Biomedical subjects

Yuhong Yang

Publications and source records attributed to Yuhong Yang.

13 recordsLinked to original sources

PubMed2006

Transcriptional activator TSRF1 reversely regulates pathogen resistance and osmotic stress tolerance in tobacco.

Increasing evidences show that ethylene-responsive factor (ERF) proteins regulate plant stress response and the interaction of different stress responsive pathways through interacting with different cis-acting elements, even other transcription factors. Here, we report a transcriptional activator TSRF1, which was previously demonstrated to regulate plant resistance to Ralstonia solanacearum, reversely regulates pathogen resistance and osmotic stress tolerance in tobacco. Sequence analysis revealed that TSRF1 contains a putative transcriptional activation domain. Using yeast two hybrid system we evidenced that this activation domain is essential for activating the expression of reporter gene. To confirm the broad-spectrum pathogen resistance of TSRF1 we observed that over-expressing TSRF1 enhances the resistance to Pseudomonas syringae and Botrytis cinerea in both tobacco and tomato plants, but RNA interference of TSRF1 in tomato plants decreases the resistance to these pathogens, unraveling the positive regulation of TSRF1 in plant pathogen infections. The expression of TSRF1 in response to NaCl and mannitol suggests the possible functions of TSRF1 in osmotic stress responses, but the physiological tests indicate that expressing TSRF1 in tobaccos decreases tolerance to NaCl or mannitol during germination and seedling root development, and this result was consistent with PEG6000 treatment with mature tobacco seedlings, indicating the negative modulation of TSRF1 in osmotic stress response. Therefore, our research reveals that transcriptional activator TSRF1 reversely regulates plant pathogen resistance and osmotic stress response.

Amino Acid Sequence↗

PubMed2006

CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop.

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. In this study, we show that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1a(L) strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbt(L) mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ~32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1a(L) strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1a(L) strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the cka(RIP) strain, which carries the disruption of the catalytic subunit of casein kinase II. In the cka(RIP) strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop.

Amino Acid Sequence↗

PubMed2004

Combining nearest neighbor classifiers versus cross-validation selection.

Various discriminant methods have been applied for classification of tumors based on gene expression profiles, among which the nearest neighbor (NN) method has been reported to perform relatively well. Usually cross-validation (CV) is used to select the neighbor size as well as the number of variables for the NN method. However, CV can perform poorly when there is considerable uncertainty in choosing the best candidate classifier. As an alternative to selecting a single "winner,'' we propose a weighting method to combine the multiple NN rules. Four gene expression data sets are used to compare its performance with CV methods. The results show that when the CV selection is unstable, the combined classifier performs much better.

Journal Article↗

PubMed2004

Distinct roles for PP1 and PP2A in the Neurospora circadian clock.

Phosphorylation of the Neurospora circadian clock protein FREQUENCY by several kinases promotes its degradation and is important for the function of the circadian feedback loop. Here, we show that FRQ is less stable in a ppp-1 (catalytic subunit of PP1) mutant, resulting in its advanced phase and short period. In contrast, FRQ stability is not altered in a rgb-1 (a regulatory subunit of PP2A) mutant, but levels of frq protein and mRNA are low, resulting in a low-amplitude and long-period oscillation of the clock. Furthermore, PP1 and PP2A expressed in Neurospora can dephosphorylate the endogenous FRQ in vitro, suggesting that these two phosphatases may differentially regulate FRQ and, consequently, the behavior of the circadian clock.

Catalytic Domain↗

PubMed2004

Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum.

Ethylene responsive factors (ERFs) are important in regulating plant pathogen resistance, abiotic stress tolerance and plant development. Recent studies have greatly enlarged the ERF protein family and revealed more important roles of ERFs in plants. Here, we report our finding of a tomato ERF protein TSRF1, which is transcriptionally up-regulated by ethylene, salicylic acid, or Ralstonia solanacearum strain BJ1057 infection. Biochemical analysis indicates that TSRF1 specifically interacts in vitro with the GCC box, an element present in the promoters of many pathogenesis-related (PR) genes. Further investigation evidences that TSRF1 activates in vivo the expression of reporter beta-glucuronidase gene controlled by GCC box. More importantly, overexpressing TSRF1 in tobacco and tomato constitutively activates the expression of PR genes, and subsequently enhancing transgenic plant resistance to the bacterial wilt caused by Ralstonia solanacearum strain BJ1057. Therefore our investigation not only extends the functions of ERF proteins in plant resistance to R. solanacearum, but also provides further clues to understanding the mechanism of host regulatory proteins in response to the infection of pathogens.

Amino Acid Sequence↗

PubMed2003

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.

Amino Acid Sequence↗

PubMed2003

Functional conservation of light, oxygen, or voltage domains in light sensing.

In Neurospora, the flavin adenine dinucleotide-containing protein WHITE COLLAR-1 is the blue-light photoreceptor for the circadian clock and other light responses. The putative chromophore-binding domain of WC-1, its light, oxygen, or voltage (LOV) domain, is similar to the LOV domains found in the plant phototropins, the Neurospora VIVID (VVD) protein, and the Arabidopsis FKF1 and its related proteins. Studies of the plant phototropins have identified 11 flavin-contacting residues that are also conserved in the LOV domains of WC-1, VVD, and FKF1. In this study, by mutating the putative WC-1 flavin-binding sites, we show that these sites are important for the light function of the protein, suggesting that the WC-1 LOV domain adapts a structure similar to that of the phototropin LOV domains. By creating a Neurospora strain in which the LOV domain of WC-1 is swapped with that of VVD, we show that the LOV domain of VVD partially replaces the function of the WC-1 LOV domain, suggesting that VVD is a wc-dependent photoreceptor in Neurospora. Furthermore, we show that the Neurosporastrains containing a chimeric WC-1 protein with the LOV domain from FKF1 or phot1 can also sense light, suggesting that FKF1 and its related proteins are light sensors in Arabidopsis. Taken together, our data suggest that these LOV domains are structurally similar protein modules involved in blue-light sensing.

Amino Acid Sequence↗

PubMed2003

Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock.

FREQUENCY (FRQ), a key component of the Neurospora circadian clock, is progressively phosphorylated after its synthesis. Previously, we identified casein kinase II (CKII) as a kinase that phosphorylates FRQ. Disruption of the catalytic subunit of CKII abolishes the clock function; it also causes severe defects in growth and development. To further establish the role of CKII in clock function, one of the CKII regulatory subunit genes, ckb1, was disrupted in Neurospora. In the ckb1 mutant strain, FRQ proteins are hypophosphorylated and more stable than in the wild-type strain, and circadian rhythms of conidiation and FRQ protein oscillation were observed to have long periods but low amplitudes. These data suggest that phosphorylation of FRQ by CKII regulates FRQ stability and the function of the circadian feedback loop. In addition, mutations of several putative CKII phosphorylation sites of FRQ led to hypophosphorylation of FRQ and long-period rhythms. Both CKA and CKB1 proteins are found in the cytoplasm and in the nucleus, but their expressions and localization are not controlled by the clock. Finally, disruption of a Neurospora casein kinase I (CKI) gene, ck-1b, showed that it is not required for clock function despite its important role in growth and developmental processes. Together, these data indicate that CKII is an important component of the Neurospora circadian clock.

Biological Clocks↗

PubMed2002

WHITE COLLAR-1, a multifunctional neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2.

WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors, are the positive elements of the circadian feedback loops in Neurospora. In addition, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. Recently, we identified WC-1 as the blue light photoreceptor responsible for these light responses. In this study, we show that the formation of the FRQ-WC complex in vivo, a step critical in closing the circadian negative feedback loop, requires WC-1. In addition, we show that WC-1 negatively regulates the expression of wc-2 at the level of the transcription, forming another interacting loop. In a wc-1 mutant, we demonstrate that there is alternative protein initiation of WC-1, and the requirements of WC-1 for the light induction of frq and other genes differ significantly, suggesting the existence of different WC complexes in the cell. Consistent with this interpretation, our results show that there are at least two different types of WC-1/WC-2 complexes in vivo, and that the larger WC-1/WC-2 complex contains more than one WC-1 molecule. Using a series of wc-1 mutants, we show that the WC-1 PASC domain and its C-terminal region are essential for the formation of the WC-1/WC-2 complex. Functional analyses reveal that the DNA-binding domain of WC-1 is required only for the activation of frq in the dark and not for the light function of the protein, confirming that WC-1 is a multifunctional protein with separable protein domains.

