The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi.

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.

Contrasting effector profiles between bacterial colonisers of kiwifruit reveal redundant roles converging on PTI-suppression and RIN4.

Testing effector knockout strains of the Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) for reduced in planta growth in their native kiwifruit host revealed a number of nonredundant effectors that contribute to Psa3 virulence. Conversely, complementation in the weak kiwifruit pathogen P. syringae pv. actinidifoliorum (Pfm) for increased growth identified redundant Psa3 effectors. Psa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus (ΔEEL; 10 effectors) were significant contributors to bacterial colonisation of the host and were additive in their effects on virulence. Four of the EEL effectors (HopD1a, AvrB2b, HopAW1a and HopD2a) redundantly contribute to virulence through suppression of pattern-triggered immunity (PTI). Important Psa3 effectors include several redundantly required effectors early in the infection process (HopZ5a, HopH1a, AvrPto1b, AvrRpm1a and HopF1e). These largely target the plant immunity hub, RIN4. This comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease.

Comparison of the pathway structures influencing the temporal response of salicylate and jasmonate defence hormones in Arabidopsis thaliana.

Defence phytohormone pathways evolved to recognize and counter multiple stressors within the environment. Salicylic acid responsive pathways regulate the defence response to biotrophic pathogens whilst responses to necrotrophic pathogens, herbivory, and wounding are regulated via jasmonic acid pathways. Despite their contrasting roles in planta, the salicylic acid and jasmonic acid defence networks share a common architecture, progressing from stages of biosynthesis, to modification, regulation, and response. The unique structure, components, and regulation of each stage of the defence networks likely contributes, in part, to the speed, establishment, and longevity of the salicylic acid and jasmonic acid signaling pathways in response to hormone treatment and various biotic stressors. Recent advancements in the understanding of the Arabidopsis thaliana salicylic acid and jasmonic acid signaling pathways are reviewed here, with a focus on how the structure of the pathways may be influencing the temporal regulation of the defence responses, and how biotic stressors and the many roles of salicylic acid and jasmonic acid in planta may have shaped the evolution of the signaling networks.

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance.

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of ΦPsa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T = 9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage resistance is overcome by mutations in a tail fibre and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374-like phages that informs their use as antimicrobials against Psa.

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta.

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors-AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a -suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

Rapid Methodologies for Assessing Pseudomonas syringae pv. actinidiae Colonization and Effector-Mediated Hypersensitive Response in Kiwifruit.

The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here, we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens, and a transient reporter eclipse assay for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the P. fluorescens and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes.

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit.

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex.

The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l- and d-rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o-methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

Transcriptome analysis of Eucalyptus grandis genotypes reveals constitutive overexpression of genes related to rust (Austropuccinia psidii) resistance.

Key Message A resistant E. grandis genotype showed a constitutive overexpression of genes related to resistance to myrtle rust caused by A. psidii. Abstract Myrtle rust caused by Austropuccinia psidii is considered one of the most important fungal diseases affecting Eucalyptus spp. plantations in Brazil. Although the selection and planting of resistant eucalypt genotypes have been the major strategies to manage the disease in Brazil, the molecular mechanisms involved in resistance are still unclear. In this study, we evaluated the gene expression profile of two contrasting Eucalyptus grandis genotypes in resistance level to rust by RNA-Seq. The two genotypes showed a very different background gene expression level even without A. psidii infection. The resistant genotype had a constitutive overexpression of a large number of protein-coding genes compared to the susceptible genotype. These genes were mainly associated with signal transduction, photosynthesis, regulation and response to salicylic acid (SA), and protein kinase leucine-rich receptors (PK-LRR). PK-LRR and SA mediated disease resistance are well known to be effective against obligate biotroph pathogens, such as A. psidii. In addition, at 24 h after infection, the susceptible genotype was able to activate some response, however, several resistance-related proteins had their expression level reduced with A. psidii infection. Here, we present the first analysis of E. grandis genotypes transcriptomes infected by A. psidii and it reveals a constitutive overexpression of several resistance-related genes in the resistant genotype compared to the susceptible one. Our findings have the potential to be used as candidate molecular markers for resistance to myrtle rust.

