In vitro analysis of His-Asp phosphorelays in Aspergillus nidulans: the first direct biochemical evidence for the existence of His-Asp phosphotransfer systems in filamentous fungi

His-Asp phosphorelays are widespread signal transduction mechanisms in bacteria, fungi, and higher plants. In order to investigate a His-Asp phosphorelay network in filamentous fungi, which has been genetically characterized in part, we attempted to construct an in vitro phosphotransfer network in Aspergillus nidulans comprising all the necessary components. As a first step, we established an in vitro phosphotransfer system with a histidine-containing phosphotransmitter YpdA, a response regulator SrrA, and a bacterial histidine kinase ArcB as a phosphate donor. We demonstrated the phosphotransfer from ArcB to A. nidulans YpdA and the subsequent transfer from YpdA to SrrA. This is the first direct biochemical evidence for the presence of the phosphotransfer system in filamentous fungi. Furthermore, a retrograde phosphorylation from YpdA to FphA, a histidine kinase similar to bacterial phytochrome, was found. The overall picture of the His-Asp phosphorelays in A. nidulans is discussed based on the results of the in vitro study.     

The SskA and SrrA response regulators are implicated in oxidative stress responses of hyphae and asexual spores in the phosphorelay signaling network of Aspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated DeltasskA and DeltasrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from DeltasskA were hypersensitive not only to treatment with H(2)O(2), but also to treatment at aberrantly low (4 degrees C) and high (50 degrees C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from DeltasskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.     

Histidine-containing phosphotransfer (HPt) signal transducers implicated in His-to-Asp phosphorelay in Arabidopsis

His to Asp phosphorelay signal transduction mechanisms involve three types of widespread signaling components: a sensor His-kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) domain. In Arabidopsis, several sensor His-kinases have recently been discovered (e.g., ETR1 and CKI1) through extensive genetic studies. Furthermore, a recent search for response regulators in this higher plant revealed that it possesses a group of response regulators (ARR-series), each of which exhibits the phospho-accepting receiver function. However, no signal transducer containing the HPt domain has been reported. Here we identify three distinct Arabidopsis genes (AHP1 to AHP3), each encoding a signal transducer containing a HPt domain. Both in vivo and in vitro evidence that each AHP can function as a phospho-transmitting HPt domain with an active histidine site was obtained by employing both the Escherichia coli and yeast His-Asp phosphorelay systems. It was demonstrated that AHP1 exhibits in vivo ability to complement a mutational lesion of the yeast YPD1 gene, encoding a typical HPt domain involved in an osmosensing signal transduction. It was also demonstrated that AHPs can interact in vitro with ARRs through the His-Asp phosphotransfer reaction. It was thus suggested that the uncovered sensors-AHPs-ARRs lineups may play important roles in propagating environmental stimuli through the multistep His-Asp phosphorelay in Arabidopsis.     

Characterization of NikA Histidine Kinase and Two Response Regulators with Special Reference to Osmotic Adaptation and Asexual Development in Aspergillus nidulans

Aspergillus nidulans has many histidine-to-aspartate (His-Asp) phosphorelay components, including 15 histidine kinases (HKs), four response regulators (RRs), and a histidine-containing phosphotransfer intermediate (HPt). Of these, NikA (HK) is highly conserved in many filamentous fungi. It has been found that NikA is responsible for the responses of filamentous fungi to fungicides such as iprodione and fludioxonil. Two RRs, SskA and SrrA, are also involved in the fungicide response, providing a typical example of the His-Asp phosphorelay system, in which NikA functions as a sensor upstream of SskA and SrrA in response to fungicides. To gain further insight into the physiological roles of the NikA-SskA/SrrA phosphorelay system, we constructed a pair of ΔnikAΔsskA and ΔnikAΔsrrA double mutants. Here we provide evidence regarding the crucial involvement of the NikA-SskA/SrrA phosphorelay system in both osmotic adaptation and asexual development, including conidia formation. Based on these results, a general insight into the A. nidulans His-Asp phosphorelay network is also discussed.     

