Cloning and Characterization of the <Emphasis Type="Italic">BLR2</Emphasis>, the Homologue of the Blue-Light Regulator of <Emphasis Type="Italic">Neurospora crassa</Emphasis> WC-2, in the Phytopathogenic Fungus <Emphasis Type="Italic">Bipolaris oryzae</Emphasis>

Bipolaris oryzae is a filamentous ascomycetous fungus that causes brown leaf spot disease in rice. We isolated and characterized BLR2, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white collar-2 (WC-2). The deduced amino acid sequence of the BLR2 showed significant homology to other fungal blue-light regulator proteins in the Per-Arnt-Sim (PAS) protein–protein interaction domain, nuclear localization signal, and GATA zinc finger DNA-binding domains. The BLR2-silenced transformants hardly produced conidia in the subsequent dark condition after near-ultraviolet (NUV) irradiation. Furthermore, the BLR2-silenced transformants suppressed the photolyase (PHR1) gene expression enhanced by NUV irradiation. These results indicate that BLR2 is necessary not only for conidial formation, but also for NUV radiation-enhanced photolyase gene expression in B. oryzae. The DDBJ accession number for the sequence reported in this paper is AB282674.     

Trichoderma atroviride PHR1, a fungal photolyase responsible for DNA repair, autoregulates its own photoinduction

The photolyases, DNA repair enzymes that use visible and long-wavelength UV light to repair cyclobutane pyrimidine dimers (CPDs) created by short-wavelength UV, belong to the larger photolyase-cryptochrome gene family. Cryptochromes (UVA-blue light photoreceptors) lack repair activity, and sensory and regulatory roles have been defined for them in plants and animals. Evolutionary considerations indicate that cryptochromes diverged from CPD photolyases before the emergence of eukaryotes. In prokaryotes and lower eukaryotes, some photolyases might have photosensory functions. phr1 codes for a class I CPD photolyase in Trichoderma atroviride. phr1 is rapidly induced by blue and UVA light, and its photoinduction requires functional blue light regulator (BLR) proteins, which are White Collar homologs in Trichoderma. Here we show that deletion of phr1 abolished photoreactivation of UVC (200 to 280 nm)-inhibited spores and thus that PHR1 is the main component of the photorepair system. The 2-kb 5' upstream region of phr1, with putative light-regulated elements, confers blue light regulation on a reporter gene. To assess phr1 photosensory function, fluence response curves of this light-regulated promoter were tested in null mutant (Deltaphr1) strains. Photoinduction of the phr1 promoter in Deltaphr1 strains was >5-fold more sensitive to light than that in the wild type, whereas in PHR1-overexpressing lines the sensitivity to light increased about 2-fold. Our data suggest that PHR1 may regulate its expression in a light-dependent manner, perhaps through negative modulation of the BLR proteins. This is the first evidence for a regulatory role of photolyase, a role usually attributed to cryptochromes.     

Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3

Species of the mycoparasitic fungal genus Hypocrea/Trichoderma are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridis peptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridis showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1 (regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Galpha-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridis occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.     

Redox and light regulation of gene expression in photosynthetic prokaryotes

All photosynthetic organisms control expression of photosynthesis genes in response to alterations in light intensity as well as to changes in cellular redox potential. Light regulation in plants involves a well-defined set of red- and blue-light absorbing photoreceptors called phytochrome and cryptochrome. Less understood are the factors that control synthesis of the plant photosystem in response to changes in cellular redox. Among a diverse set of photosynthetic bacteria the best understood regulatory systems are those synthesized by the photosynthetic bacterium Rhodobacter capsulatus. This species uses the global two-component signal transduction cascade, RegB and RegA, to anaerobically de-repress anaerobic gene expression. Under reducing conditions, the phosphate on RegB is transferred to RegA, which then activates genes involved in photosynthesis, nitrogen fixation, carbon fixation, respiration and electron transport. In the presence of oxygen, there is a second regulator known as CrtJ, which is responsible for repressing photosynthesis gene expression. CrtJ responds to redox by forming an intramolecular disulphide bond under oxidizing, but not reducing, growth conditions. The presence of the disulphide bond stimulates DNA binding activity of the repressor. There is also a flavoprotein that functions as a blue-light absorbing anti-repressor of CrtJ in the related bacterial species Rhodobacter sphaeroides called AppA. AppA exhibits a novel long-lived photocycle that is initiated by blue-light absorption by the flavin. Once excited, AppA binds to CrtJ thereby inhibiting the repressor activity of CrtJ. Various mechanistic aspects of this photocycle will be discussed.     

Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media

Large-scale production of recombinant spider silk proteins is a long-term goal for their industrial use. Therefore, we have recently developed a process for bacterial production. Due to a highly repetitive gene sequence of spider silks, the host strain E. coli BLR(DE3) was employed since it shows no homologue recombination. Although perfectly suited for production in full media, the BLR strain does not grow in cost-effective minimal media, indicating a previously not reported L: -isoleucine auxotrophy. We provide evidence that mutated threonine deaminase is likely responsible for the detected auxotrophy of BLR.     

