Correlation Between Induced Resistance and Host Fluorescence in Barley Inoculated with Erysiphe graminis

Resistance was induced in barley by virulent and avirulent Erysiphe graminis f. sp. hordei and by E. graminis f. sp. tritici. The diameters of fluorescent haloes and papillae at the site of the primary germ tube of the inducers were larger until 12 hours after inoculation with E g. f. sp. tritici than in the corresponding periods after inoculation with E. g. f. sp. hordei. Fluorescence at the site of appressorial lobes of the inducers developed, gradually from 10 and 12 hours after inoculation with E. g. f. sp. hordei and E. g. f. sp. tritici, respectively. Penetration success of single infection units of the challenger was reduced in cells with inducer fluorescence, suggesting that resistance is principally localized to cells previously attacked by the inducer. But penetration success was independent of the average distance to the ten nearest inducer fluorescences, and penetration failures did also occur in cells without inducer fluorescence, suggesting that the induced resistance to some extent is translocated to other epidermal cells. The average diameter of the fluorescent papillae at the site of unsuccessful challenger infection units increased as a result of induced resistance.     

Genetic similarity of flag shoot and ascospore subpopulations of Erysiphe necator in Italy

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled (theta = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (theta = 0.138, P = 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge.     

Genetic Similarity of Flag Shoot and Ascospore Subpopulations of Erysiphe necator in Italy

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled (θ = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (θ = 0.138, P = 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge.     

小麦全基因组抗赤霉病QTL关联位点特异性SSR标记的筛选、等位变异及效应解析

赤霉病是小麦主要的流行病害之一。借助标记辅助选择将不同数量性状基因座（quantitative trait loci，QTL）聚合是防治赤霉病有效且环保的方法，可以从源头上控制赤霉病并降低籽粒中毒素含量。抗赤霉病QTL在小麦全基因组均有分布，但除了Fhb1、Fhb2等少数位点有比较可靠的鉴别标记，绝大部分位点缺乏有效的位点特异性鉴别标记。简单重复序列（simple sequence repeat，SSR）标记多态性丰富，可以区分自然群体中不同等位变异，方便用于标记辅助育种。基于此，搜集了不同文献中报道的与赤霉病关联的SSR标记386个，并用这些标记构建全基因组赤霉病抗性QTL一致性图谱，接着对这些关联标记进行拷贝数分析，进而选择位点内的单拷贝SSR标记，将这些单拷贝标记在156个品种组成的自然群体中进行扩增，并与三季大田和三季温室环境下赤霉病抗性进行关联，筛选与赤霉病抗性关联的单拷贝SSR标记，明确这些标记在自然群体中的有效等位变异和效应。结果表明，共8个单拷贝SSR标记至少在两季试验中与表型显著关联(P<0.05)，涉及2B、2D、3B、5A、5B、6A、6D、7A染色体，有5个单拷贝标记位点存在有效等位变异。中国地方品种和日本品种携带更多的有利变异，且有利等位变异数目越多的品种赤霉病抗性越好。研究分析的QTL位点及其关联的单拷贝SSR标记可用于赤霉病抗病育种，有利于提高品种赤霉病抗性水平和育种效率。     

两种存活蛋白变异剪接体在HeLa细胞中的表达定位及相互作用

存活蛋白(survivin)是重要的肿瘤相关抗原基因,在肿瘤的发生发展中 起着重要的作用. 除了标准的剪接形式外,它至少还编码2种变异剪接产物—存活蛋白-2B 和存活蛋白-ΔEx3,这2个变异剪接体所编码的蛋白具有不同的生物学功能.为研究这2个变 异剪接体在肿瘤细胞中的相互作用情况,本实验利用增强型青色荧光蛋白（enhanced cyan fluorescent protein, ECFP）和增强型黄色荧光蛋白（enhanced yellow fluorescent protein, EYFP）分别标记存活蛋白-2B 和存活蛋白-ΔEx3.首先通过激光共聚焦扫描显微镜观 察它们的细胞定位;同时利用荧光共振能量转移（fluorescence resonance energy transf er, FRET）技术研究两者在细胞内的相互作用情况.研究结果表明, 存活蛋白-2B主要分布 在细胞质中,而存活蛋白-ΔEx3则主要分布在细胞核内,少量分布在细胞质中;FRET分析结 果显示,两者仅在细胞质中存在着很弱的相互作用,表明两者很可能是通过某种间接的方式发 挥功能上的相互调节作用.本研究为进一步探讨存活蛋白变异剪接体的生物学功能及相互作用 机制奠定了基础.     

