in the calmodulin-binding domain of the enzyme. The site of the NO effect on the enzyme is intriguing; since the basal enzyme activity of the different AC isozymes did not vary, it is unlikely that NO action could be targeted to the catalytic site of the enzyme.
The basal activities of type I and VI recombinant enzymes are not altered by NO, suggesting that, in their basal states, these isoforms share a conformation that precludes susceptibility to NO. On this basis, we speculate that this inhibition is due to a change in V max , with no change in the K m of the substrate.
Although it has been reported that NO did not inhibit cAMP production by the AC I isotype 32 , those experiments were performed on insect cell membranes. It is interesting to observe that both enzymes, AC I and AC VI, share a conformation that precludes any susceptibility to NO 33 , suggesting that SNP might be nitrosylating two cysteine residues, thus modifying their catalytic activity.
A possible means of testing this hypothesis is to induce a mutation of the residues in genetically modified AC isoforms. Each step in the life cycle is triggered by environmental cues like temperature, light and humidity reviewed in [35] , [36].
Perception and subsequent processing of signals from a variable environment is based on a cascade comprising of receptors and intracellular modules that are able to transmit and amplify a signal to downstream effectors. Second messengers play a decisive role in these cascades. Here, we analyzed the ubiquitous second messenger cAMP for its function in virulence, vegetative growth, secondary metabolism, and sexual reproduction.
A prevalent secondary metabolite of F. It is harmful to humans and animals and is, in contrast to ZEA, a virulence factor for the infection of wheat. DON biosynthesis can be induced in vitro by several additives to media such as polyamines [37] , [38] , H 2 O 2 [39] , and cobalt chloride [40].
Furthermore, its biosynthesis is influenced by ambient conditions like temperature [41] , and pH [42]. The underlying regulation processes and signaling cascades are complex and not fully revealed.
Our results indicate that cAMP signaling cascade represent a superior regulatory framework for DON biosynthesis, since the Fgac1 deletion mutants were deficient in DON biosynthesis both in planta and in vitro.
The in-planta results are of limited impact, as the mutant was not able to penetrate and colonize the plant. Due to this lack of invasive growth, the areas of high DON induction within the wheat floret, i. Recent studies in F.
This suggests that, under DON inducing conditions, none of the G-protein subunits activate the adenylyl cyclase. It cannot be ruled out that functional redundancy in the G alpha subunits contribute to the minor phenotypes observed for single deletion mutants in the subunits compared to the deletion of the unique adenylyl cyclase. Deletion of the F.
These results draw a picture of a complicated crosstalk between different regulators that act specifically in response to certain environmental cues see also table S2. Intruigingly, the mutants were also unable to penetrate wheat epidermal cells. This correlation strongly suggests that the formation of complex infection structures is prerequisite for successful colonization of wheat tissue. On wheat floral organs, the mutant was hypersporulating which represents a dramatic morphogenetic switch in the lifestyle from pathogenic to vegetative growth.
It seems plausible that the transition from runner hyphae to complex infection structures on wheat floral leaves is triggered by cAMP levels. Elevated cAMP levels in wild-type infected wheat samples, as proven by the ELISA analysis, may influence the formation of compound appressoria, while depletion of intracellular cAMP favors sporulation during wheat infection. Therefore, the pathogenic lifestyle occurs and early sporulation is suppressed. However, comprehensive histology of the infection process of F.
Deletion of tri5 , encoding a trichodien synthase in F. It is proposed that both chemical and physical stimuli are involved in the induction of appressorium formation in M. Adenylyl cyclase deletion mutants of M. We, so far, did not identify conditions to induce compound appressoria formation in vitro on agar plates or artificial surfaces M.
Boenisch, unpublished results. Hence, specific and as-yet unknown compounds present on natural surfaces seem to be necessary for compound appressoria formation in F. Exogenous nutrient sources such as plant exudates or sucrose generally promote an increase in branching frequency during infection cushion development of R. Furthermore, the availability of nutrients is discussed to be necessary for the production of mucilage in R.
We also observed the formation of mucilage surrounding the wild-type, but not the mutant hyphae that colonize the host surface fig. Hence, it is feasible that a defective sensing of a certain nutritional environment together with the absence of mucilage in the mutant strain contributes to the lack of infection structures. The lack of mature perithecia on wheat straw might be due to a dysfunctional sensing of the environment by the deletion mutant.
Deletion of a G alpha subunit in U. It is tempting to speculate that a dysfunctional sensing of plant-derived nutrients or certain patterns on the plant surface lead to the sterility phenotype on wheat nodes. Carrot agar, in this regard, might contain substances that bypass the adenylyl cyclase pathway and, thereby, facilitate perithecia formation.
The observation that the mutant produces conidia during epiphytic growth on detached wheat floral leaves and on solid substrates like CM, but not in submersed culture, substantiates the hypothesis of an influence of nutrient sensing, since also conidia production is nutrient-dependent [53]. Interestingly, adenylyl cyclase knock-out mutants of N. Also, a high abundance of hyphal fusion events anastomoses is regarded as an indicator for nutrient starvation conditions see [55] and references therein.
