Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria

Powdery mildew fungi are biotrophic pathogens that require living plant cells for their growth and reproduction. Elaboration of a specialized cell called a haustorium is essential for their pathogenesis, providing a portal into host cells for nutrient uptake and delivery of virulence effectors. Haustoria are enveloped by a modified plant plasma membrane, the extrahaustorial membrane (EHM), and an extrahaustorial matrix (EHMx), across which molecular exchange must occur, but the origin and composition of this interfacial zone remains obscure. Here we present a method for isolating Golovinomyces orontii haustoria from Arabidopsis leaves and an ultrastructural characterization of the haustorial interface. Haustoria were progressively encased by deposits of plant cell wall polymers, delivered by secretory vesicles and multivesicular bodies (MVBs) that ultimately become entrapped within the encasement. The EHM and EHMx were not labelled by antibodies recognizing eight plant cell wall and plasma membrane antigens. However, plant resistance protein RPW8.2 was specifically recruited to the EHMs of mature haustoria. Fungal cell wall-associated molecular patterns such as chitin and β-1,3-glucans were exposed at the surface of haustoria. Fungal MVBs were abundant in haustoria and putative exosome vesicles were detected in the paramural space and EHMx, suggesting the existence of an exosome-mediated secretion pathway.     

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.     

CVTree在454高通量测序分析菌群结构中的应用

目的探究将基于短串频度的CVTree方法用于反映菌群结构的16S rRNA基因的454高通量测序数据分析的可行性,为快速分析高通量菌群结构数据提供新的方法。方法对一个四世同堂的中国家庭7名成员肠道菌群和不同基因型及饮食类型的小鼠肠道菌群用454高通量方法获得16S rRNA基因的V3区的测序数据,用CVTree的方法进行菌群结构的比较分析。结果通过选取合适的短串长度,CVTree的方法能准确检测到各样本间的聚类关系,其结果与之前文献报道的基于Unifrac算法的结果相一致。结论CVTree能快速、有效地处理16S rRNA基因的454高通量测序数据,实现对不同菌群结构相似性的比较分析。     

454高通量焦磷酸测序法鉴定膜生物反应器膜污染优势菌种

【目的】对诱发膜-生物反应器(Membrane bioreactors,MBR)膜污染的优势菌种进行研究。【方法】利用454高通量焦磷酸测序法对MBR污泥混合液样品与膜污染物样品中微生物信息进行统计,并对两组样品的Chao丰度指数与Shannon生物多样性指数计算,对测序结果进行系统发育学分析。【结果】从污泥混合液样品与膜污染物样品中获得9 353与7 504条优化序列,发现膜污染物中微生物丰度与多样性均高于污泥混合样品。借助基因频谱对OTU分布特点进行统计,表明源于污泥混合液中的微生物在膜表面定殖生长过程中发生了种群变化,在膜面污染物样品中,β-变形菌纲丰度显著降低,α-变形菌纲、γ-变形菌纲与Phycisphaerae在微生物种群结构中比重增加。【结论】454焦磷酸测序分析表明,黄色单胞菌(Xanthomonadaceae),嗜热厌氧杆菌(Thermoanaerobacter),Phycisphaera以及2株尚未培养出的细菌(Candidate_division_TM7及Candidate_division_OD1)是诱发MBR膜污染的优势菌种(微生物丰度1%)。诱发膜污染的细菌既包括了黏性高、表面疏水的种类(如γ-变形菌),从而引发细菌在膜表面的定殖,也包括了代谢能力强的物种(如Candidate_division_OD1)可以确保种间递氢顺畅。     

Single-nucleotide polymorphism analysis by pyrosequencing

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.     

Seasonal variations in the occurrence of Golovinomyces orontii and Podosphaera xanthii,causal agents of cucurbit powdery mildew in Northern Italy

Powdery mildew, caused by Golovinomyces orontii and Podosphaera xanthii, is a widespread disease that causes important losses in cucurbit production. To determine the aetiology and the epidemiology of cucurbit powdery mildew disease in the North of Italy, observations on the occurrence of the main disease‐causing fungal species were conducted during the 2010, 2011 and 2012 growing seasons. Samples of infected leaves of zucchini, melon and pumpkin plants, either from field or greenhouse crops, were collected every 15–18 days from May to September/October. To identify the fungal species, both morphological observations based on the asexual stage and molecular identifications by a Multiplex‐PCR reaction with species‐specific primers were performed. Climatic parameters of temperature and relative humidity were also monitored. Pearson's correlation coefficient and Principal Component Analysis showed a negative significant correlation between the two species, and a peculiar epidemiological behaviour was also observed: the earlier infections were caused by G. orontii, which was the predominant species till the end of June–middle of July. At this time, this species progressively decreased in frequency and was replaced by P. xanthii that became the main species infecting cucurbits till the end of the growing season. As the two species have different ecological requirements, these seasonal variations in the cucurbit powdery mildew species composition could possibly be explained by the influence of temperature and relative humidity on the pathogen epidemiology during the growing season but also by the different overwintering strategies adopted by the two species.     