Microfluidic PCR Combined with Pyrosequencing for Identification of Allelic Variants with Phenotypic Associations among Targeted Salmonella Genes

A novel targeted massive parallel sequencing approach identified genetic variation in eight known or predicted fimbrial adhesins for 46 Salmonella strains. The results highlight associations between specific adhesin alleles, host species, and antimicrobial resistance. The differentiation of allelic variants has potential applications for diagnostic microbiology and epidemiological investigations.     

Distinctions among Allelic Variants Associated with Chromosome 3 Inversions in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

Efforts were made to discriminate new genetic variants among electrophoretic alleles that are associated with chromosome 3 inversions of Drosophila pseudoobscura and D. persimilis. Apparent genetic similarities for electrophoretic alleles between these two species and among the common inversions they carry were reexamined by altering gel concentration and buffer pH. At the amylase locus, the 1.09 electrophoretic allele could be further separated into two allelic classes that differentiated the WT and KL arrangements. Similarly, the 0.84 electrophoretic allele was divided into two allelic classes, one characteristic of the Santa Cruz phylad arrangements, TL and SC, and the other found in strains of the Standard phylad arrangements and CH. Uncommon amylase alleles proved to be different alleles in the two species. No new allelic variants, however, could be found among strains with the amylase 1.00 allele, the commonest allele in the Standard phylad of both species. No major new allelic variation was detected for acid phosphatase-3 and larval protein-10 that revealed any further differentiation among species or inversions. Variation at all three loci in strains of the Bogota population remained genetically similar to variation in strains of mainland D. pseudoobscura.     

Association between Allelic Variants of the Genes Involved in Glucocorticoids Metabolism and Asthma

Russian Journal of Genetics - Bronchial asthma (BA) is a common severe and disabling multifactorial disease. Up to 50–60% of differences in sensitivity to therapy in patients with BA is due...     

芒果炭疽病菌β-微管蛋白基因的克隆及其与多菌灵抗药性发生的关系

参照豆科合萌属 (Aeschynomene)作物炭疽病菌的tub1和tub2基因序列设计了 2对引物 ,分别从芒果 (Man gifera)炭疽病菌对多菌灵 (MBC)田间抗药性 (MBCR)和敏感 (MBCS)的菌株中扩增 β_微管蛋白基因。结果只有以tub2为参照设计的引物扩增到了特异片段。进一步对全基因进行了克隆和测序。该基因序列全长 1344bp ,编码4 4 7aa ,其核苷酸和氨基酸序列与豆科合萌属炭疽病菌的tub2基因高度同源。对芒果炭疽病菌抗、感菌株 β_微管蛋白氨基酸序列进行比较分析 ,发现第 181、2 37和 36 3位氨基酸发生了突变 ,而其它位置 (如第 198位或 2 0 0位 )均不变     

Dynamics of CYP51: implications for function and inhibitor design

Sterol 14α‐demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane‐bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane‐bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen‐bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes. Copyright © 2015 John Wiley & Sons, Ltd.     

