Desiccation survival in an Antarctic nematode: molecular analysis using expressed sequenced tags

<p>Abstract</p> <p>Background</p> <p>Nematodes are the dominant soil animals in Antarctic Dry Valleys and are capable of surviving desiccation and freezing in an anhydrobiotic state. Genes induced by desiccation stress have been successfully enumerated in nematodes; however we have little knowledge of gene regulation by Antarctic nematodes which can survive multiple environmental stresses. To address this problem we investigated the genetic responses of a nematode species, <it>Plectus murrayi</it>, that is capable of tolerating Antarctic environmental extremes, in particular desiccation and freezing. In this study, we provide the first insight into the desiccation induced transcriptome of an Antarctic nematode through cDNA library construction and suppressive subtractive hybridization.</p> <p>Results</p> <p>We obtained 2,486 expressed sequence tags (ESTs) from 2,586 clones derived from the cDNA library of desiccated <it>P. murrayi</it>. The 2,486 ESTs formed 1,387 putative unique transcripts of which 523 (38%) had matches in the model-nematode <it>Caenorhabditis elegans</it>, 107 (7%) in nematodes other than <it>C. elegans</it>, 153 (11%) in non-nematode organisms and 605 (44%) had no significant match to any sequences in the current databases. The 1,387 unique transcripts were functionally classified by using Gene Ontology (GO) hierarchy and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results indicate that the transcriptome contains a group of transcripts from diverse functional areas. The subtractive library of desiccated nematodes showed 80 transcripts differentially expressed during desiccation stress, of which 28% were metabolism related, 19% were involved in environmental information processing, 28% involved in genetic information processing and 21% were novel transcripts. Expression profiling of 14 selected genes by quantitative Real-time PCR showed 9 genes significantly up-regulated, 3 down-regulated and 2 continuously expressed in response to desiccation.</p> <p>Conclusion</p> <p>The establishment of a desiccation EST collection for <it>Plectus murrayi</it>, a useful model in assessing the structural, physiological, biochemical and genetic aspects of multiple stress tolerance, is an important step in understanding the genome level response of this nematode to desiccation stress. The type of transcript analysis performed in this study sets the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in nematodes.</p

Expression Profiling of Cucumis sativus in Response to Infection by Pseudoperonospora cubensis

The oomycete pathogen, Pseudoperonospora cubensis, is the causal agent of downy mildew on cucurbits, and at present, no effective resistance to this pathogen is available in cultivated cucumber (Cucumis sativus). To better understand the host response to a virulent pathogen, we performed expression profiling throughout a time course of a compatible interaction using whole transcriptome sequencing. As described herein, we were able to detect the expression of 15,286 cucumber genes, of which 14,476 were expressed throughout the infection process from 1 day post-inoculation (dpi) to 8 dpi. A large number of genes, 1,612 to 3,286, were differentially expressed in pair-wise comparisons between time points. We observed the rapid induction of key defense related genes, including catalases, chitinases, lipoxygenases, peroxidases, and protease inhibitors within 1 dpi, suggesting detection of the pathogen by the host. Co-expression network analyses revealed transcriptional networks with distinct patterns of expression including down-regulation at 2 dpi of known defense response genes suggesting coordinated suppression of host responses by the pathogen. Comparative analyses of cucumber gene expression patterns with that of orthologous Arabidopsis thaliana genes following challenge with Hyaloperonospora arabidopsidis revealed correlated expression patterns of single copy orthologs suggesting that these two dicot hosts have similar transcriptional responses to related pathogens. In total, the work described herein presents an in-depth analysis of the interplay between host susceptibility and pathogen virulence in an agriculturally important pathosystem

Alternative Splicing of a Multi-Drug Transporter from Pseudoperonospora cubensis Generates an RXLR Effector Protein That Elicits a Rapid Cell Death

Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors

Transcriptional profiling of trait deterioration in the insect pathogenic nematode Heterorhabditis bacteriophora