Circadian Rhythm↗

PubMed2002

White collar-1, a DNA binding transcription factor and a light sensor.

Blue light regulates many physiological processes in fungi, but their photoreceptors are not known. In Neurospora crassa, all light responses depend on the Per-Arnt-Sim (PAS) domain-containing transcription factor white collar-1 (wc-1). By removing the WC-1 light, oxygen, or voltage domain, a specialized PAS domain that binds flavin mononucleotide in plant phototropins, we show that light responses are abolished, including light entrainment of the circadian clock. However, the WC-1-mediated dark activation of frq remains normal in this mutant, and the circadian clock can be entrained by temperature. Furthermore, we demonstrate that the purified Neurospora WC-1-WC-2 protein complex is associated with stoichiometric amounts of the chromophore flavin-adenine dinucleotide. Together, these observations suggest that WC-1 is the blue-light photoreceptor for the circadian clock and other light responses in Neurospora.

Amino Acid Sequence↗

PubMed2002

Regulation of the Neurospora circadian clock by casein kinase II.

Phosphorylation of clock proteins represents an important mechanism regulating circadian clocks. In Neurospora, clock protein FREQUENCY (FRQ) is progressively phosphorylated over time, and its level decreases when it is extensively phosphorylated. To identify the kinase phosphorylating FRQ and to understand the function of FRQ phosphorylation, a FRQ-phosphorylating kinase was purified and identified as casein kinase II (CKII). Disruption of the catalytic subunit gene of CKII in Neurospora resulted in hypophosphorylation and increased levels of FRQ protein. In addition, the circadian rhythms of frq RNA, FRQ protein, and clock-controlled genes are abolished in the CKII mutant. Our data suggest that the phosphorylation of FRQ by CKII may have at least three functions; it decreases the stability of FRQ, reduces the protein complex formation between FRQ and the WHITE COLLAR proteins, and is important for the closing of the Neurospora circadian negative feedback loop. Taken together, our results suggest that CKII is an important component of the Neurospora circadian clock.

Biological Clocks↗

PubMed2002

PAS domain-mediated WC-1/WC-2 interaction is essential for maintaining the steady-state level of WC-1 and the function of both proteins in circadian clock and light responses of Neurospora.

In the frq-wc-based circadian feedback loops of Neurospora, two PAS domain-containing transcription factors, WHITE COLLAR-1 (WC-1) and WC-2, form heterodimeric complexes that activate the transcription of frequency (frq). FRQ serves two roles in these feedback loops: repressing its own transcription by interacting with the WC complex and positively upregulating the levels of WC-1 and WC-2 proteins. We report here that the steady-state level of WC-1 protein is independently regulated by both FRQ and WC-2 through different posttranscriptional mechanisms. The WC-1 level is extremely low in wc-2 knockout strains, and this low level of expression is independent of wc-1 transcription and FRQ protein expression. In addition, our data show that the PAS domain of WC-2 mediates the interactions of this protein with both WC-1 and FRQ in vivo. Such interactions are essential for maintaining the steady-state level of WC-1 and the proper function of WC-1 and WC-2 in circadian clock and light responses.

Circadian Rhythm↗

PubMedDate not supplied

Optical spectroscopy to investigate the structure of regenerated Bombyx mori silk fibroin in solution.

Fluorescence and circular dichroism spectroscopy were used to monitor the conformational transition of regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions under different conditions. According to the analysis of fluorescence spectra using anilinonaphthalene-8-sulfonic acid magnesium salt (ANS) as an external probe, the destruction of the hydrophobic core prior to the secondary structure change suggests that this collapse may initiate the conformational transition from random coil to beta-sheet for RSF. The temperature dependence of the structural changes of RSF, detected by both fluorescence spectroscopy and circular dichroism, shows a reversible process upon heating and recooling, with the midpoint around 45 degrees C. The results also indicate that most of the tryptophan (Trp) residues contained in silk fibroin are concentrated on the surface of the unfolded protein. However, they will change their location in the highly ordered structure (e.g., becoming more homogeneous) with the conformational transition of silk fibroin. Moreover, our studies also suggest that the presence of water plays a crucial role during the structure changes of fibroin.

Anilino Naphthalenesulfonates↗