Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, <Emphasis Type="Italic">AtSRK2C</Emphasis> and <Emphasis Type="Italic">AtGolS2</Emphasis>

Recent environmental issues have increased the demand for woody biomass as a renewable resource for industry and energy. For a stable supply of woody biomass, it is critical to decrease the effects of abiotic stresses, such as drought and salinity, which hinder plant growth. For the goal to develop practical stress-tolerant trees, we generated transgenic poplar plants (P. tremula × tremuloides), in which a key Arabidopsis regulatory factor involved in stress responses, SNF1-related protein kinase 2C (AtSRK2C), or galactinol synthase 2 (AtGolS2), was overexpressed. Both types of transgenic poplar plants displayed higher tolerance to abiotic stresses, in comparison with nontransgenic plants, indicating that AtSRK2C and AtGolS2 can function in the abiotic stress response pathway of poplar. We also examined the expression profiles of ten poplar genes putatively homologous to well-known Arabidopsis stress response genes and found that several of the poplar genes showed different responses to abiotic stress from their Arabidopsis counterparts. Whereas the overexpression of AtSRK2C in transgenic Arabidopsis plants was reported to upregulate the expression of endogenous genes, the overexpression of AtSRK2C or AtGolS2 in transgenic poplar did not. Taken together, our findings suggest that the details of the underlying molecular mechanisms of the abiotic stress response may differ, but that the key regulatory factors in Arabidopsis and poplar have common features and are effective molecular targets for further breeding to enhance abiotic stress tolerance in poplar.     

Mechanism of dimerization and structural features of human LI-cadherin

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca²⁺-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.     

A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.     

Improved performance of column chromatography by arginine: dye-affinity chromatography

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.     

Ⅲ型效应子GALA对青枯菌在两种寄主植物上致病性的影响

[目的]研究Ⅲ型效应子GALAs对青枯菌OE1-1在不同寄主植物致病性上的影响。[方法]构建青枯菌OE1-1的多种GALA缺失突变体,通过根切和叶片注射等方法研究GALAs对青枯菌OE1-1致病力和细胞内增殖能力的影响。[结果]GALA多基因缺失突变体对寄主烟草的致病力减弱,在烟草体内细菌繁殖能力较野生型明显降低,但在寄主番茄上不影响其致病性。[结论]GALA效应子对青枯菌OE1-1在烟草植株致病性上展现协同作用。     

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.     

A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties

The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane.