Venn diagram of the type III effector gene complements of <i>Pseudomonas avellanae</i> species.

<p>The diagram is based on the comparison of the same complement of other sequenced plant pathogenic pseudomonads and showing full identity in the reciprocal comparison. The effector proteins conserved among the seven strains are indicated in the centre of the diagram.</p

Phylogenetic relationships among representative strains of phytopathogenic <i>Pseudomonas</i> species and <i>P. syringae</i> pathovars.

<p>The phylogenetic tree was constructed using 6,579 concatenated nucleotides of seven housekeeping genes (<i>argS, dnaQ, gltA, gyrB, recA, rpoB</i> and <i>rpoD</i>) with bootstrap values greater than 65 per cent (1,000 replicates) shown at the nodes. The phylogenetic relationships were inferred using the maximum likelihood (ML) method and the GTR + I + G as the best model with the PHYLIP package. Strain members of genomospecies 8 (<i>P. avellanae</i>) sensu Gardan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075794#B5" target="_blank">5</a>], including also <i>P</i>. <i>s</i>. pv. <i>morsprunorum</i> M302280, are shown in red, whereas strain members of genomospecies 2 (P. <i>s</i>. pv. <i>lachrymans</i> M302278) and 3 (P. <i>s</i>. pv. <i>tomato</i>) are in blue. <i>P. fluorescens</i> A506 and <i>P</i><i>. putida</i> UW4 were included as outgroups.</p

A Genomic Redefinition of <i>Pseudomonas avellanae</i> species

<div><p>The circumscription of bacterial species is a complex task. So far, DNA-DNA hybridization (DDH), 16S rRNA gene sequencing, and multiocus sequence typing analysis (MLSA) are currently the preferred techniques for their genetic determination. However, the average nucleotide identity (ANI) analysis of conserved and shared genes between two bacterial strains based on the pair-wise genome comparisons, with support of the tetranucleotide frequency correlation coefficients (TETRA) value, has recently been proposed as a reliable substitute for DDH. The species demarcation boundary has been set to a value of 95-96% of the ANI identity, with further confirmation through the assessment of the corresponding TETRA value. In this study, we performed a genome-wide MLSA of 14 phytopathogenic pseudomonads genomes, and assessed the ANI and TETRA values of 27 genomes, representing seven out of the nine genomospecies of <i>Pseudomonas</i> spp. sensu Gardan <i>et alii</i>, and their phylogenetic relationships using maximum likelihood and Bayesian approaches. The results demonstrate the existence of a well demarcated genomic cluster that includes strains classified as <i>P. avellanae, P. syringae</i> pv. <i>theae</i>, <i>P.</i> s. pv. <i>actinidiae</i> and one <i>P.</i> s. pv. <i>morsprunorum</i> strain all belonging to the single species <i>P. avellanae</i>. In addition, when compared with <i>P. avellanae</i>, five strains of <i>P.</i> s. pv. <i>tomato</i>, including the model strain DC3000, and one <i>P.</i> s. pv. <i>lachrymans</i> strain, appear as very closely related to <i>P. avellanae</i>, with ANI values of nearly 96% as confirmed by the TETRA analysis. Conversely, one representative strain, previously classified as <i>P. avellanae</i> and isolated in central Italy, is a genuine member of the <i>P. syringae</i> species complex and can be defined as <i>P.</i> s. pv. <i>avellanae</i>. Currently. The core and pan genomes of <i>P. avellanae</i> species consist of 3,995 and 5,410 putative protein-coding genes, respectively.</p> </div

Split network of the concatenated 6,579 nucleotides for 29 <i>Pseudomonas</i> spp. genomes.

<div><p>A) Strains of genomospecies 3 (P. <i>s</i>. pv. <i>tomato</i>) and 2 (P. <i>s</i>. pv. <i>lachrymans</i>) and genomospecies 8 (<i>P. avellanae</i>, P. <i>s</i>. pv. <i>actinidiae, P</i>. <i>s</i>. pv. <i>theae</i>) sensu Gardan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075794#B5" target="_blank">5</a>] as well as <i>P</i>. <i>s</i>. pv. <i>morsprunorum</i> M30228, clustered apart from the other representative strains of genomospecies 1, 4, 6 and 9. Bootstraps values higher than 65% are shown.</p> <p>B) Particular of the split network of Figure 5A regarding strains of <i>Pseudomonas avellanae</i> species and the closely-related <i>P. syringae</i> pv. <i>tomato</i> and <i>P</i>. <i>s</i>. pv. <i>lachrymans</i> strains. Bootstraps values higher than 65% are shown.</p></div

Graphical representation of the average nucleotide identity (ANI) comparison between genomes of <i>Pseudomonas</i> strains.

<p>Graphical representation of the average nucleotide identity (ANI) comparison between genomes of <i>Pseudomonas</i> strains.</p

Bayesian phylogenetic tree showing relationships among representative strains of phytopathogenic <i>Pseudomonas</i> species and <i>P. syringae</i> pathovars.

<p>The phylogenetic tree was constructed using 2,193 concatenated amino acid of seven housekeeping genes (<i>argS, dnaQ, gltA, gyrB, recA, rpoB</i> and <i>rpoD</i>) with bootstrap values (100,000 generations) shown at the nodes. Strain members of genomospecies 8 (<i>P. avellanae</i>) sensu Gardan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075794#B5" target="_blank">5</a>], including also <i>P</i>. <i>s</i>. pv. <i>morsprunorum</i> M302280, are shown in red, whereas strain members of genomospecies 2 (P. <i>s</i>. pv. <i>lachrymans</i> M302278) and 3 (P. <i>s</i>. pv. <i>tomato</i>) are in blue. <i>P. fluorescens</i> A506 and <i>P</i><i>. putida</i> UW4 were included as outgroups. To note that strains of genomospecies 2 and 3 are embedded into strains of genomospecies 8. The interior node values of the tree are clade credibility values based on the posterior credibility values produced by MrBayes. </p

The core genome of <i>Pseudomonas avellanae</i> species.

<p>Each strain is represented by an oval that is colored according to the current and traditional strain determination. The number of orthologous coding sequences (CDSs) shared by all strains (i.e., the core genome) is in the center (i.e., 3,995). Numbers in the non-overlapping portions of each oval show the number of CDSs unique to each strain.</p

The pan genome of <i>Pseudomonas avellanae</i> species.

<p>The <i>P. avellanae</i> pan genome consists of 5,410 ORFs. The graphic shows also the total putative proteins and protein family number found for each strain.</p

Transcriptome reprogramming of resistant and susceptible peach genotypes during <i>Xanthomonas arboricola</i> pv. <i>pruni</i> early leaf infection

<div><p>Bacterial spot caused by <i>Xanthomonas arboricola</i> pv. <i>pruni</i> (Xap) is a major threat to <i>Prunus</i> species worldwide. The molecular mechanisms of peach resistance to Xap during early leaf infection were investigated by RNA-Seq analysis of two <i>Prunus persica</i> cultivars, ‘Redkist’ (resistant), and ‘JH Hale’ (susceptible) at 30 minutes, 1 and 3 hours-post-infection (hpi). Both cultivars exhibited extensive modulation of gene expression at 30 mpi, which reduced significantly at 1 hpi, increasing again at 3 hpi. Overall, 714 differentially expressed genes (DEGs) were detected in ‘Redkist’ (12% at 30 mpi and 1 hpi and 88% at 3 hpi). In ‘JH Hale’, 821 DEGs were identified (47% at 30 mpi and 1 hpi and 53% at 3 hpi). Highly up-regulated genes (fold change > 100) at 3 hpi exhibited higher fold change values in ‘Redkist’ than in ‘JH Hale’. RNA-Seq bioinformatics analyses were validated by RT-qPCR. In both cultivars, DEGs included genes with putative roles in perception, signal transduction, secondary metabolism, and transcription regulation, and there were defense responses in both cultivars, with enrichment for the gene ontology terms, ‘immune system process’, ‘defense response’, and ‘cell death’. There were particular differences between the cultivars in the intensity and kinetics of modulation of expression of genes with putative roles in transcriptional activity, secondary metabolism, photosynthesis, and receptor and signaling processes. Analysis of differential exon usage (DEU) revealed that both cultivars initiated remodeling their transcriptomes at 30 mpi; however, ‘Redkist’ exhibited alternative exon usage for a greater number of genes at every time point compared with ‘JH Hale’. Candidate resistance genes (<i>WRKY</i>-like, <i>CRK</i>-like, <i>Copper amine oxidase</i>-like, and <i>TIR-NBS-LRR</i>-like) are of interest for further functional characterization with the aim of elucidating their role in <i>Prunus</i> spp. resistance to Xap.</p></div

Transcriptome remodeling in terms of numbers of regulated genes in ‘Redkist’ (resistant) and ‘JH Hale’ (susceptible) cultivars during a time course (30 mpi, 1, and 3 hpi) after Xap infection.

<p>Transcriptome remodeling in terms of numbers of regulated genes in ‘Redkist’ (resistant) and ‘JH Hale’ (susceptible) cultivars during a time course (30 mpi, 1, and 3 hpi) after Xap infection.</p