Polymorphisms of CYP51A1 from Cholesterol Synthesis: Associations with Birth Weight and Maternal Lipid Levels and Impact on CYP51 Protein Structure

We investigated the housekeeping cytochrome P450 CYP51A1 encoding lanosterol 14α-demethylase from cholesterol synthesis that was so far not directly linked to human disorders. By direct sequencing of CYP51A1 in 188 women with spontaneous preterm delivery and 188 unrelated preterm infants (gestational age <37 weeks) we identified 22 variants where 10 are novel and rare. In infants there were two novel CYP51A1 variants where damaging effects of p.Tyr145Asp from the substrate recognition region, but not p.Asn193Asp, were predicted by PolyPhen2 and SIFT. This was confirmed by molecular modeling showing that Tyr145Asp substitution results in changed electrostatic potential of the CYP51 protein surface and lengthened distance to the heme which prevents hydrogen bonding. The CYP51 Tyr145Asp mutation is rare and thus very interesting for further structure/function relationship studies. From the 12 identified known variants rs6465348 was chosen for family based association studies due to its high minor allele frequency. Interestingly, this CYP51A1 common variant associates with small for gestational age weight in newborns (p = 0.028) and lower blood total cholesterol and low density lipoprotein cholesterol levels in mothers in 2nd trimester of pregnancy (p = 0.042 and p = 0.046 respectively). Our results indicate a new link between a cholesterol synthesis gene CYP51A1 and pregnancy pathologies.     

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.     

Molecular dynamic modeling of CYP51B in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylase (CYP51), the key enzyme in sterol biosynthesis pathway, is an important target protein of cholesterol-lowering agents, antifungal drugs, and herbicides. CYP51B enzyme is one of the CYP51 family members. In the present study, we have chosen four representative inhibitors of CYP51B: diniconazole (Din), fluconazole (Flu), tebuconazole (Teb), and voriconazole (Vor), and launched to investigate the binding features of CYP51B-inhibitor and gating characteristics via molecular docking and molecular dynamics (MD) simulations. The results show that the ranking of binding affinities among these inhibitors is Din > Teb > Vor > Flu. Din shows the strongest binding affinity, whereas Flu shows the weakest binding affinity. More importantly, based on the calculated binding free energy results, we hypothesize that the nonpolar interactions are the most important contributors, and three key residues (Thr77, Ala258, and Lys454) play crucial role in protein-inhibitor binding. Besides, residue Phe180 may play a molecular switch role in the process of the CYP51B-Teb and CYP51B-Vor binding. Additionally, Tunnel analysis results show that the major tunnel of Din, Flu, and Teb is located between helix K, FG loop, and β4 hairpin (Tunnel II).The top ranked possible tunnel (Tunnel II) corresponds to Vor exits through helix K, F and helix J. This study further revealed the CYP51B relevant structural characteristics at the atomic level as well as provided a basis for rational design of new and more efficacious antifungal agents.     

Grapevine powdery mildew (Erysiphe necator): a fascinating system for the study of the biology, ecology and epidemiology of an obligate biotroph

