Honour C. McCann

Genomic Analysis of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Provides Insight into the Origins of an Emergent Plant Disease

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries – even millennia – ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Host–pathogen interplay and the evolution of bacterial effectors

Many bacterial pathogens require a type III secretion system (T3SS) and suite of type III secreted effectors (T3SEs) to successfully colonize their hosts, extract nutrients and consequently cause disease. T3SEs, in particular, are key components of the bacterial arsenal, as they function directly inside the host to disrupt or suppress critical components of the defence network. The development of host defence and surveillance systems imposes intense selective pressures on these bacterial virulence factors, resulting in a host–pathogen co‐evolutionary arms race. This arms race leaves its genetic signature in the pattern and structure of natural genetic variation found in T3SEs, thereby permitting us to infer the specific evolutionary processes and pressures driving these interactions. In this review, we summarize our current knowledge of T3SS‐mediated host–pathogen co‐evolution. We examine the evolution of the T3SS and the T3SEs that traverse it, in both plant and animal pathosystems, and discuss the processes that maintain these important pathogenicity determinants within pathogen populations. We go on to examine the possible origins of T3SEs, the mechanisms that give rise to new T3SEs and the processes that underlie their evolution.

Identification of innate immunity elicitors using molecular signatures of natural selection

The innate immune system is an ancient and broad-spectrum defense system found in all eukaryotes. The detection of microbial elicitors results in the up-regulation of defense-related genes and the elicitation of inflammatory and apoptotic responses. These innate immune responses are the front-line barrier against disease because they collectively suppress the growth of the vast majority of invading microbes. Despite their critical role, we know remarkably little about the diversity of immune elicitors. To address this paucity, we reasoned that hosts are more likely to evolve recognition to “core” pathogen proteins under strong negative selection for the maintenance of essential cellular functions, whereas repeated exposure to host–defense responses will impose strong positive selective pressure for elicitor diversification to avoid host recognition. Therefore, we hypothesized that novel bacterial elicitors can be identified through these opposing forces of natural selection. We tested this hypothesis by examining the genomes of six bacterial phytopathogens and identifying 56 candidate elicitors that have an excess of positively selected residues in a background of strong negative selection. We show that these positively selected residues are atypically clustered, similar to patterns seen in the few well-characterized elicitors. We then validated selected candidate elicitors by showing that they induce Arabidopsis thaliana innate immunity in functional (virulence suppression) and cellular (callose deposition) assays. These finding provide targets for the study of host–pathogen interactions and applied research into alternative antimicrobial treatments.

Evolution, Genomics and Epidemiology of Pseudomonas syringae.

A remarkable shift in our understanding of plant-pathogenic bacteria is underway. Until recently, nearly all research on phytopathogenic bacteria was focused on a small number of model strains, which provided a deep, but narrow, perspective on plant-microbe interactions. Advances in genome sequencing technologies have changed this by enabling the incorporation of much greater diversity into comparative and functional research. We are now moving beyond a typological understanding of a select collection of strains to a more generalized appreciation of the breadth and scope of plant-microbe interactions. The study of natural populations and evolution has particularly benefited from the expansion of genomic data. We are beginning to have a much deeper understanding of the natural genetic diversity, niche breadth, ecological constraints and defining characteristics of phytopathogenic species. Given this expanding genomic and ecological knowledge, we believe the time is ripe to evaluate what we know about the evolutionary dynamics of plant pathogens.

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.

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.