GFP-tagged expression analysis revealed that some histidine kinases of Aspergillus nidulans show temporally and spatially different expression during the life cycle

His-Asp phosphorelays are signal transduction mechanisms widely found in both prokaryotes and eukaryotes. The phosphorelay comprises three types of signal transducers: a sensor with histidine kinase (HK), a response regulator containing a phospho-accepting receiver (RR), and a histidine-containing phosphotransmitter (HPt). In this study, we examined HK expression using a green fluorescent protein (GFP) reporter driven by HK promoters in Aspergillus nidulans. All the transformants showed fluorescence derived from GFP in a submerged culture, although some of them were very weak, indicating that all the promoters were active. As judged by the fluorescence of transformants grown on a culture plate on which sexual development was induced, promoters of fphA, hk-8-2, and hk-8-5 preferentially functioned in conidial heads, the promoter of phkA preferentially functioned in cleistothecia, and the promoters of tcsB and nikA function in both conidial heads and cleistothecia. These results indicate that at least some HKs of A. nidulans showed temporally and spatially different expression during the cell cycle.     

Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia.

Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. The His-Asp phosphorelay components include "sensor histidine kinase (HK)", "phosphotransfer intermediate (HPt)", and "response regulator (RR)". With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay. It was revealed that M. loti has 47 HKs, 1 HPts, and 58 RRs; B. japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S. meliloti has 40 HKs, 1 HPt, and 58 RRs. These His-Asp phosphorelay components were extensively compiled and characterized. The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation.     

The RcsCB His-Asp phosphorelay system is essential to overcome chlorpromazine-induced stress in Escherichia coli

The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.     

Response regulators SrrA and SskA are central components of a phosphorelay system involved in stress signal transduction and asexual sporulation in Aspergillus nidulans

Among eukaryotes, only slime molds, fungi, and plants contain signal transduction phosphorelay systems. In filamentous fungi, multiple sensor kinases appear to use a single histidine-containing phosphotransfer (HPt) protein to relay signals to two response regulators (RR). In Aspergillus nidulans, the RR SskA mediates activation of the mitogen-activated protein kinase SakA in response to osmotic and oxidative stress, whereas the functions of the RR SrrA were unknown. We used a genetic approach to characterize the srrA gene as a new member of the skn7/prr1 family and to analyze the roles of SrrA in the phosphorelay system composed of the RR SskA, the HPt protein YpdA, and the sensor kinase NikA. While mutants lacking the HPt protein YpdA are unviable, mutants lacking SskA (DeltasskA), SrrA (DeltasrrA), or both RR (DeltasrrA DeltasskA) are viable and differentially affected in osmotic and oxidative stress responses. Both RR are involved in osmostress resistance, but DeltasskA mutants are more sensitive to this stress, and only SrrA is required for H(2)O(2) resistance and H(2)O(2)-mediated induction of catalase CatB. In contrast, both RR are individually required for fungicide sensitivity and calcofluor resistance and for normal sporulation and conidiospore viability. The DeltasrrA and DeltasskA sporulation defects appear to be related to decreased mRNA levels of the key sporulation gene brlA. In contrast, conidiospore viability defects do not correlate with the activity of the spore-specific catalase CatA. Our results support a model in which NikA acts upstream of SrrA and SskA to transmit fungicide signals and to regulate asexual sporulation and conidiospore viability. In contrast, NikA appears dispensable for osmotic and oxidative stress signaling. These results highlight important differences in stress signal transmission among fungi and define a phosphorelay system involved in oxidative and osmotic stress, cell wall maintenance, fungicide sensitivity, asexual reproduction, and spore viability.     