Comparative genomic and transcriptomic analyses reveal different pathogenicity-related genes among three eucalyptus fungal pathogens.

Ceratocystis fimbriata is an important plant pathogen known to cause Ceratocystis Wilt (CW), a prevalent fungal disease known to affect Eucalyptus spp. plantations in Brazil. To better understand the molecular mechanisms related to pathogenicity in eucalyptus, we generated a high-quality assembly and annotation of the Ce. fimbriata LPF1912 isolate (LPF1912) genome, as well as the first transcriptome of LPF1912 from 16 eucalyptus clones at three infection incubation periods (12, 18, and 24 h). The LPF1912 genome assembly contains 805 scaffolds, totaling 31.8 Mb, with 43% of the genome estimated to be coding sequence comprised of 7,390 protein-coding genes of which 626 (8.5%) were classified as secreted proteins, 120 ribosomal RNAs, and 532 transfer RNAs. Comparative genomic analysis among three eucalyptus fungal pathogens (Ce. fimbriata, Ce. eucalypticola, and Calonectria pseudoreteaudii), showed high similarity in the proteome (21.81%) and secretome (52.01%) of LPF1912 and Ce. eucalypticola. GO annotation of pathogenicity-related genes of LPF1912 and Ce. eucalypticola, revealed enrichment in cell wall degrading enzymes (CWDEs), and lipid/cutin metabolism for Ca. pseudoreteaudii. Additionally, a transcriptome analysis between resistant and susceptible eucalyptus clones to CW infection indicated that a majority (11) of LPF1912 differentially expressed genes had GO terms associated with enzymatic functions, such as the polygalacturonase gene family, confirming the crucial role of CWDEs for Ce. fimbriata pathogenicity. Finally, our genomic and transcriptomic analysis approach provides a better understanding of the mechanisms involved in Ce. fimbriata pathogenesis, as well as a framework for further studies.

Construction of a kiwifruit yeast two-hybrid cDNA library to identify host targets of the Pseudomonas syringae pv. actinidiae effector AvrPto5.

OBJECTIVE: Bacterial canker is a destructive disease of kiwifruit caused by the Gram-negative bacterium Pseudomonas syringae pv. actinidiae (Psa). To understand the disease-causing mechanism of Psa, a kiwifruit yeast two-hybrid cDNA library was constructed to identify putative host targets of the Psa Type Three Secreted Effector AvrPto5. RESULTS: In this study, we used the Mate & Plate™ yeast two-hybrid library method for constructing a kiwifruit cDNA library from messenger RNA of young leaves. The constructed library consisted of 2.15 × 106 independent clones with an average insert size of 1.52 kb. The screening of the kiwifruit yeast two-hybrid cDNA library with Psa AvrPto5 revealed the interaction of a V-type proton ATPase subunit-H, a proline rich-protein and heavy metal-associated isoprenylated plant protein 26. Among these, heavy metal-associated isoprenylated plant protein 26 showed a positive interaction with Psa AvrPto5 as both prey and bait.

Re-programming of Pseudomonas syringae pv. actinidiae gene expression during early stages of infection of kiwifruit.

