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July 7, 2019

Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.


July 7, 2019

A gapless genome sequence of the fungus Botrytis cinerea.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, we here report a gapless, near-finished genome sequence for B. cinerea strain B05.10. The assembly comprises 18 chromosomes and was confirmed by an optical map and a genetic map based on ~75 000 SNP markers. All chromosomes contain fully assembled centromeric regions, and 10 chromosomes have telomeres on both ends. The genetic map consisted of 4153 cM and comparison of genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that confer resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that UTRs of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress. This article is protected by copyright. All rights reserved. © 2016 BSPP AND JOHN WILEY & SONS LTD.


July 7, 2019

Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi.

While recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes.Overall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source.The competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from the phyllosphere. Noteworthy was the strong antagonistic activity of the soil yeast C. subhashii, which had so far only been described from a clinical sample and not been studied with respect to biocontrol. Based on binary competition assays and growth analyses (e.g., on different carbon sources, growth in root exudates), C. subhashii was identified as a competitive and antagonistic soil yeast with potential as a novel biocontrol agent against plant pathogenic fungi.


July 7, 2019

Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data.

The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present.We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and then performs read correction and de novo assembly using Sprai. Organelle-PBA completes the assembly process with the additional step of scaffolding by SSPACE-LongRead. The program then detects the chloroplast inverted repeats and reassembles and re-orients the assembly based on the organelle origin of the reference. We have evaluated the performance of the software using PacBio reads from different species, read coverage, and reference genomes. Finally, we present the assembly of two novel chloroplast genomes from the species Picea glauca (Pinaceae) and Sinningia speciosa (Gesneriaceae).Organelle-PBA is an easy-to-use Perl-based software pipeline that was written specifically to assemble mitochondrial and chloroplast genomes from whole genome PacBio reads. The program is available at https://github.com/aubombarely/Organelle_PBA .


July 7, 2019

Identification of small RNAs in extracellular vesicles from the commensal yeast Malassezia sympodialis.

Malassezia is the dominant fungus in the human skin mycobiome and is associated with common skin disorders including atopic eczema (AE)/dermatitis. Recently, it was found that Malassezia sympodialis secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses. Extracellular vesicles from different cell-types including fungi have been found to deliver functional RNAs to recipient cells. In this study we assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ when M. sympodialis is cultured at normal human skin pH versus the elevated pH present on the skin of patients with AE. The total number and the protein concentration of the released MalaEx harvested after 48?h culture did not differ significantly between the two pH conditions nor did the size of the vesicles. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides consistently present in the MalaEx. The levels of small RNAs were not significantly differentially expressed between the two different pH conditions indicating that they are not influenced by the elevated pH level observed on the AE skin.


July 7, 2019

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.


July 7, 2019

Genome sequence of a unique Magnaporthe oryzae RMg-Dl isolate from India that causes blast disease in diverse cereal crops, obtained using PacBio single-molecule and Illumina HiSeq2500 sequencing.

The whole-genome assembly of a unique rice isolate from India, Magnaporthe oryzae RMg-Dl that causes blast disease in diverse cereal crops is presented. Analysis of the 34.82 Mb genome sequence will aid in better understanding the genetic determinants of host range, host jump, survival, pathogenicity, and virulence factors of M. oryzae. Copyright © 2017 Kumar et al.


July 7, 2019

Genomic sequence of ‘Candidatus Liberibacter solanacearum’ haplotype C and its comparison with haplotype A and B genomes.

Haplotypes A and B of ‘Candidatus Liberibacter solanacearum’ (CLso) are associated with diseases of solanaceous plants, especially Zebra chip disease of potato, and haplotypes C, D and E are associated with symptoms on apiaceous plants. To date, one complete genome of haplotype B and two high quality draft genomes of haplotype A have been obtained for these unculturable bacteria using metagenomics from the psyllid vector Bactericera cockerelli. Here, we present the first genomic sequences obtained for the carrot-associated CLso. These two genomic sequences of haplotype C, FIN114 (1.24 Mbp) and FIN111 (1.20 Mbp), were obtained from carrot psyllids (Trioza apicalis) harboring CLso. Genomic comparisons between the haplotypes A, B and C revealed that the genome organization differs between these haplotypes, due to large inversions and other recombinations. Comparison of protein-coding genes indicated that the core genome of CLso consists of 885 ortholog groups, with the pan-genome consisting of 1327 ortholog groups. Twenty-seven ortholog groups are unique to CLso haplotype C, whilst 11 ortholog groups shared by the haplotypes A and B, are not found in the haplotype C. Some of these ortholog groups that are not part of the core genome may encode functions related to interactions with the different host plant and psyllid species.


July 7, 2019

Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


July 7, 2019

Complete genome sequences of six copper-resistant Xanthomonas strains causing bacterial spot of solaneous plants, belonging to X. gardneri, X. euvesicatoria, and X. vesicatoria, using long-read technology.

Xanthomonas vesicatoria, Xanthomonas euvesicatoria, and Xanthomonas gardneri cause bacterial spot disease. Copper has been applied since the 1920s as part of integrated management programs. The first copper-resistant strains were reported some decades later. Here, we fully sequenced six Xanthomonas strains pathogenic to tomato and/or pepper and having a copper-resistant phenotype. Copyright © 2017 Richard et al.


July 7, 2019

Draft genome sequence of Karnal bunt pathogen (Tilletia indica) of wheat provides insights into the pathogenic mechanisms of quarantined fungus.

Karnal bunt disease in wheat is caused by hemibiotrophic fungus, Tilletia indica that has been placed as quarantine pest in more than 70 countries. Despite its economic importance, little knowledge about the molecular components of fungal pathogenesis is known. In this study, first time the genome sequence of T. indica has been deciphered for unraveling the effectors’ functions of molecular pathogenesis of Karnal bunt disease. The T. indica genome was sequenced employing hybrid approach of PacBio Single Molecule Real Time (SMRT) and Illumina HiSEQ 2000 sequencing platforms. The genome was assembled into 10,957 contigs (N50 contig length 3 kb) with total size of 26.7 Mb and GC content of 53.99%. The number of predicted putative genes were 11,535, which were annotated with Gene Ontology databases. Functional annotation of Karnal bunt pathogen genome and classification of identified effectors into protein families revealed interesting functions related to pathogenesis. Search for effectors’ genes using pathogen host interaction database identified 135 genes. The T. indica genome sequence and putative genes involved in molecular pathogenesis would further help in devising novel and effective disease management strategies including development of resistant wheat genotypes, novel biomarkers for pathogen detection and new targets for fungicide development.


July 7, 2019

AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15(T), isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.


July 7, 2019

Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C.

The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.


July 7, 2019

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism.

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3?Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host.


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