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.

A New TaqMan Real-Time PCR Assay for Detecting the Blueberry Pathogen Monilinia vaccinii-corymbosi.

Monilinia vaccinii-corymbosi (Mvc) is an important fungal pathogen of blueberry, causing mummy berry disease. While the symptoms of the advanced stages of the disease can be obvious, diagnosing its early stages can be challenging. To facilitate fast and sensitive screening of asymptomatic or latently infected plant material for Mvc, we developed a specific TaqMan real-time PCR assay targeting the internal transcribed spacer (ITS) region. The assay was shown to be specific to Mvc and did not cross react with any of the other tested Monilinia species or other blueberry pathogens. Using the multicopy ITS region ensured high analytical sensitivity, enabling very low concentrations of Mvc DNA (0.1 pg) to be detected both in water and host DNA matrix. Comparable results were obtained in interlaboratory testing, showing that the assay is robust, and can be effectively used in other laboratories. Assay sensitivity was also confirmed on infected plant tissue, showing that it is effective in detecting the pathogen in infected asymptomatic stem tissue, as well as infected tissue that was mixed with healthy tissue at a ratio of 1:10 by weight. The assay was duplexed with a plant internal control (cytochrome oxidase gene) for simultaneous amplification of the pathogen and plant internal control in a single reaction. This new diagnostic tool can be used for sensitive and rapid screening of blueberry plants for the presence of Mvc in many different settings, e.g., for breeding programs, research, or biosecurity diagnostics.

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.

Comparison of the complete genome sequence of two closely related isolates of 'Candidatus Phytoplasma australiense' reveals genome plasticity.

BACKGROUND: 'Candidatus Phytoplasma australiense' is associated with at least nine diseases in Australia and New Zealand. The impact of this phytoplasma is considerable, both economically and environmentally. The genome of a NZ isolate was sequenced in an effort to understand its pathogenicity and ecology. Comparison with a closely related Australian isolate enabled us to examine mechanisms of genomic rearrangement. RESULTS: The complete genome sequence of a strawberry lethal yellows (SLY) isolate of 'Candidatus Phytoplasma australiense' was determined. It is a circular genome of 959,779 base pairs with 1126 predicted open reading frames. Despite being 80 kbp larger than another 'Ca. Phytoplasma australiense' isolate PAa, the variation between housekeeping genes was generally less than 1% at a nucleotide level. The difference in size between the two isolates was largely due to the number and size of potential mobile units (PMUs), which contributed to some changes in gene order. Comparison of the genomes of the two isolates revealed that the highly conserved 5' UTR of a putative DNA-directed RNA polymerase seems to be associated with insertion and rearrangement events. Two types of PMUs have been identified on the basis of the order of three to four conserved genes, with both PMUs appearing to have been present in the last common ancestor of 'Ca. Phytoplasma asteris' and 'Ca. Phytoplasma australiense'. Comparison with other phytoplasma genomes showed that modification methylases were, in general, species-specific. A putative methylase (xorIIM) found in 'Ca. Phytoplasma australiense' appeared to have no analogue in any other firmicute, and we believe has been introduced by way of lateral gene transfer. A putative retrostransposon (ltrA) analogous to that found in OY-M was present in both isolates, although all examples in PAa appear to be fragments. Comparative analysis identified highly conserved 5' and 3' UTR regions of ltrA, which may indicate how the gene is excised and inserted. CONCLUSIONS: Comparison of two assembled 'Ca. Phytoplasma australiense' genomes has shown they possess a high level of plasticity. This comparative analysis has yielded clues as to how rearrangements occur, and the identification of sets of genes that appear to be associated with these events.

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.

Phylogenetic Analysis of "Candidatus Phytoplasma australiense" Reveals Distinct Populations in New Zealand.

ABSTRACT The phytoplasma "Candidatus Phytoplasma australiense" has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16-23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and "Candidatus Phytoplasma australiense". Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.

Complete DNA Sequence of Pseudomonas syringae pv. actinidiae, the Causal Agent of Kiwifruit Canker Disease.

Pseudomonas syringae pv. actinidiae is the causal agent of bacterial canker of kiwifruit, a disease that has rapidly spread worldwide. We have fully sequenced and assembled the chromosomal and plasmid DNA from P. syringae pv. actinidiae ICMP 18884 using the PacBio RS II platform.

Association of "Candidatus Phytoplasma australiense" with Sudden Decline of Cabbage Tree in New Zealand.

Sudden decline of the New Zealand cabbage tree (Cordyline australis) results in the rapid death of affected plants within months of first external symptoms becoming apparent. Symptoms, which have been observed in saplings and mature trees, include vascular discoloration and leaf yellowing followed by leaf desiccation and eventual plant collapse. Previous work failed to link the disease with any causal agent. A phytoplasma has now been detected in all symptomatic saplings and some symptomatic trees tested, using one-step and nested polymerase chain reaction (PCR) to amplify portions of the 16S rRNA gene. This phytoplasma was not detected in nonsymptomatic plants. Phytoplasma DNA was found in shoot and rhizome apices, leaves and wood tissue of saplings, and in the rhizome apex and trunk tissues of adult trees. Sequencing of the PCR products from selected samples indicated that the phytoplasma is "Candidatus Phytoplasma australiense." Phytoplasma cells were detected by transmission electron microscopy in phloem sieve tubes of the rhizomes of affected saplings. One sapling with early symptoms recovered after injection with tetracycline antibiotic, but two saplings with advanced symptoms did not recover. It is concluded that "Candidatus Phytoplasma australiense" is present in symptomatic plants and is the cause of sudden decline.

Phytoplasma plasmid DNA extraction.

Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense".

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.