BACKGROUND: Pseudomonas syringae is a widespread bacterial species complex that includes a number of significant plant pathogens. Amongst these, P. syringae pv. actinidiae (Psa) initiated a worldwide pandemic in 2008 on cultivars of Actinidia chinensis var. chinensis. To gain information about the expression of genes involved in pathogenicity we have carried out transcriptome analysis of Psa during the early stages of kiwifruit infection. RESULTS: Gene expression in Psa was investigated during the first five days after infection of kiwifruit plantlets, using RNA-seq. Principal component and heatmap analyses showed distinct phases of gene expression during the time course of infection. The first phase was an immediate transient peak of induction around three hours post inoculation (HPI) that included genes that code for a Type VI Secretion System and nutrient acquisition (particularly phosphate). This was followed by a significant commitment, between 3 and 24 HPI, to the induction of genes encoding the Type III Secretion System (T3SS) and Type III Secreted Effectors (T3SE). Expression of these genes collectively accounted for 6.3% of the bacterial transcriptome at this stage. There was considerable variation in the expression levels of individual T3SEs but all followed the same temporal expression pattern, with the exception of hopAS1, which peaked later in expression at 48 HPI. As infection progressed over the time course of five days, there was an increase in the expression of genes with roles in sugar, amino acid and sulfur transport and the production of alginate and colanic acid. These are both polymers that are major constituents of extracellular polysaccharide substances (EPS) and are involved in biofilm production. Reverse transcription-quantitative PCR (RT-qPCR) on an independent infection time course experiment showed that the expression profile of selected bacterial genes at each infection phase correlated well with the RNA-seq data. CONCLUSIONS: The results from this study indicate that there is a complex remodeling of the transcriptome during the early stages of infection, with at least three distinct phases of coordinated gene expression. These include genes induced during the immediate contact with the host, those involved in the initiation of infection, and finally those responsible for nutrient acquisition.

The ecological genetics of Pseudomonas syringae from kiwifruit leaves.

Interactions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurring Pseudomonas syringae isolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae (Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenic Psa and commensal P. syringae PG3a isolated from the same leaf was tested using reciprocal invasion from rare assays in vitro and in planta. P. syringae G33C (PG3a) inhibited Psa NZ54, while the presence of Psa NZ54 enhanced the growth of P. syringae G33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system of Psa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations.

Specific Hypersensitive Response-Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato.

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

A bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response.

Type-III secreted effectors (T3Es) play critical roles during bacterial pathogenesis in plants. Plant recognition of certain T3Es can trigger defence, often accompanied by macroscopic cell death, termed the hypersensitive response (HR). Economically important species of kiwifruit are susceptible to Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker. Although Psa is non-pathogenic in Arabidopsis thaliana, we observed that a T3E, HopZ5 that is unique to a global outbreak clade of Psa, triggers HR and defence in Arabidopsis accession Ct-1. Ws-2 and Col-0 accessions are unable to produce an HR in response to Pseudomonas-delivered HopZ5. While Ws-2 is susceptible to virulent bacterial strain Pseudomonas syringae pv. tomato DC3000 carrying HopZ5, Col-0 is resistant despite the lack of an HR. We show that HopZ5, like other members of the YopJ superfamily of acetyltransferases that it belongs to, autoacetylates lysine residues. Through comparisons to other family members, we identified an acetyltransferase catalytic activity and demonstrate its requirement for triggering defence in Arabidopsis and Nicotiana species. Collectively, data herein indicate that HopZ5 is a plasma membrane-localized acetyltransferase with autoacetylation activity required for avirulence.

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range.

BACKGROUND: Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. RESULTS: Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes. CONCLUSIONS: Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Origin and Evolution of the Kiwifruit Canker Pandemic.

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose.

Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

Evolution of copper resistance in the kiwifruit pathogen Pseudomonas syringae pv. actinidiae through acquisition of integrative conjugative elements and plasmids.

Horizontal gene transfer can precipitate rapid evolutionary change. In 2010 the global pandemic of kiwifruit canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) reached New Zealand. At the time of introduction, the single clone responsible for the outbreak was sensitive to copper, however, analysis of a sample of isolates taken in 2015 and 2016 showed that a quarter were copper resistant. Genome sequences of seven strains showed that copper resistance - comprising czc/cusABC and copABCD systems - along with resistance to arsenic and cadmium, was acquired via uptake of integrative conjugative elements (ICEs), but also plasmids. Comparative analysis showed ICEs to have a mosaic structure, with one being a tripartite arrangement of two different ICEs and a plasmid that were isolated in 1921 (USA), 1968 (NZ) and 1988 (Japan), from P. syringae pathogens of millet, wheat and kiwifruit respectively. Two of the Psa ICEs were nearly identical to two ICEs isolated from kiwifruit leaf colonists prior to the introduction of Psa into NZ. Additionally, we show ICE transfer in vitro and in planta, analyze fitness consequences of ICE carriage, capture the de novo formation of novel recombinant ICEs, and explore ICE host-range.