Few plant pathogens have had a more profound effect on the evolution of disease management than Erysiphe necator, which causes grapevine powdery mildew. When the pathogen first spread from North America to England in 1845, and onwards to France in 1847, 'germ theory' was neither understood among the general populace nor even generally accepted within the scientific community. Louis Pasteur had only recently reported the microbial nature of fermentation, and it would be another 30 years before Robert Koch would publish his proofs of the microbial nature of certain animal diseases. However, within 6 years after the arrival of the pathogen, nearly 6 million grape growers in France were routinely applying sulphur to suppress powdery mildew on nearly 2.5 million hectares of vineyards (Campbell, 2006). The pathogen has remained a focus for disease management efforts ever since. Because of the worldwide importance of the crop and its susceptibility to the disease, and because conventional management with modern, organic fungicides has been compromised on several occasions since 1980 by the evolution of fungicide resistance, there has also been a renewed effort worldwide to explore the pathogen's biology and ecology, its genetics and molecular interactions with host plants, and to refine current and suggest new management strategies. These latter aspects are the subject of our review. Taxonomy: The most widely accepted classification follows. Family Erysiphaceae, Erysiphe necator Schw. [syn. Uncinula necator (Schw.) Burr., E. tuckeri Berk., U. americana Howe and U. spiralis Berk. & Curt; anamorph Oidium tuckeri Berk.]. Erysiphe necator var. ampelopsidis was found on Parthenocissus spp. in North America according to Braun (1987), although later studies revealed isolates whose host range spanned genera, making the application of this taxon somewhat imprecise (Gadoury and Pearson, 1991). The classification of the genera before 1980 was based on features of the mature ascocarp: (i) numbers of asci; and (ii) morphology of the appendages, in particular the appendage tips. The foregoing has been supplanted by phylogeny inferred from the internal transcribed spacer (ITS) of ribosomal DNA sequences (Saenz and Taylor, 1999), which correlates with conidial ontogeny and morphology (Braun et al., 2002). Host range: The pathogen is obligately parasitic on genera within the Vitaceae, including Vitis, Cissus, Parthenocissus and Ampelopsis (Pearson and Gadoury, 1992). The most economically important host is grapevine (Vitis), particularly the European grape, V. vinifera, which is highly susceptible to powdery mildew. Disease symptoms and signs: In the strictest sense, macroscopically visible mildew colonies are signs of the pathogen rather than symptoms resulting from its infection, but, for convenience, we describe the symptoms and signs together as the collective appearance of colonized host tissues. All green tissues of the host may be infected. Ascospore colonies are most commonly found on the lower surface of the first-formed leaves near the bark of the vine, and may be accompanied by a similarly shaped chlorotic spot on the upper surface. Young colonies appear whitish and those that have not yet sporulated show a metallic sheen. They are roughly circular, ranging in size from a few millimetres to a centimetre or more in diameter, and can occur singly or in groups that coalesce to cover much of the leaf. Senescent colonies are greyish, and may bear cleistothecia in various stages of development. Dead epidermal cells often subtend the colonized area, as natural mortality in the mildew colony, the use of fungicides, mycoparasites or resistance responses in the leaf result in the deaths of segments of the mildew colony and infected epidermal cells. Severely affected leaves usually senesce, develop necrotic blotches and fall prematurely. Infection of stems initially produces symptoms similar to those on leaves, but colonies on shoots are eventually killed as periderm forms, producing a dark, web-like scar on the cane (Gadoury et al., 2011). Inflorescences and berries are most susceptible when young, and can become completely coated with whitish mildew. The growth of the berry epidermal tissue stops when severely infected, which may result in splitting as young fruit expand. Berries in a transitional stage between susceptible and resistant (generally between 3 and 4 weeks after anthesis) develop diffuse, nonsporulating mildew colonies only visible under magnification. Diffuse colonies die as berries continue to mature, leaving behind a network of necrotic epidermal cells (Gadoury et al., 2007). Survival over winter as mycelium in buds results in a distinctive foliar symptom. Shoots arising from these buds may be heavily coated with fungal growth, stark white in colour and stand out like white flags in the vine, resulting in the term 'flag shoots'. More commonly, colonization of a flag shoot is less extensive, and infection of a single leaf, or of leaves on one side of the shoot only, is observed (Gadoury et al., 2011).     

Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery.     

Sequence Variants of the POMC Gene and Their Associations with Body Composition in Children

We investigated POMC sequence variants in 242 overweight and nonoverweight African‐American and white children and examined the associations between body composition and POMC polymorphisms. Three novel polymorphisms and two previously identified sequence variants were found: A7301G, A7429G, and C8246T were all in untranslated regions. A 9‐bp (AGC AGC GGC) duplication/insertion was found between positions 7677 and 7678, and one normal‐weight African‐American girl had a 45‐bp triple duplication/insertion at this location. Compared with whites, African‐American children were significantly more likely to have polymorphisms A7301G, A7429G, and the 9‐bp insertion. However, there were no significant associations between any of the polymorphisms and body composition. Five African‐American subjects who were homozygous for A7429G had a trend (p = 0.08) for a greater BMI‐SD score (5.3 ± 5.3 kg/m²) compared with wild‐type children (BMI‐SD score, 2.4 ± 3.2 kg/m²) or heterozygotes (BMI‐SD score, 2.7 ± 3.7 kg/m²). However, there were no differences in BMI‐SD score for A7429G when African American subjects were studied separately and both gender and height were taken into account. The contribution of the POMC gene variants we studied to pediatric‐onset obesity seems to be limited.