A MAPK-positive feedback mechanism for BLR1 signaling propels retinoic acid-triggered differentiation and cell cycle arrest

MAPK signaling is required for retinoic acid (RA)-triggered G(0) cell cycle arrest and cell differentiation, but the mechanism is not well defined. In this study, RA is found to cause MAPK activation with sustained association of RAF to MEK or ERK, leading to a MAPK-dependent accumulation of p21(Waf1/Cip1) and binding to CDK2 blocking G(1)/S transition. BLR1, a chemokine receptor, was found to function as a critical component of RA-triggered MAPK signaling. Unlike wild-type parental cells, RA-treated BLR1 knock-out cells failed to show RAF and consequential MEK and ERK phosphorylation, failed to accumulate CDK inhibitors that control G(1)/S transition, and failed to differentiate and arrest in response to RA, whereas ectopically overexpressing BLR1 enhanced MAPK signaling and caused accelerated RA-induced differentiation and arrest. Ectopic overexpression of RAF enhanced BLR1 expression in response to RA, whereas inhibition of RAF or MEK by inhibitors or knockdown of RAF by short interfering RNA diminished RA-induced BLR1 expression and attenuated differentiation and growth arrest. Ectopic expression of the RAF CR3, the catalytically active domain, in the BLR1 knock-out restored RA-induced MAPK activation and the ability to differentiate and arrest, indicating that RAF effects MAPK signaling by BLR1 to propel differentiation/arrest. Taken together, RA induces cell differentiation and growth arrest through activation of a novel MAPK pathway with BLR1 as a critical component in a positive feedback mechanism that may contribute to the prolonged MAPK signaling propelling RA-induced cell cycle arrest and differentiation.     

基因串联原核高效表达胸腺肽α1

人工合成含有SD序列、胸腺肽α1(Tα1)基因、纯化标签和酶切位点的DNA序列作为构建元件,利用同尾酶将其依次克隆到pET-32a(+)中,得到含有1-8个不同重复数目的含SD序列基因的串联表达载体,利用PCR初步鉴定和进一步的测序证实序列完全正确。进而利用lacZ基因作为指示基因,将带有SD序列的lacZ基因克隆到不同Tα1基因串数载体末尾,利用蓝白斑实验验证启动子后不同SD序列的效率,证实串联载体中的各个SD序列均能有效启动翻译。在此基础上对各串表达载体进行摇瓶发酵,SDS-PAGE电泳检测证实各串均可正确表达Tα1,且为可溶性表达。本文为小肽原核高效表达提出了新方法,有望成为小肽高效表达的新途径。     

Plant blue-light receptors

Plants have several blue-light receptors, which regulate different aspects of growth and development. Recent studies have identified three such receptors: cryptochrome 1, cryptochrome 2 and phototropin. Cryptochromes 1 and 2 are photolyase-like receptors that regulate hypocotyl growth and flowering time; phototropin mediates phototropism in response to blue light. In addition, phytochrome A has also been found to mediate various blue-light responses. Although the signal-transduction mechanisms of blue-light receptors remain largely unclear, phototropin is probably a protein kinase that regulates cytoplasmic calcium concentrations, whereas the cryptochromes might regulate anion-channel activity and changes in gene expression.     

不同波长光源对新蚜虫疠霉产孢能力的影响及霉菌蓝光受体基因的克隆和表达分析

光在调控真菌的多种生理过程中发挥着重要作用。为探究光照对新蚜虫疠霉Pandora neoaphidis产孢的影响,本文研究了不同波长光源(蓝光、绿光、白光、红光、黄光)和无光黑暗条件对新蚜虫疠霉分生孢子弹射能力的影响,通过cDNA末端的快速扩增(RACE)对新蚜虫疠霉的蓝光受体蛋白基因pnwc-1进行克隆并对其进行生物信息学分析,利用qRT-PCR对蓝光光源不同照射时长下pnwc-1的表达量进行了定量分析。结果表明,蓝色光源(波长460–465nm)照射后的新蚜虫疠霉菌丝产生的分生孢子数量显著高于其他波长光源,排序为:蓝光>绿光>白光>红光>黄光>无光。另外,分析克隆获得的全长为2 423bp的pnwc-1基因发现,其编码的蛋白具有蓝光受体蛋白典型的保守结构域,同源比对结果显示新蚜虫疠霉与接合菌门真菌归为一类但相对独立。qRT-PCR的定量分析结果表明随着照射时长增加,蓝光处理能显著提高pnwc-1的表达量,而且pnwc-1的相对表达量与累积产孢量存在正相关(R2=0.9798)。本研究为后续蓝光及其受体基因功能的深入研究提供了实验基础,并促进以新蚜虫疠霉为代表的虫霉目真菌在害虫生物防治中的应用。     

Cryptochrome 1 contributes to blue-light sensing in pea

Cryptochromes are widespread in higher plants but their physiological roles as blue-light photoreceptors have been examined in relatively few species. Screening in a phyA null mutant background has identified several blue-light response mutants in pea (Pisum sativum), including one that carries a substitution of a highly conserved glycine residue in the N-terminal photolyase-homologous domain of the pea CRY1 gene. Analyses of cry1, phyA, and phyB mutants show that all three photoreceptors contribute to seedling photomorphogenesis under high-irradiance blue light, whereas phyA is the main photoreceptor active under low irradiances. Triple phyA phyB cry1 mutants grown under high-irradiance blue light are indistinguishable from dark-grown wild-type plants in length and leaf expansion but show a small residual response to higher-irradiance white light. Monogenic cry1 mutants have little discernable phenotype at the seedling stage, but later in development are more elongated than wild-type plants. In addition, the loss of cry1 moderates the short-internode phenotype of older phyA mutants, suggesting an antagonism between phyA and cry1 under some conditions. Pea cry1 has a small inhibitory effect on flowering under long and short days. However, the phyA cry1 double mutant retains a clear promotion of flowering in response to blue-light photoperiod extensions, indicating a role for one or more additional blue-light photoreceptors in the control of flowering in pea.