Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant–pathogen interactions in gymnosperm forest trees, including kauri

Severe Community-acquired Pneumonia Due to Staphylococcus aureus, 2003–04 Influenza Season

S. aureus community-acquired pneumonia has been reported from 9 states

Estimating HIV Incidence among Adults in Kenya and Uganda: A Systematic Comparison of Multiple Methods

CITATION: Kim, A. A. et al. 2011. Estimating HIV incidence among adults in Kenya and Uganda : a systematic comparison of multiple methods. PLos ONE, 6(3): e17535, doi:10.1371/journal.pone.0017535.The original publication is available at http://journals.plos.org/plosoneBackground: Several approaches have been used for measuring HIV incidence in large areas, yet each presents specific challenges in incidence estimation. Methodology/Principal Findings: We present a comparison of incidence estimates for Kenya and Uganda using multiple methods: 1) Epidemic Projections Package (EPP) and Spectrum models fitted to HIV prevalence from antenatal clinics (ANC) and national population-based surveys (NPS) in Kenya (2003, 2007) and Uganda (2004/2005); 2) a survey-derived model to infer age-specific incidence between two sequential NPS; 3) an assay-derived measurement in NPS using the BED IgG capture enzyme immunoassay, adjusted for misclassification using a locally derived false-recent rate (FRR) for the assay; (4) community cohorts in Uganda; (5) prevalence trends in young ANC attendees. EPP/Spectrum-derived and survey-derived modeled estimates were similar: 0.67 [uncertainty range: 0.60, 0.74] and 0.6 [confidence interval: (CI) 0.4, 0.9], respectively, for Uganda (2005) and 0.72 [uncertainty range: 0.70, 0.74] and 0.7 [CI 0.3, 1.1], respectively, for Kenya (2007). Using a local FRR, assay-derived incidence estimates were 0.3 [CI 0.0, 0.9] for Uganda (2004/2005) and 0.6 [CI 0, 1.3] for Kenya (2007). Incidence trends were similar for all methods for both Uganda and Kenya. Conclusions/Significance: Triangulation of methods is recommended to determine best-supported estimates of incidence to guide programs. Assay-derived incidence estimates are sensitive to the level of the assay's FRR, and uncertainty around high FRRs can significantly impact the validity of the estimate. Systematic evaluations of new and existing incidence assays are needed to the study the level, distribution, and determinants of the FRR to guide whether incidence assays can produce reliable estimates of national HIV incidence.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017535Publisher's versio

The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed

Targeted Gene Mutations in the Forest Pathogen <i>Dothistroma septosporum</i> Using CRISPR/Cas9

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens

Fungi decaying the wood of fallen beech (Nothofagus) trees in the South Island of New Zealand.

In order to extend present knowledge of communities of wood decay fungi in native forests, basidiomycetes and ascomycetes were isolated from within 15 fallen stems in beech (Nothofagus, Nothofagaceae) forests in the South Island of New Zealand. Fungal species were identified as precisely as possible using traditional culturing and molecular approaches. The internal distribution of species within stems was determined. Common fungi that occupied significant portions of stems were Ganoderma applanatum sensu Wakefield, Australoporus tasmanicus, Inonotus nothofagi, Pleurotus purpureo-olivaceus and an unidentified hymenochaetaceous species. Richness and diversity of basidiomycete species were greater in stems of red beech (Nothofagus fusca) and silver beech (N. menziesii) than in those of matai (Prumnopitys taxifolia, Podocarpaceae) and tawa (Beilschmiedia tawa, Lauraceae), as determined from earlier studies in podocarp hardwood and beech indigenous forests. There was greater similarity in the species composition of basidiomycete fungi colonising the three beech species compared with those colonising rimu (Dacrydium cupressinum, Podocarpaceae), tawa and matai. Based on observations in this study and on international research on the effects of selective logging on basidiomycete biodiversity, the decision to restrict to 50% the extraction of wood following storm damage in beech forests on the South Island West Coast appears to have been appropriate.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Development of a Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Dothistroma septosporum

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.Forestry, Faculty ofNon UBCForest and Conservation Sciences, Department ofReviewedFacult

Reduced Virulence of an Introduced Forest Pathogen over 50 Years

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys

Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains

Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.Supplementary Material: Fig. S1 Predicted duplications and deletions in chromosomes 1‐14 for 18 strains of D. septosporum.Fig. S2 Initial evidence for a reciprocal chromosome translocation in the NZE10 genome. (A) Assembled contigs from the SLV genome were aligned with NZE10 reference chromosomes (scaffolds). Two contigs (circled) mapped to both chromosomes 5 and 13 of the NZE10 reference genome. This was found in many of the other genome sequences. (B) Visualisation of reads from the ALP3 genome mapped onto a region of chromosome 13 show a gap, in which mate pairs are mapped to chromosome 5.Fig. S3 A reciprocal translocation involving chromosomes 5 and 13 in the NZE10 genome. (A) The reciprocal translation was centred on an identical sequence (GCGCGGT) found at positions 1459800‐1459806 in NZE10 chromosome 5 and 717926‐717932 in chromosome 13. Chromosomes 5 and 13 are shaded grey and pale blue respectively with ends coloured to distinguish the two arms in each case. Coloured sequences surrounding the breakpoint indicate which arm they are from. (B) In strains from regions other than Australasia, the two long sections of NZE10 chromosomes 5 and 13 are joined to make a 2.2 Mb chromosome and two short sections to make a 1.4 Mb chromosome. Sequences around the common 7 bp sequence are shown for strain ALP3 as an example. (C, D) Pairs of divergently transcribed genes straddle the breakpoints on NZE10 chromosomes 5 (C) and 13 (D). A GC content of about 70% was seen at the breakpoint regions (50 bp sliding window) as shown by the %GC (blue) profiles.Fig. S4 Alignment of pathway regulator AflR from 19 D. septosporum strains. Amino acid changes compared to strain NZE10 are highlighted in blue (these sites are also variant between AflR sequences of D. septosporum , Cladosporium fulvum , Aspergillus parasiticus and Aspergillus nidulans ; (Chettri et al ., 2013)) or in green (at sites conserved between those four species). The Zn2Cys6 zinc binuclear domain is highlighted in pink; the linker sequence thought to determine DNA binding specificity in grey; the acidic glutamine rich motif in yellow and C terminal arginine residues implicated in AflJ binding in red.Fig. S5 Secondary structure predictions for AflR from D. septosporum NZE10 and ALP3. Pairwise alignment predicted by HHpred. The arrow indicates the location of the N349K polymorphism in ALP3.Table S1 Transposable elements in the Dothistroma septosporum genomes.Table S2 Genes deleted in the 18 genomes compared to Dothistroma septosporum NZE10.Table S3 Genes deleted from chromosome 14 and their expression levels in NZE10.Table S4 Single Nucleotide Polymorphisms (SNPs) in dothistromin genes, grouped by dothistromin gene loci.Table S5 Deleted genes on Dothistroma septosporum chromosome 12.Table S6 Gene duplications predicted by CNV (copy number variant) analysis.Table S7 (a) Polymerase Chain Reaction (PCR) primers used for verification of 5:13 translocation (b) Primers used for copy number variant (CNV) verification (quantitative PCR [qPCR]).The Bio‐Protection Research Centre of New Zealand, Scion and Massey University.https://onlinelibrary.wiley.com/journal/13643703pm2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog