The application of the CRISPR-Cas9 system in Pseudomonas syringae pv. actinidiae.

Introduction. Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world.Aim. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species.Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa.Results. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of S. pyogenes, which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site.Conclusion. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.

Genomic analyses of Pseudomonas syringae pv. actinidiae isolated in Korea suggest the transfer of the bacterial pathogen via kiwifruit pollen.

Introduction. The bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa), has emerged as a major threat to kiwifruit cultivation throughout the world. One pandemic strain (from the Psa3 group) has occurred in various geographical regions. It is important to understand how this pathogen is being transmitted.Aim. Although Psa has been found in Korea since 1992, the isolates were until recently of a distinct type (Psa2). Recently, the more virulent Psa3 type has been detected. The purpose of this study was to describe the variety of Psa3 now found in Korea.Methodology. Strains were isolated from kiwifruit plants in Korea and from pollen imported into Korea from New Zealand. The genomes of 10 isolates were sequenced using the Illumina platform and compared to the completely assembled genomes of pandemic Psa3 strains from New Zealand and China. Comparisons were also made with pandemic strains from Chile and non-pandemic Psa3 isolates from China.Results. Six of the 10 Psa3 isolates from Korea show a clear relationship with New Zealand isolates. Two isolates show a distinct relationship to isolates from Chile; one further isolate has a sequence that is highly similar to that of M228, a strain previously isolated in China; and the last isolate belongs to the Psa3 group, but is not a member of the pandemic lineage.Conclusion. This analysis establishes that there have been multiple routes of transmission of the Psa3 pandemic strain into Korea. One route has involved the importation of pollen from New Zealand. A second route probably involves importation from Chile.

The Genome Sequence of M228, a Chinese Isolate of Pseudomonas syringae pv. actinidiae, Illustrates Insertion Sequence Element Mobility.

We present here the complete genome sequence of M228, a Chinese biovar 3 strain of Pseudomonas syringae pv. actinidiae, a bacterial pathogen of kiwifruit. A comparison of the insertion sequence (IS) profile of M228 with that of ICMP18708, a New Zealand isolate of P. syringae pv. actinidiae, provided insight into the evolutionary history of IS elements within biovar 3.

Single-Phase PacBio De Novo Assembly of the Genome of the Chytrid Fungus Batrachochytrium dendrobatidis, a Pathogen of Amphibia.

Here, we present an updated genome assembly of the diploid chytrid fungus Batrachochytrium dendrobatidis strain RTP6. This strain is part of the global panzootic lineage (BdGPL) and was isolated in Dunedin, New Zealand. The assembly was generated using PacBio long-read and Illumina short-read data, allowing for the accurate phasing of heterozygosities.

Comparison between complete genomes of an isolate of Pseudomonas syringae pv. actinidiae from Japan and a New Zealand isolate of the pandemic lineage.

The modern pandemic of the bacterial kiwifruit pathogen Pseudomonas syringae pv actinidiae (Psa) is caused by a particular Psa lineage. To better understand the genetic basis of the virulence of this lineage, we compare the completely assembled genome of a pandemic New Zealand strain with that of the Psa type strain first isolated in Japan in 1983. Aligning the two genomes shows numerous translocations, constrained so as to retain the appropriate orientation of the Architecture Imparting Sequences (AIMs). There are several large horizontally acquired regions, some of which include Type I, Type II or Type III restriction systems. The activity of these systems is reflected in the methylation patterns of the two strains. The pandemic strain carries an Integrative Conjugative Element (ICE) located at a tRNA-Lys site. Two other complex elements are also present at tRNA-Lys sites in the genome. These elements are derived from ICE but have now acquired some alternative secretion function. There are numerous types of mobile element in the two genomes. Analysis of these elements reveals no evidence of recombination between the two Psa lineages.

Complete Genome Sequence of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Biovar 5, Originating from Japan.

We present the first complete genome sequence of a copper-resistant biovar 5 strain of a bacterial pathogen of kiwifruit, Pseudomonas syringae pv. actinidiae. Comparison with the genome sequence of a copper-sensitive biovar 5 isolate indicates that copper resistance is encoded on a plasmid.

Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide.

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.

Tyrosine Recombinase Retrotransposons and Transposons.

Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.

Pseudomonas syringae pv. actinidiae from recent outbreaks of kiwifruit bacterial canker belong to different clones that originated in China.

A recently emerged plant disease, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis), is caused by Pseudomonas syringae pv. actinidiae (PSA). The disease was first reported in China and Japan in the 1980s. A severe outbreak of PSA began in Italy in 2008 and has spread to other European countries. PSA was found in both New Zealand and Chile in 2010. To study the evolution of the pathogen and analyse the transmission of PSA between countries, genomes of strains from China and Japan (where the genus Actinidia is endemic), Italy, New Zealand and Chile were sequenced. The genomes of PSA strains are very similar. However, all strains from New Zealand share several single nucleotide polymorphisms (SNPs) that distinguish them from all other PSA strains. Similarly, all the PSA strains from the 2008 Italian outbreak form a distinct clonal group and those from Chile form a third group. In addition to the rare SNPs present in the core genomes, there is abundant genetic diversity in a genomic island that is part of the accessory genome. The island from several Chinese strains is almost identical to the island present in the New Zealand strains. The island from a different Chinese strain is identical to the island present in the strains from the recent Italian outbreak. The Chilean strains of PSA carry a third variant of this island. These genomic islands are integrative conjugative elements (ICEs). Sequencing of these ICEs provides evidence of three recent horizontal transmissions of ICE from other strains of Pseudomonas syringae to PSA. The analyses of the core genome SNPs and the ICEs, combined with disease history, all support the hypothesis of an independent Chinese origin for both the Italian and the New Zealand outbreaks and suggest the Chilean strains also originate from China.

PRP8 inteins in species of the genus Botrytis and other ascomycetes.

The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state.

Marine bacterial succession as a potential indicator of postmortem submersion interval.

The process of decomposition of bodies in the marine environment is poorly understood and almost nothing is currently known about the microorganisms involved. This study aimed to investigate the microbes involved in decomposition in the sea and to evaluate the potential use of marine bacterial succession for postmortem submersion interval (PMSI) estimation, for which there is currently no reliable method. Partial pig remains were completely submerged during autumn and winter and were regularly sampled to document marine bacterial colonisation and the changes in community composition over time. Five stages of decomposition were recognised, some of which exhibited characters specific for partial carrion. Marine bacteria rapidly colonised the submerged remains in a successional manner. Seasonal differences were observed for the rate of decomposition and also for several groups of colonising bacteria. Marine bacteria specific for particular PMSIs were identified. This study provides an insight into the involvement of saprophytic marine bacteria in the decomposition of mammalian remains in the sea and is the first to explore the use of marine bacterial colonisation and succession as a novel tool for PMSI estimation. We propose that with further study, marine bacterial succession will prove useful for determination of the length of time a body may have been immersed in a marine environment.

Sexual mating of Botrytis cinerea illustrates PRP8 intein HEG activity.

Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/-). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/- heterozygote, the homing endonuclease initiates intein 'homing' by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.

Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans.

Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis.

Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

Elimination of the amphibian chytrid fungus Batrachochytrium dendrobatidis by Archey's frog Leiopelma archeyi.

Archey's frog Leiopelma archeyi is a critically endangered New Zealand endemic species. The discovery of the emerging infectious disease, chytridiomycosis, in wild populations of this frog raised concern that this disease may drive the species to extinction. Twelve wild-caught Archey's frogs naturally infected with the amphibian chytrid fungus Batrachochytrium dendrobatidis were monitored in captivity by observing clinical signs, measuring weight gain, and performing repeated PCR tests. Eight frogs were treated with topical chloramphenicol, without PCR results being available, for B. dendrobatidis at the day of entry of the frog into the trial. Eleven of the 12 frogs (92%) cleared their infection within 3 mo of capture, even though they were held at 15 degrees C and in high humidity, conditions that are ideal for the survival and propagation of B. dendrobatidis. B. dendrobatidis in the remaining frog tested positive for the fungus was eliminated after treatment with topical chloramphenicol. None of the 8 frogs exposed to chloramphenicol showed any acute adverse reactions. Archey's frog appears to have a low level of susceptibility to the clinical effects of chytridiomycosis. Individual frogs can eliminate B. dendrobatidis and Archey's frog can apparently be treated with topical chloramphenicol with no acute adverse reactions. However, the small number of specimens treated here requires that more extensive testing be done to confirm the safety of chloramphenicol. The significance of the amphibian chytrid fungus for wild populations of Archey's frog needs to be determined by a longitudinal study in an infected wild population to correlate the presence of B. dendrobatidis in individual frogs. Such a study should occur over a period of at least 3 yr with clinical assessment and monitoring of survival, growth and body condition parameters.

LINE-like retrotransposition in Saccharomyces cerevisiae.

Over one-third of human genome sequence is a product of non-LTR retrotransposition. The retrotransposon that currently drives this process in humans is the highly abundant LINE-1 (L1) element. Despite the ubiquitous nature of L1's in mammals, we still lack a complete mechanistic understanding of the L1 replication cycle and how it is regulated. To generate a genetically amenable model for non-LTR retrotransposition, we have reengineered the Zorro3 retrotransposon, an L1 homolog from Candida albicans, for use in the budding yeast Saccharomyces cerevisiae. We found that S. cerevisiae, which has no endogenous L1 homologs or remnants, can still support Zorro3 retrotransposition. Analysis of Zorro3 mutants and insertion structures suggest that this is authentic L1-like retrotransposition with remarkable resemblance to mammalian L1-mediated events. This suggests that S. cerevisiae has unexpectedly retained the basal host machinery required for L1 retrotransposition. This model will also serve as a powerful system to study the cell biology of L1 elements and for the genetic identification and characterization of cellular factors involved in L1 retrotransposition.

A yeast model for target-primed (non-LTR) retrotransposition.

BACKGROUND: Target-primed (non-LTR) retrotransposons, such as the human L1 element, are mobile genetic elements found in many eukaryotic genomes. They are often present in large numbers and their retrotransposition can cause mutations and genomic rearrangements. Despite their importance, many aspects of their replication are not well understood. RESULTS: We have developed a yeast model system for studying target-primed retrotransposons. This system uses the Zorro3 element from Candida albicans. A cloned copy of Zorro3, tagged with a retrotransposition indicator gene, retrotransposes at a high frequency when introduced into an appropriate C. albicans host strain. Retrotransposed copies of the tagged element exhibit similar features to the native copies, indicating that the natural retrotransposition pathway is being used. Retrotransposition is dependent on the products of the tagged element's own genes and is highly temperature-regulated. The new assay permits the analysis of the effects of specific mutations introduced into the cloned element. CONCLUSION: This Zorro3 retrotransposition assay system complements previously available target-primed retrotransposition assays. Due to the relative simplicity of the growth, manipulation and analysis of yeast cells, the system should advance our understanding of target-primed retrotransposition.

Preceding hydrophobic and beta-branched amino acids attenuate splicing by the CnePRP8 intein.

As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the alpha-complementation peptide abolished the peptide's ability to restore beta-galactosidase activity, while an active variant allowed complementation. This alpha-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a beta-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.

Sequence requirements for splicing by the Cne PRP8 intein.

The dependence of protein splicing on conserved residues of the Cne PRP8 intein was assessed by alanine scanning mutagenesis in a foreign protein context. Corroboration was obtained for the involvement of residues at the splice junctions and of the conserved threonine and histidine of motif B. Five additional residues were identified as absolutely required for splicing. Variant W151A displayed premature C-terminal cleavage, not seen with other Cne PRP8 mutants. We propose a model whereby W151 acts to prevent premature C-terminal cleavage, favoring complete splicing as opposed to two disjointed cleavage events.

The nuclear-encoded inteins of fungi.

An intein is a protein sequence embedded within a precursor protein that is excised during protein maturation. Inteins were first found encoded in the VMA gene of Saccharomyces cerevisiae. Subsequently, they have been found in diverse organisms (eukaryotes, archaea, eubacteria and viruses). The VMA intein has been found in various saccharomycete yeasts but not in other fungi. Different inteins have now been found widely in the fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins. A protein distantly related to inteins, but closely related to metazoan hedgehog proteins, has been described from Glomeromycota. Many of the newly described inteins contain homing endonucleases and some of these are apparently active. The enlarged fungal intein data set permits insight into the evolution of inteins, including the role of horizontal transfer in their persistence. The diverse fungal inteins provide a resource for biotechnology using their protein splicing or homing endonuclease capabilities.