Rapid radiations of both kiwifruit hybrid lineages and their parents shed light on a two-layer mode of species diversification

Reticulate speciation caused by interspecific hybridization is now recognized as an important mechanism in the creation of biological diversity. However, depicting the patterns of phylogenetic networks for lineages that have undergone interspecific gene flow is challenging. Here we sequenced 25 taxa representing natural diversity in the genus Actinidia with an average mapping depth of 26× on the reference genome to reconstruct their reticulate history. We found evidence, including significant gene tree discordance, cytonuclear conflicts, and changes in genome-wide heterozygosity across taxa, collectively supporting extensive reticulation in the genus. Furthermore, at least two separate parental species pairs were involved in the repeated origin of the hybrid lineages, in some of which a further phase of syngameon was triggered. On the basis of the elucidated hybridization relationships, we obtained a highly resolved backbone phylogeny consisting of taxa exhibiting no evidence of hybrid origin. The backbone taxa have distinct demographic histories and are the product of recent rounds of rapid radiations via sorting of ancestral variation under variable climatic and ecological conditions. Our results suggest a mode for consecutive plant diversification through two layers of radiations, consisting of the rapid evolution of backbone lineages and the formation of hybrid swarms derived from these lineages.

The ecological genetics of Pseudomonas syringae residing on the kiwifruit leaf surface

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. ORIGINALITY-SIGNIFICANT STATEMENT Bacterial pathogen populations are often studied with little consideration of co-occurring microbes and yet interactions between pathogens and commensals can affect both population structure and disease progression. A fine-scale sampling of commensals present on kiwifruit leaves during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae reveals a clonal population structure. A new clade of non-pathogenic P. syringae (PG3a) appears to be associated with kiwifruit on a global scale. The presence of PG3a on kiwifruit has significant effects on the outcome of infection by P. syringae pv. actinidiae. This emphasises the value of studying the effect of co-occurring bacteria on pathogen-plant interactions.

TGFam-Finder: An optimal solution for target-gene family annotation in eukaryotic genomes

Whole genome annotation errors that omit essential protein-coding genes hinder further research. We developed Target Gene Family Finder (TGFam-Finder), an optimal tool for structural annotation of protein-coding genes containing target domain(s) of interest in eukaryotic genomes. Large-scale re-annotation of 100 publicly available eukaryotic genomes led to the discovery of essential genes that were missed in previous annotations. An average of 117 (346%) and 148 (45%) additional FAR1 and NLR genes were newly identified in 50 plant genomes. Furthermore, 117 (47%) additional C2H2 zinc finger genes were detected in 50 animal genomes including human and mouse. Accuracy of the newly annotated genes was validated by RT-PCR and cDNA sequencing in human, mouse and rice. In the human genome, 26 newly annotated genes were identical with known functional genes. TGFam-Finder along with the new gene models provide an optimized platform for unbiased functional and comparative genomics and comprehensive evolutionary study in eukaryotes.

Origin and evolution of a pandemic lineage of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa), whose emergence coincided with domestication of its host. The most recent pandemic has had a deleterious effect on kiwifruit production worldwide. In order to strengthen understanding of population structure, phylogeography and evolutionary dynamics of Psa, we sampled 746 Pseudomonas isolates from cultivated and wild kiwifruit across six provinces in China, of which 87 were Psa. Of 234 Pseudomonas isolated from wild Actinidia spp. none were identified as Psa. Genome sequencing of fifty isolates and the inclusion of an additional thirty from previous studies show that China is the origin of the recently emerged pandemic lineage. However China harbours only a fraction of global Psa diversity, with greatest diversity found in Korea and Japan. Distinct transmission events were responsible for introduction of the pandemic lineage of Psa into New Zealand, Chile and Europe. Two independent transmission events occurred between China and Korea, and two Japanese isolates from 2014 cluster with New Zealand Psa. Despite high similarity at the level of the core genome and negligible impact of within-lineage recombination, there has been substantial gene gain and loss even within the single clade from which the global pandemic arose. SIGNIFICANCE STATEMENT Bleeding canker disease of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) has come to prominence in the last three decades. Emergence has coincided with domestication of the host plant and provides a rare opportunity to understand ecological and genetic factors affecting the evolutionary origins of Psa. Here, based on genomic analysis of an extensive set of strains sampled from China and augmented by isolates from a global sample, we show, contrary to earlier predictions, that China is not the native home of the pathogen, but is nonetheless the source of the recent global pandemic. Our data identify specific transmission events, substantial genetic diversity and point to non-agricultural plants in either Japan or Korea as home to the source population.