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

Significantly enriched GO terms (Biological processes) in the susceptible cultivar ‘JH Hale’ at 30 mpi with Xap.

<p>Significantly enriched GO terms (Biological processes) in the susceptible cultivar ‘JH Hale’ at 30 mpi with Xap.</p

Relative fold change (FC) of expression levels of genes regulated in both ‘Redkist’ (resistant) and in ‘JH Hale’ (susceptible) peach cultivars after Xap infection; max FC = 2216, min FC = -49.

<p>Relative fold change (FC) of expression levels of genes regulated in both ‘Redkist’ (resistant) and in ‘JH Hale’ (susceptible) peach cultivars after Xap infection; max FC = 2216, min FC = -49.</p

Highly up-regulated genes (fold change > 100) at 3 hpi with Xap in ‘Redkist’ (resistant) and ‘JH Hale’ (susceptible) peach cultivars.

<p>Highly up-regulated genes (fold change > 100) at 3 hpi with Xap in ‘Redkist’ (resistant) and ‘JH Hale’ (susceptible) peach cultivars.</p

Distribution of frequency classes of gene expression levels (RPKM) after Xap inoculation on leaves of ‘JH Hale’ (susceptible) and ‘Redkist’ (resistant) peach cultivars.

<p>Distribution of frequency classes of gene expression levels (RPKM) after Xap inoculation on leaves of ‘JH Hale’ (susceptible) and ‘Redkist’ (resistant) peach cultivars.</p

RNA-Seq read statistics before mapping and after quality selection and trimming.

<p>RNA-Seq read statistics before mapping and after quality selection and trimming.</p

MA plot of genes differentially expressed (red dots) by two peach varieties during a time course after Xap infection.

<p>DEGs were selected by filtering based on log₂ (FC) > 2, or log₂ (FC) < -2, and FDR < 0.05.</p

RNA-Seq statistics for reads mapped to the peach genome.

<p>Data are presented as averages of two biological replicates.</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>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

Variant discovery in <i>PpeMYB25</i> (annotation refinement of <i>ppa023143m</i>).

<p>Five nectarine genotypes (‘Madonna di Agosto’, MdA; ‘Quetta’, Q; ‘Stark Red Gold’, SRG; ‘Goldmine’, G; ‘Ambra’, A) were analyzed to confirm the presence of the insertion within exon 3 of <i>PpeMYB25</i>. (A) Long-range amplification products reveal for all the accessions a fragment of about 7 kb (compared to 960 bp expected from the reference genome). (B) Double digestion results of the long-range PCR products show the same pattern for all the genotypes. (C) Position and structure of the Ty-<i>copia</i> retrotransposon deduced by the by the NGS analysis of ‘Quetta’ long-range amplicon. The insertion results in a truncated version of the R2R3-MYB protein.</p