Biology subjects

Tyler, B. M.

Publications and source records attributed to Tyler, B. M..

6 recordsLinked to original sources

bioRxiv2021

A new oomycete metabarcoding method using the rps10 gene

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing. Interpretive summaryOomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed to rapidly characterize the diversity of oomycete species found in environmental samples. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as being useful for oomycete community sequencing. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a community we synthesized in the laboratory. The amplified rps10 region is predicted to have a higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete community sequencing. The rps10 barcode and methods described herein provide an effective tool for characterizing oomycetes using environmental DNA sequencing.

microbiology↗

bioRxiv2021

Evaluation of the effects of library preparation procedure and sample characteristics on the accuracy of metagenomic profiles

Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities, or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera(R) Flex kit, perform when applied to metagenomic DNA. We measured each methods ability to produce metagenomic data that accurately represents the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral-associated, mouse-gut-associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each methods performance, indicating that careful consideration may be needed when selecting between methods, especially for low complexity communities. However, cost-effective preparation methods we assessed are generally comparable to the current gold standard Nextera(R) DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies. IMPORTANCEMetagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely-accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.

microbiology↗

bioRxiv2020

Phytophthora sojae effector Avr1d functions as E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors to host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy-roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By co-immunoprecipitation, gel infiltration and ITC assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure of Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy-roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor, negatively regulating soybean resistance against P. sojae. Altogether, this study highlights a novel virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This is the first study to unravel the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding. Significance StatementUbiquitination acts as a crucial regulator in plant immunity. Accordingly, microbial pathogens secrete effectors to hijak host ubiquitination system. However, the molecular mechanisms by which microbial effectors modulate host ubiquitination system are not yet clear. Here, we found that the Phytophthora sojae effector Avr1d physically binds to the U-box type E3 ligase GmPUB13, a susceptibility factor in soybean. The crystal structure of Avr1d in complex with GmPUB13 revealed that Avr1d occupies the binding site in GmPUB13 for the E2 ubiquitin conjugating enzyme and competes with E2 for physical binding to GmPUB13. Avr1d stabilized GmPUB13 by suppressing the self-ubiquitination activity of GmPUB13 and thereby promoting Phytophthora infection. This study provides structural basis for modulation of host targets by Phytophthora effectors.

microbiology↗

bioRxiv2019

Genomic investigation of the strawberry pathogen Phytophthora fragariae indicates pathogenicity is determined by transcriptional variation in three key races

The oomycete Phytophthora fragariae is a highly destructive pathogen of cultivated strawberry (Fragaria x ananassa), causing the root rotting disease, red core. The host-pathogen interaction has a well described gene-for-gene resistance relationship, but to date neither candidate avirulence nor resistance genes have been identified. We sequenced a set of American, Canadian and UK isolates of known race type, along with three representatives of the closely related pathogen of the raspberry (Rubus idaeus), Phytophthora rubi, and found a clear population structure, with a high degree of nucleotide divergence seen between some race types and abundant private variation associated with race types 4 and 5. In contrast, between isolates defined as UK races 1, 2 & 3 (UK1-2-3) there was no evidence of gene loss or gain; or the presence of insertions/deletions (INDELs) or Single Nucleotide Polymorphisms (SNPs) within or in proximity to putative pathogenicity genes could be found associated with race variation. Transcriptomic analysis of representative UK1-2-3 isolates revealed abundant expression variation in key effector family genes associated with pathogen race; however, further long read sequencing did not reveal any long range polymorphisms to be associated with avirulence to race UK2 or UK3 resistance, suggesting either control in trans or other stable forms of epigenetic modification modulating gene expression. This work reveals the combined power of population resequencing to uncover race structure in pathosystems and in planta transcriptomic analysis to identify candidate avirulence genes. This work has implications for the identification of putative avirulence genes in the absence of associated expression data and points towards the need for detailed molecular characterisation of mechanisms of effector regulation and silencing in oomycete plant pathogens.

genomics↗

bioRxiv2019

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly. Author summaryOomycetes are fungal-like microorganisms that belong to the stramenopiles within the Stramenopila-Alveolata-Rhizaria (SAR) supergroup. The Phytophthora oomycetes are infamous as plant killers, threatening crop production worldwide. Because of the highly repetitive nature of their genomes, assembly of oomycete genomes presents challenges that impede identification of centromeres, which are chromosomal sites mediating faithful chromosome segregation. We report long-read sequencing-based genome assembly of the Phytophthora sojae reference strain, which facilitated the discovery of centromeres. P. sojae harbors large regional centromeres fully embedded in heterochromatin, and enriched for a Copia-like transposon that is also found in discrete clusters in other oomycetes. This study provides insight into the oomycete genome organization, broadens our knowledge of the centromere structure, function and evolution in eukaryotes, and may help elucidate the high frequency of aneuploidy during oomycete reproduction.

microbiology↗

bioRxiv2016

EuMicrobedbLite: A lightweight genomic resource and analytic platform for draft oomycete genomes

We have developed EuMicrobedbLite - A light weight comprehensive genome resource and sequence analysis platform for oomycete organisms. EuMicrobedbLite is a successor of the VBI Microbial Database (VMD) that was built using the Genome Unified Schema (GUS). In this version, the GUS schema has been greatly simplified with removal of many obsolete modules and redesign of others to incorporate contemporary data. Several dependencies such as perl object layers used for data loading in VMD have been replaced with independent light weight scripts. EumicrobedbLite now runs on a powerful annotation engine developed at our lab called \"Genome Annotator Lite\". Currently this database has 26 publicly available genomes and 10 EST datasets of oomycete organisms. The browser page has dynamic tracks presenting comparative genomics analyses, coding and non-coding data, tRNA genes, repeats and EST alignments. In addition, we have defined 44,777 core conserved proteins from twelve oomycete organisms that form 2974 clusters. Synteny viewing is enabled by incorporation of the Genome Synteny Viewer (GSV) tool. The user interface has undergone major changes for ease of browsing. Queryable comparative genomics information, conserved orthologous genes and pathways are among the new key features updated in this database. The browser has been upgraded to enable user upload of GFF files for quick view of genome annotation comparisons. The toolkit page integrates the EMBOSS package and has a gene prediction tool. Annotations for the organisms are updated once every six months to ensure quality. The database resource is available at www.eumicrobedb.org.

bioinformatics↗