Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, <i>Armillaria mellea</i>: Toward a New Infection Model System

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the <i>A. mellea</i> genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (<i>n</i> = 629 unique) and secreted (<i>n</i> = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (<i>n</i> = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the <i>A. mellea</i> proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that <i>A. mellea</i> exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive <i>A. mellea</i> proteins (<i>n</i> = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms

Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, <i>Armillaria mellea</i>: Toward a New Infection Model System

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the <i>A. mellea</i> genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (<i>n</i> = 629 unique) and secreted (<i>n</i> = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (<i>n</i> = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the <i>A. mellea</i> proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that <i>A. mellea</i> exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive <i>A. mellea</i> proteins (<i>n</i> = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms

Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, <i>Armillaria mellea</i>: Toward a New Infection Model System

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the <i>A. mellea</i> genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (<i>n</i> = 629 unique) and secreted (<i>n</i> = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (<i>n</i> = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the <i>A. mellea</i> proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that <i>A. mellea</i> exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive <i>A. mellea</i> proteins (<i>n</i> = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms

Germline Transgenesis and Insertional Mutagenesis in <em>Schistosoma mansoni</em> Mediated by Murine Leukemia Virus

<div><p>Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, <em>Schistosoma mansoni</em>, S. <em>japonicum</em> and <em>S. haematobium</em>. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of <em>S. mansoni</em> after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from <em>in vitro</em> laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. </p> <h3>Database accession</h3><p>Sequence data from this study have been submitted to the European Nucleotide Archive (<a href="http://www.ebi.ac.uk/embl">http://www.ebi.ac.uk/embl</a>) under accession number ERP000379.</p> </div

Rescue of transgenic schistosomula in the presence of G418 (geneticin).

<p>Panel A: Experimental design. <i>In vitro</i> laid eggs (IVLE) were transduced with MLV virions and hatched six days later. The miracidia were employed to infect snails and ∼40 days later the cercariae were collected. The presence of the transgene in the cercarial genomic DNA was verified by qPCR and Illumina sequencing. Schistosomula were obtained by transformation of cercariae (cercarial transformation, CT) and cultured in 250 µg/ml G418 as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002820#ppat.1002820-Rinaldi1" target="_blank">[29]</a>. Culture media including G418 were replaced every second day for eight days. Control schistosomula from wild type schistosomes (i.e. non-transgenic) were included. Panel B: Left top; representative micrographs taken on days 4, 6 and 8 of transgenic schistosomula (derived from snails infected with MLV transduced eggs/miracidia) cultured in G418, as indicated. Left bottom; representative micrographs taken by days 4, 6 and 8 of control (i.e. non-transgenic) schistosomula cultured in G418, as indicated. Right; survival of transgenic schistosomules derived from snails infected with MLV transduced eggs/miracidia (green) and control wild type schistosomula (blue), cultured in G418.</p

Schematic overview of approaches employed to introduce retroviral transgenes into the germline of <i>Schistosoma mansoni</i>.

<p>A: Left panel, micrograph showing viable schistosome eggs recovered from livers of <i>S. mansoni</i>-infected mice. Center panel, schematic representation of the MLV virion inoculated into the culture media. Right panel, micrograph of eggs laid <i>in vitro</i> by cultured mix sexed adults of <i>S. mansoni</i>; B: outline of germline transmission in <i>S. mansoni</i>. From the single-celled zygote in the newly fertilized egg that is released by the female schistosome, germ cells are propagated through the intra-snail developmental stages. Within the snail, daughter sporocysts arise from the germ cells of the mother sporocyst, and eventually cercariae develop from the daughter sporocysts. C: Representative micrographs of the embryogenesis of the <i>S. mansoni</i> egg; numbers below the images indicate the embryonic stage according to the staging system of Jurberg et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002820#ppat.1002820-Jurberg1" target="_blank">[42]</a>. The zygotic stage (also termed stage 0) occurs inside the female worm. In culture, development from stages 0 to 8 takes about one week. Triangles above (green) and below (red) the eggs indicate the efficiency of snail infection and transgene copy number using transgenesis approaches targeting LE and IVLE, respectively. In regard to the green triangle, IVLE were cultured for seven days, until stage 8, before they were induced to hatch. *Pertaining to the red triangle, transgene copy numbers were measured in genomic DNA from cercariae.</p

Locations of MLV retroviral transgene integrations within the genome of <i>Schistosoma mansoni</i>.

*<p>proportion of sequence content.</p><p>‘Somatic’ refers to schistosomes (adult or schistosomulum stages) transduced directly with pseudotyped MLV virions. ‘Germline’ refers to the schistosomula derived as progeny from schistosome eggs transduced with virions. Somatic 5′, Somatic 3′, etc. refer to Illumina libraries constructed from regions of the schistosome genome flanking the 5′- or 3′-LTR of the retrovirus.</p><p>‘Promoter/5′-UTR regions’, ≤3 kb upstream of first exon of genes.</p

Construction of Illumina libraries from virion transduced schistosomes.

<p>A: Schematic representation of a representative MLV retrovirus construct integrated into the gDNA isolated from MLV-transduced organisms. The retrovirus cassette included the firefly luciferase reporter gene (LUC) driven by the <i>S. mansoni</i> actin 1.1 gene promoter (<i>Sm</i>ACT), and flanked by the 5′- and 3′-long terminal inverted repeats of the murine leukemia virus (LTR). The cassette also included the gene endowing neomycin resistance (<i>neoR</i>) and the psi motif (ψ) involved in packaging the viral DNA; B: Mechanical fragmentation of the genomic DNA; C: Repair of the fragment ends, adenylation, ligation of the Illumina adapters, and two rounds of semi-nested PCR (colored primers represent the sequencing primers); D: Size selection of the 5′-end (5′) and 3′-end (3′) libraries and gel extraction. The fragment selected from 300 bp to 500 bp (i) was employed to generate the libraries. A higher fragment (ii) was purified and stored as back up. The gel extracted and purified libraries were quantified by qPCR and loaded into Illumina flowcells. L, molecular size standards (ladder).</p

Competitive indices of defined null mutants of <i>S.</i> Typhimurium lacking candidate virulence-associated loci identified by TraDIS.

<p><i>P</i> values are shown in parentheses. The range of TraDIS fitness scores obtained in the chicken experiment is also shown for each gene, with the fraction of significantly attenuated mutants shown in parentheses.</p

Circular diagrams of the <i>S.</i> Typhimurium SL1344 chromosome and plasmids (not to scale), showing the near random distribution and high density of transposon insertions mapped by TraDIS.

<p>The inner two rings indicate the positions of annotated genes, coloured according to their GC content (blue = low, yellow = intermediate, red = high). The outer ring indicates the number of transposon-flanking sequence reads obtained at each position, with peaks corresponding to the presence of a transposon insertion.</p