Seasonal trends in the biomass and structure of bryophyte-associated fungal communities explored by 454 pyrosequencing

? Bryophytes are a dominant vegetation component of the boreal forest, but little is known about their associated fungal communities, including seasonal variation within them. ? Seasonal variation in the fungal biomass and composition of fungal communities associated with three widespread boreal bryophytes was investigated using HPLC assays of ergosterol and amplicon pyrosequencing of the internal transcribed spacer 2 (ITS2) region of rDNA. ? The bryophyte phyllosphere community was dominated by Ascomycota. Fungal biomass did not decline appreciably in winter (P?=?0.272). Significant host-specific patterns in seasonal variation of biomass were detected (P?=?0.003). Although seasonal effects were not the primary factors structuring community composition, collection date significantly explained (P?=?0.001) variation not attributed to locality, host, and tissue. Community homogenization and a reduction in turnover occurred with the onset of frost events and subzero air and soil temperatures. Fluctuations in the relative abundance of particular fungal groups seem to reflect the nature of their association with mosses, although conclusions are drawn with caution because of potential methodological bias. ? The moss-associated fungal community is dynamic, exhibiting seasonal turnover in composition and relative abundance of different fungal groups, and significant fungal biomass is present year-round, suggesting a winter-active fungal community.     

Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria

Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection.     

Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing

AFLP‐based genome scans are widely used to study the genetics of adaptation and to identify genomic regions potentially under selection. However, this approach usually fails to detect the actual genes or mutations targeted by selection owing to the difficulties of obtaining DNA sequences from AFLP fragments. Here, we combine classical AFLP outlier detection with 454 sequencing of AFLP fragments to obtain sequences from outlier loci. We applied this approach to the study of resistance to Bacillus thuringiensis israelensis (Bti) toxins in the dengue vector Aedes aegypti. A genome scan of Bti‐resistant and Bti‐susceptible A. aegypti laboratory strains was performed based on 432 AFLP markers. Fourteen outliers were detected using two different population genetic algorithms. Out of these, 11 were successfully sequenced. Three contained transposable elements (TEs) sequences, and the 10 outliers that could be mapped at a unique location in the reference genome were located on different supercontigs. One outlier was in the vicinity of a gene coding for an aminopeptidase potentially involved in Bti toxin‐binding. Patterns of sequence variability of this gene showed significant deviation from neutrality in the resistant strain but not in the susceptible strain, even after taking into account the known demographic history of the selected strain. This gene is a promising candidate for future functional analysis.     

DNA methylation analysis by pyrosequencing

Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides through the enzymatic conversion of released pyrophosphate into a proportional light signal. Quantitative measures are of special importance for DNA methylation analysis in various developmental and pathological situations. Analysis of DNA methylation patterns by pyrosequencing combines a simple reaction protocol with reproducible and accurate measures of the degree of methylation at several CpGs in close proximity with high quantitative resolution. After bisulfite treatment and PCR, the degree of each methylation at each CpG position in a sequence is determined from the ratio of T and C. The process of purification and sequencing can be repeated for the same template to analyze other CpGs in the same amplification product. Quantitative epigenotypes are obtained using this protocol in approximately 4 h for up to 96 DNA samples when bisulfite-treated DNA is already available as the starting material.     

Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time‐intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185‐bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per‐amplicon‐variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi‐quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus‐specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.     

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

Changes in the root-associated fungal communities along a primary succession gradient analysed by 454 pyrosequencing

We investigated changes in the root-associated fungal communities associated with the ectomycorrhizal herb Bistorta vivipara along a primary succession gradient using 454 amplicon sequencing. Our main objective was to assess the degree of variation in fungal richness and community composition as vegetation cover increases along the chronosequence. Sixty root systems of B. vivipara were sampled in vegetation zones delimited by dated moraines in front of a retreating glacier in Norway. We extracted DNA from rinsed root systems, amplified the ITS1 region using fungal-specific primers and analysed the amplicons using 454 sequencing. Between 437 and 5063 sequences were obtained from each root system. Clustering analyses using a 98.5% sequence similarity cut-off yielded a total of 470 operational taxonomic units (OTUs), excluding singletons. Between eight and 41 fungal OTUs were detected within each root system. Already in the first stage of succession, a high fungal diversity was present in the B. vivipara root systems. Total number of OTUs increased significantly along the gradient towards climax vegetation, but the average number of OTUs per root system stayed unchanged. There was a high patchiness in distribution of fungal OTUs across root systems, indicating that stochastic processes to a large extent structure the fungal communities. However, time since deglaciation had impact on the fungal community structure, as a systematic shift in the community composition was observed along the chronosequence. Ectomycorrhizal basidiomycetes were the dominant fungi in the roots of B. vivipara, when it comes to both number of OTUs and number of sequences.