The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.     

Host surface properties affect prepenetration processes in the barley powdery mildew fungus

The initial contact between Blumeria graminis f.sp. hordei and its host barley (Hordeum vulgare) takes place on epicuticular waxes at the surfaces of aerial plant organs. Here, the extent to which chemical composition, crystal structure and hydrophobicity of cuticular waxes affect fungal prepenetration processes was explored. The leaf surface properties of barley eceriferum (cer) wax mutants were characterized in detail. Barley leaves and artificial surfaces were used to investigate the early events of fungal infection. Even after epicuticular waxes had been stripped away, cer mutant leaf surfaces did not affect fungal prepenetration properties. Removal of total leaf cuticular waxes, however, resulted in a 20% reduction in conidial germination and differentiation. Two major components of barley leaf wax, hexacosanol and hexacosanal, differed considerably in their ability to effectively trigger conidial differentiation on glass surfaces. While hexacosanol, attaining a maximum hydrophobicity with contact angles of no more than 80 degrees, proved to be noninductive, hexacosanal significantly stimulated differentiation in c. 50% of B. graminis conidia, but only at contact angles > 80 degrees. These results, together with an observed inductive effect of highly hydrophobic, wax-free artificial surfaces, provide new insights into the interplay of physical and chemical surface cues involved in triggering prepenetration processes in B. graminis.     

大豆抗病基因同源序列的克隆与分析

已克隆的植物抗病基因序列存在一些相对保守的结构区域.利用根据核苷酸结合位点(NBS)结构域扩增所获得的大豆抗病基因同源片段为混合探针,进行大豆cDNA文库筛选.通过筛库和5'RACE-PCR扩增后,获得一全长基因KR3.KR3的长度为2353 bp,编码636个氨基酸.KR3蛋白在结构上与烟草抗花叶病毒N基因蛋白有较高的同源性,具有Toll/白细胞介素-1受体(TIR)、NBS等抗病基因的分子特征.Southern杂交显示KR3在基因组中为低拷贝;RT-PCR分析表明,该基因的表达受外源水杨酸的诱导.     

大豆抗病基因同源序列的克隆与分析

已克隆的植物抗病基因序列存在一些相对保守的结构区域。利用根据核苷酸结合位点(NBS)结构域扩增所获得的大豆抗病基因同源片段为混合探针,进行大豆cDNA文库筛选。通过筛库和5′RAcE-PcR扩增后,获得一全长基因KR3。KR3的长度为2353 bp,编码636个氨基酸。KR3蛋白在结构上与烟草抗花叶病毒N基因蛋白有较高的同源性,具有Toll／白细胞介素-1受体(TIR)、NBS等抗病基因的分了特征。Southern 杂交显KR3在基因组中为低拷贝：RT-PCR分析表明,该基因的表达受外源水杨酸的诱导。     

荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色效果比较*

比较了荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色的效果。荧光素钠法中样品处理只需20min.左右,具有直接、快速的特点;荧光指示剂对病菌分生孢子萌发及菌丝生长无抑制作用,主要沉集于活菌体的隔膜和细胞质部位,使病菌产生明显的亮绿荧光和清晰的细胞轮廓,亮绿荧光衰退期为7min.;借助荧光显微镜可以观察病菌在小麦叶表的发展过程,区别活菌体和失活菌体。考马斯亮蓝法包括传统的组织学染色步骤,经过改进后的样品处理过程需要40min.左右;染色后使寄主组织呈现淡蓝色,病菌菌体染成深蓝色;该方法可以观察病菌在小麦叶表和被侵染细胞内部发育形成的结构,包括孢子发育形成的初生芽管、附着胞芽管、成熟附着胞以及在寄主细胞内形成的初生吸器原体、成熟的指状体吸器和次生吸器。     

Genetics of Powdery Mildew Resistance in Barley

A comprehensive, critical review on the present knowledge regarding the genetics of resistance of barley to the powdery mildew fungus is presented. The review deals with six kinds of resistance: Race-specific resistance; Mlo resistance; partial resistance; induced resistance; passive resistance; and non-host resistance. Most of the sections are subdivided into: phenotype of the interaction; resistance mechanisms; and genetics. A distinction is made between three groups of genes involved in the defense of plants to diseases: those that serve exclusively to mediate resistance; those that are mobilized to strengthen the plants' defense; and those that serve exclusively functions other than disease defense, but may bring about resistance. The more than 200 gene symbols assigned to race-specific mildew resistance genes over time are summarized and revised to 85 symbols that may be considered valid.     

海岛棉NBS类型抗病基因类似物的起源、多样性及进化

利用已克隆植物的R基因NBS序列中保守模体合成简并引物,以海岛棉品系Pima 90(Gossypium barba—dense)基因组DNA为模板进行PCR扩增,通过T／A克隆、测序和序列比较分析共得到31条RGAs,其中19条具有连续的ORF。利用海岛棉的31条RGAs与GenBank中陆地棉种质系M—249(Gossypium hirsutum)的RGAs进行了比较分析,RGAs可分为两大类：其中第Ⅰ类全部为陆地棉的RGAs;第Ⅱ类分别包括了陆地棉和海岛棉的RGAs。同时对海岛棉RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明海岛棉RGAs可分为TIR(Drosophila Toll or human inter—leukin receptor—1ike)和non—FIR两类,与前人所报道的R基因进化一致。对19条具有连续ORF的RGAs进行了结构分析,结果表明它们包括P—loop、Kin—2、“PLAL”及Meyers等所定义的RNBS—A、B、C3个模体。结果表明,可能海岛棉NBS类型抗病基因类似物和其他物种具有同样的起源和进化机制。     

XBP-1和Erα存在相互作用

雌激素受体α(Erα)是乳腺癌治疗的靶标和预后的指标。在乳腺癌中,人X盒结合蛋白1(XBP-1)与Erα共表达,并在一些乳腺肿瘤中过表达。这些结果提示,XBP-1与Erα可能存在相互作用。XBP-1由于剪切方式不同,产生两种形式的XBP-1,即XBP-1S和XBP-1U。体外GST沉淀实验表明,XBP-1S和XBP-1U均能结合Erα,XBP-1S的结合能力大于XBP-1U。体内免疫共沉淀实验也表明,XBP-1S和XBP-1U以激素不依赖的方式与Erα结合。Erα通过其DNA结合结构域与XBP-1S和XBP-1U结合,而XBP-1S和XBP-1U通过其N末端的亮氨酸拉链结构域和C末端的转录激活结构域与Erα相互作用。这些结果提示,XBP-1S和XBP-1U可能通过与Erα的相互作用参与雌激素受体信号途径。     

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector‐triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc‐finger‐like structure with a novel interleaved zinc‐binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P‐mediated recognition. The first zinc‐coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid‐binding and chromatin‐associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non‐conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.     

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα₁₃ downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα₁₃, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα₁₃. Rgnef co-immunoprecipitated with activated Gα₁₃Q226L but not Gα₁₂Q229L. The Rgnef C-terminal (CT, 1279–1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα₁₃-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα₁₃. Point mutations of Rgnef-CT residues disrupt association with active Gα₁₃ but not Gα_q. These results show that Rgnef functions as an effector of Gα₁₃ signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.     

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac₂PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac₂PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac₂PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac₂PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac₂PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress β-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac₂PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac₂PIM and/or MDP.