Hyphal fusions are believed to facilitate genetic exchange in a parasexual cycle, but can also be used to build up a hyphal network in order to acquire resources reviewed in [56]. In the present study, we were able to shade some light on the complex regulation pattern that involves hyphal differentiation, DON production and virulence. Table S2 provides an overview on phenotypes observed after deletion of adenylyl cyclases in other phytopathogenic fungi.
This compendium underscores the diverse role of cAMP in plant pathogens. While in F. Similarly, also other developmental processes like sexual and asexual propagation and secondary metabolism are affected to varying extents by cAMP depletion in different plant pathogens.
In this study, it became obvious that cAMP is an essential second messenger for proper development of F. Furthermore, we provide strong indications pointing towards a novel functional dependence of compound appressoria formation and wheat epidermis penetration by F. The results suggest that, at least on wheat, elevated cAMP levels favour pathogenic development in terms of infection structures and DON biosynthesis, while depletion of cAMP promotes conidiation and filamentous growth.
Moreover, cAMP plays a role in the transduction of environmental cues, since sexual reproduction and conidiation phenotypes depend on growth conditions. Cyclic AMP is dispensable for maize infection. Here, other as-yet unknown factors support pathogenicity. Replacement and Southern hybridization strategy for Fgac1. Deletion of Fgac1 2 by homologous recombination using a replacement fragment excised from pRSdelta Fgac1 using restriction enzyme Bam HI and Kpn I 1 3: genotype of disrupted strains.
Flanking regions are indicated as bold black lines. The gene flanks were fused to a hygromycin resistance cassette, consisting of the resistance gene hygromycin B phosphotransferase, hph , the gpdA promoter P -gpdA , and trpC terminator T trpC of A. Primer binding sites for PCR are indicated as small arrows numbering refers to table S1. The regions used as probes for Southern analysis is represented by the dashed line.
Scheme not to scale. DNA of the mutant and wild type strain was digested using Hin dIII for probe 1 and Dra I for probe 2 , separated on agarose gels, blotted on membranes and probed with a DIG-labelled probe for a fragment of the flanking region of Fgac1 probe 1 and for a gene-internal fragment of Fgac1 probe 2. Probe 1 hybridized with the DNA of the disruption mutant bps and the wild type bps.
Probe 2 only gave a signal in the wild type. Deletion of Fgac1 was verified in one mutant analyzed after single spore purification using primers 5 and 6. Agar plates were inoculated with mycelial plugs from 3-day-old cultures.
Conidia production assay. The assay was performed with three replicates each. Ascospore viability assay. Ascospores that land on the agar gave rise to new colonies indicating that they were viable. Assay for necrotic lesions development. The wild type evokes necrotic lesions after 5 days postinoculation dpi. Infection assay on dissected wheat paleas. CLSM micrographs of cross-sections at 10 days postinoculation dpi.
Summary of phenotypes of adenylyl cyclase mutants in other plant fungal pathogens. We thank B. Hadeler and C. Doormann for critical reading of the manuscript. We also thank E. Woelken, K. Dehn, R. Walter, and Dr. Friedrich for technical support with electron microscopy. Furthermore, we are grateful to Prof. Voigt for providing the LSM microscope used in this study.
Wrote the paper: JB WS. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Introduction Fusarium graminearum is the main causal agent of Fusarium head blight FHB disease of small grain cereals like wheat Triticum aestivum and barley Hordeum vulgare and of ear rot of maize.
Transformation of F. Results Generation of Mutants A comparative database survey using known orthologues of adenylyl cyclases led to the identification of the putative F. Download: PPT. Figure 1. Figure 2. DON concentrations in wheat heads and under in-vitro induction conditions. The F. Figure 3. Perithecia development on detached wheat nodes A and carrot agar B. Discussion The entire life cycle of F.
Disruption of cAMP Signaling Interferes with Sexual Reproduction, Conidiation and Hyphal Fusion The lack of mature perithecia on wheat straw might be due to a dysfunctional sensing of the environment by the deletion mutant. Figure 9. Proposed model: how FgAC1 influence numerous physiological functions like vegetative growth, sexual reproduction, DON biosynthesis, conidiation, and pathogenicity in F.
Supporting Information. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Table S1. Primers used in this study. Table S2. Acknowledgments We thank B. References 1. BMC Plant Biol View Article Google Scholar 2. Hanoune J, Defer N Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol — View Article Google Scholar 4. Biochem Soc Trans — View Article Google Scholar 5.
Arch Microbiol — View Article Google Scholar 6. J Biosci Bioeng — View Article Google Scholar 7. Microbiology — View Article Google Scholar 8.
Regulation of intracellular concentrations of cyclic AMP is largely a result in controlling adenylyl cyclase. Adenylyl cyclases are integral membrane proteins that consist of two bundles of six transmembrane segments.
Two catalytic domains extend as loops into the cytoplasm, as depicted in the figure to the right. A soluble non-membrane bound form of adenylyl cyclase has recently been characterized in mammalian sperm.
This form of the enzyme appears to be activated by bicarbonate ion. There are at least nine isoforms of adenylyl cyclase, discovered by cloning of full-length cDNAs. These enzymes differ considerably in regulatory properties and are differentially expressed among tissues, adding support to observations that support a very complex model of interactions that regulate cyclic AMP production.
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