Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions.

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions. mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure.     

A new alkalitolerant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast strain is a promising model for dissecting properties and regulation of Na<Superscript>+</Superscript>-dependent phosphate transport systems

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na⁺-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na⁺-dependent systems specifically activated by Na⁺. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na⁺/P_i-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH 9.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1607–1615.Original Russian Text Copyright © 2004 by Zvyagilskaya, Persson.     

水稻细胞质雄性不育系与保持系的呼吸途径比较

比较水稻细胞质雄性不育系珍汕97A及其保持系珍汕97B之幼穗、叶片和花药的呼吸代谢。结果表明：不育系花药的总呼吸速率、抗氰呼吸所占总呼吸比例和细胞色素氧化酶（COD）、苹果酸脱氢酶（MDH）和6－磷酸葡萄糖脱氢酶（6－PGDH）活性低于保持系;幼穗中则仅6－PGDH低于保持系;叶片间各指标均无差异。Na3PO4可抑制保持系的叶片和幼穗及不育系叶片总呼吸的30％,而仅能抑制不育系幼穗总呼吸的24％;丙二酸则抑制不育系与保持系的叶片和幼穗的总呼吸的70％。     

The impacts of cytoplasmic incompatibility factor (cifA and cifB) genetic variation on phenotypes

Wolbachia are maternally transmitted, intracellular bacteria that can often selfishly spread through arthropod populations via cytoplasmic incompatibility (CI). CI manifests as embryonic death when males expressing prophage WO genes cifA and cifB mate with uninfected females or females harboring an incompatible Wolbachia strain. Females with a compatible cifA-expressing strain rescue CI. Thus, cif-mediated CI confers a relative fitness advantage to females transmitting Wolbachia. However, whether cif sequence variation underpins incompatibilities between Wolbachia strains and variation in CI penetrance remains unknown. Here, we engineer Drosophila melanogaster to transgenically express cognate and non-cognate cif homologs and assess their CI and rescue capability. Cognate expression revealed that cifA;B native to D. melanogaster causes strong CI, and cognate cifA;B homologs from two other Drosophila-associated Wolbachia cause weak transgenic CI, including the first demonstration of phylogenetic type 2 cifA;B CI. Intriguingly, non-cognate expression of cifA and cifB alleles from different strains revealed that cifA homologs generally contribute to strong transgenic CI and interchangeable rescue despite their evolutionary divergence, and cifB genetic divergence contributes to weak or no transgenic CI. Finally, we find that a type 1 cifA can rescue CI caused by a genetically divergent type 2 cifA;B in a manner consistent with unidirectional incompatibility. By genetically dissecting individual CI functions for type 1 and 2 cifA and cifB, this work illuminates new relationships between cif genotype and CI phenotype. We discuss the relevance of these findings to CI’s genetic basis, phenotypic variation patterns, and mechanism.     

BRASSINOSTEROID-SIGNALLING KINASES 7 and 8 associate with the FLS2 immune receptor and are required for flg22-induced PTI responses

Pattern-triggered immunity (PTI) is typically initiated in plants by recognition of pathogen- or damage-associated molecular patterns (PAMP/DAMPs) by cell surface-localized pattern recognition receptors (PRRs). Here, we investigated the role in PTI of Arabidopsis thaliana brassinosteroid-signalling kinases 7 and 8 (BSK7 and BSK8), which are members of the receptor-like cytoplasmic kinase subfamily XII. BSK7 and BSK8 localized to the plant cell periphery and interacted in yeast and in planta with FLS2, but not with other PRRs. Consistent with a role in FLS2 signalling, bsk7 and bsk8 single and bsk7,8 double mutant plants were impaired in several immune responses induced by flg22, but not by other PAMP/DAMPs. These included resistance to Pseudomonas syringae and Botrytis cinerea, reactive oxygen species accumulation, callose deposition at the cell wall, and expression of the defence-related gene PR1, but not activation of MAP kinases and expression of the FRK1 and WRKY29 genes. bsk7, bsk8, and bsk7,8 plants also displayed enhanced susceptibility to P. syringae and B. cinerea. Finally, BSK7 and BSK8 variants mutated in their myristoylation site or in the ATP-binding site failed to complement defective phenotypes of the corresponding mutants, suggesting that localization to the cell periphery and kinase activity are critical for BSK7 and BSK8 functions. Together, these findings demonstrate that BSK7 and BSK8 play a role in PTI initiated by recognition of flg22 by interacting with the FLS2 immune receptor.     