Biochemical characterization of a putative cytokinin-responsive His-kinase, CKI1, from Arabidopsis thaliana

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". Results from recent intensive studies suggested that in the higher plant Arabidopsis thaliana, His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we characterized, in vitro, the putative cytokinin-responsive CKI1 His-kinase, in terms of His-Asp phosphorelays. It was demonstrated for the first time that the receiver domain in this sensor exhibits a strong phosphohistidine phosphatase activity toward some Arabidopsis HPt phosphotransmitters (AHP1 and AHP2), suggesting the functional importance of the receiver domain for a resumed interaction of the sensor His-kinase with other His-Asp phosphorelay components.     

Characterization of the NikA histidine kinase implicated in the phosphorelay signal transduction of Aspergillus nidulans, with special reference to fungicide responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His) 蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径。在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统。最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用。     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.     

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.     

A new class of signal transducer in His-Asp phosphorelay systems

Nitrate transport activity of the LtnT permease of the cyanobacterium Synechococcus elongatus is activated when LtnA, a response regulator without an effector domain, is phosphorylated by LtnB, a hybrid histidine kinase. We identified a protein (LtnC) that is required for activation of LtnT. LtnC consists of an N-terminal histidine-containing phosphoacceptor (HisKA) domain, a receiver domain, and a unique C-terminal domain found in some cyanobacterial proteins. Because LtnC lacks an ATP-binding kinase domain of a histidine kinase, it is incapable of autophosphorylation, but LtnC is phosphorylated by LtnA. The histidine residue in the HisKA domain but not the aspartate residue in the receiver domain is essential for phosphorylation of LtnC and activation of LtnT. LtnC phosphorylation leads to oligomerization of the protein. Fusion of the C-terminal domain of LtnC to glutathione S-transferase, which forms oligomers, also activates LtnT, suggesting that oligomerization of the LtnC C-terminal domain causes LtnT activation. These results indicate that the C-terminal domain of LtnC acts as an effector domain that directs the output of the signal from the phosphorelay system. The two-step (His-Asp-His) phosphorelay system, composed of the LtnB, LtnA, and LtnC proteins, is distinct from the known phosphorelay systems, namely, the typical two-component system (His-Asp) and the multistep phosphorelay system (His-Asp-His-Asp), because the HisKA domain of LtnC is the terminal phosphoacceptor that determines the signal output. LtnC is a new class of signal transducer in His-Asp phosphorelay systems that contains a HisKA domain and an effector domain.     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His)蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径.在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统.最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用.     

Cytokinins: metabolism and function in plant adaptation to environmental stresses

In plants, the cytokinin (CK) phytohormones regulate numerous biological processes, including responses to environmental stresses, via a complex network of CK signaling. By an unknown mechanism, stress signals are perceived and transmitted through the His-Asp phosphorelay, an important component of the CK signal transduction pathway, triggering CK-responsive genes. Because of the intensive crosstalk between CKs and abscisic acid (ABA), modulation of CK levels and their signal transduction affects both ABA-dependent and ABA-independent pathways, enabling plant adaptation to adverse conditions. This review presents our current understanding of the functions of CKs and CK signaling in the regulation of plant adaptation to stress. Biotechnological strategies based on the modulation of CK levels have been examined with the aim of stabilizing agriculture yields.     

His-Asp phosphorelay signaling: a communication avenue between plants and their environment

His-Asp phosphorelay systems have been recently discovered in plants and have emerged as some of the most important signaling systems. The phosphorelay systems in plants include components with sensor (His-protein kinase) domains, His-containing phosphotransfer (HPt) domains, and receiver (response regulator) domains. Recent studies implicate phosphorelay systems in sensing and propagating signals from a wide variety of external and/or internal stimuli such as ethylene, cytokinin, and osmolarity. In maize and Arabidopsis, some response regulators are up-regulated by both cytokinins and nitrate. These findings imply that the His-Asp phosphorelay may operate in an inorganic nitrogen-signaling pathway mediated by cytokinin in plants.