Background: The success of a biological control agent depends on key traits, particularly reproductive potential, environmental tolerance, and ability to be cultured. These traits can deteriorate rapidly when the biological control agent is reared in culture. Trait deterioration under laboratory conditions has been widely documented in the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora (Hb) but the specific mechanisms behind these genetic processes remain unclear. This research investigates the molecular mechanisms of trait deterioration of two experimental lines of Hb, an inbred line (L5M) and its original parental line (OHB). We generated transcriptional profiles of two experimental lines of Hb, identified the differentially expressed genes (DEGs) and validated their differential expression in the deteriorated line. Results: An expression profiling study was performed between experimental lines L5M and OHB of Hb with probes for 15,220 ESTs from the Hb transcriptome. Microarray analysis showed 1,185 DEGs comprising of 469 down- and 716 up-regulated genes in trait deteriorated nematodes. Analysis of the DEGs showed that trait deterioration involves massive changes of the transcripts encoding enzymes involved in metabolism, signal transduction, virulence and longevity. We observed a pattern of reduced expression of enzymes related to primary metabolic processes and induced secondary metabolism. Expression of sixteen DEGs in trait deteriorated nematodes was validated by quantitative reverse transcription-PCR (qRT-PCR) which revealed similar expression kinetics for all the genes tested as shown by microarray. Conclusion: As the most closely related major entomopathogen to C. elegans, Hb provides an attractive near-term application for using a model organism to better understand interspecies interactions and to enhance our understanding of the mechanisms underlying trait deterioration in biological control agents. This information could also be used to improve the beneficial traits of biological control agents and better understand fundamental aspects of nematode parasitism and mutualism

mRNA-Seq Analysis of the Pseudoperonospora cubensis Transcriptome During Cucumber (Cucumis sativus L.) Infection

Pseudoperonospora cubensis, an oomycete, is the causal agent of cucurbit downy mildew, and is responsible for significant losses on cucurbit crops worldwide. While other oomycete plant pathogens have been extensively studied at the molecular level, Ps. cubensis and the molecular basis of its interaction with cucurbit hosts has not been well examined. Here, we present the first large-scale global gene expression analysis of Ps. cubensis infection of a susceptible Cucumis sativus cultivar, ‘Vlaspik’, and identification of genes with putative roles in infection, growth, and pathogenicity. Using high throughput whole transcriptome sequencing, we captured differential expression of 2383 Ps. cubensis genes in sporangia and at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Additionally, comparison of Ps. cubensis expression profiles with expression profiles from an infection time course of the oomycete pathogen Phytophthora infestans on Solanum tuberosum revealed similarities in expression patterns of 1,576–6,806 orthologous genes suggesting a substantial degree of overlap in molecular events in virulence between the biotrophic Ps. cubensis and the hemi-biotrophic P. infestans. Co-expression analyses identified distinct modules of Ps. cubensis genes that were representative of early, intermediate, and late infection stages. Collectively, these expression data have advanced our understanding of key molecular and genetic events in the virulence of Ps. cubensis and thus, provides a foundation for identifying mechanism(s) by which to engineer or effect resistance in the host

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Conservation and Loss of a Putative Iron Utilization Gene Cluster among Genotypes of Aspergillus flavus

Iron is an essential component for growth and development. Despite relative abundance in the environment, bioavailability of iron is limited due to oxidation by atmospheric oxygen into insoluble ferric iron. Filamentous fungi have developed diverse pathways to uptake and use iron. In the current study, a putative iron utilization gene cluster (IUC) in Aspergillus flavus was identified and characterized. Gene analyses indicate A. flavus may use reductive as well as siderophore-mediated iron uptake and utilization pathways. The ferroxidation and iron permeation process, in which iron transport depends on the coupling of these two activities, mediates the reductive pathway. The IUC identified in this work includes six genes and is located in a highly polymorphic region of the genome. Diversity among A. flavus genotypes is manifested in the structure of the IUC, which ranged from complete deletion to a region disabled by multiple indels. Molecular profiling of A. flavus populations suggests lineage-specific loss of IUC. The observed variation among A. flavus genotypes in iron utilization and the lineage-specific loss of the iron utilization genes in several A. flavus clonal lineages provide insight on evolution of iron acquisition and utilization within Aspergillus section Flavi. The potential divergence in capacity to acquire iron should be taken into account when selecting A. flavus active ingredients for biocontrol in niches where climate change may alter iron availability

Pythium iwayamai DAOM BR242034 Genome Assembly and Annotation

Pythium iwayamai DAOM BR242034 Genome Assembly and Annotation Contents: piw_functional_annotation.txt; piw.maker.proteins.fasta; piw.maker.transcripts.fasta; piw_contigs_asm.fasta; piw_contigs_asm.maker.gff

Pythium vexans DAOM BR484 Genome Assembly and Annotation

Pythium vexans DAOM BR484 Genome Assembly and Annotation Contents: pve1_func_anno.list; pve.maker.proteins.fasta; pve.maker.transcripts.fasta; pve_contigs_asm.fasta; pve_contigs_asm.maker.gff