Wild soybean SNARE proteins BET1s mediate the subcellular localization of the cytoplasmic receptor-like kinases CRCK1s to modulate salt stress responses

Plants have evolved numerous receptor-like kinases (RLKs) that modulate environmental stress responses. However, little is known regarding soybean (Glycine max) RLKs. We have previously identified that Glycine soja Ca²⁺/CAM-binding RLK (GsCBRLK) is involved in salt tolerance. Here, we report that soluble NSF attachment protein receptor proteins BET1s mediate subcellular localization of calmodulin-binding receptor-like cytoplasmic kinases CRCK1s to modulate salt stress responses. Direct interaction between GsCBRLK and GsBET11a was initially identified via yeast two-hybrid and bimolecular fluorescence complementation assays. Further analysis demonstrated conserved interaction between BET1s and CRCK1s. GsCBRLK interacted with all BET1 proteins in wild soybean (Glycine soja) and Arabidopsis, and GsBET11a strongly associated with GsCRCK1a–1d, but slightly with AtCRCK1. In addition, GsBET11a interacted with GsCBRLK via its C-terminal transmembrane domain (TMD), where the entire TMD, not the sequence, was critical for the interaction. Moreover, the N-terminal variable domain (VD) of GsCBRLK was responsible for interacting with GsBET11a, and the intensity of interaction between GsCBRLK/AtCRCK1 and GsBET11a was dependent on VD. Furthermore, GsBET11a was able to mediate the GsCBRLK subcellular localization via direct interaction with VD. Additionally, knockout of AtBET11 or AtBET12 individually did not alter GsCBRLK localization, while GsBET11a expression caused partial internalization of GsCBRLK from the plasma membrane (PM). We further suggest the necessity of GsCBRLK VD for its PM localization via N-terminal truncation assays. Finally, GsBET11a was shown to confer enhanced salt stress tolerance when overexpressed in Arabidopsis and soybean. These results revealed the conserved and direct interaction between BET1s and CRCK1s, and suggested their involvement in salt stress responses.     

Evolution of Wolbachia cytoplasmic incompatibility types

The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects.     

玉米新选细胞质雄性不育系小孢子发育的细胞学观察及DNA甲基化分析

细胞质雄性不育在高等植物中普遍存在,是杂种优势利用的重要工具,为推动植物杂种优势的利用发挥了重要作用。文章以本课题组前期新选的玉米细胞质雄性不育系A1、A2及保持系18(红)为材料,利用石蜡切片技术对不育材料小孢子发育过程进行细胞学观察,采用高效液相色谱法(HPLC)对不同发育时期的叶片及不同发育时期的雄穗DNA进行甲基化分析,从细胞学和表观遗传学角度了解不育系A1、A2的败育机制。结果表明：不育材料A1、A2小孢子发生败育的主要时期为四分体时期至单核小孢子中期。在不育系A2中还存在另一种败育方式,即在花粉母细胞时期表现出败育特征。甲基化分析结果表明,保持系18(红)的叶片DNA甲基化水平从苗期到拔节期迅速上升,而不育系A1、A2叶片DNA甲基化水平基本保持不变;保持系雄穗DNA甲基化水平表现为从花粉母细胞时期到双核期逐渐升高,而不育材料A1、A2从花粉母细胞时期到双核期的雄穗DNA甲基化水平表现为先上升后下降的趋势,达到最高峰的时期均出现在小孢子发育的四分体时期。从小孢子发育的细胞学观察结果可以发现,小孢子败育的主要时期往往具有较高的甲基化水平,推测DNA甲基化水平变化可能与不育材料A1、A2的花粉败育有关。