Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes

Abstract

Background: Plum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes.

Results: Here, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as Prunus armeniaca PPVres MATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1.

Conclusions: Accordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.

Keywords: Apricot; MATHd; PPV resistance; Plum Pox virus; Potyvirus; Prunus; Silencing.

Fig. 1

Venn diagrams showing the number of DEGs identified comparing PPV inoculated and non-inoculated plants for each cultivar. a Total number of DEGs. b Numbers of up- and down-regulated genes upon PPV infection in ‘Canino’ and ‘Stella’ cultivars

Fig. 2

Heat map of RNA-seq expression levels for the identified PPVres locus DEGs between the resistant ‘Stella’ and the susceptible ‘Canino’ cultivars. Blue positive log fold-change (logFC) indicates higher expression in the cultivar ‘Canino’ than in ‘Stella’. Columns represent comparison between PPV inoculated (PPV+) and non-inoculated (PPV-) samples, respectively. The gene clustering is drawn on the left. Non-significant differences with p-values > 0.05 are indicated (n.s.)

Fig. 3

qRT-PCR analysis of PPVres locus MATHd genes showing differential expression according to RNA-seq data. a ParP-3. b ParP-4. c ParP-5. Normalized expression levels were obtained using the housekeeping genes Actin and Sand-like as controls. Data are means from 1 to 3 biological samples with three technical replicates for each one. Error bars represent standard deviation and different letters indicate significant differences (P < 0.05). Left: Histograms of gene-expression using PPV-inoculated (+) and non-inoculated (−) plants of ‘Canino’ (CA), ‘Goldrich’ (GO) and ‘Stella’ (ST). Right: Histograms of gene-expression of non-inoculated plants of 5 PPV susceptible (CA: ‘Canino’, CU: ‘Currot’, GI: ‘Ginesta’, KA: ‘Katy’, MI: ‘Mitger’) and 4 PPV resistant (OR: ‘Orange Red’, HA: ‘Harlayne’, GO: ‘Goldrich’, ST: ‘Stella’) cultivars. Blue lines indicate mean value obtained after removing extreme values

Fig. 4

Identification of the PPVres locus variants mediating PPV resistance in apricot. a Variant filtering of SNPs and small INDELs within the PPVres locus called using 24 apricot cultivars and 2 apricot relatives WGS. b Positions of filtered variants in the peach syntenic region (green lines) corresponding to the PPVres locus. MATHd genes cluster is indicated and peach MATHd genes absent in the apricot genome appear grey colored. Variants in ParP-3 and ParP-4 (putative orthologs of ppa022254m and ppb0221 95 m) are detailed below. The 5-bp deletion causing a frameshift mutation is labeled with an asterisk. c ParP-4 CDS and predicted amino acid sequences for the resistant (R) and susceptible (S) alleles. The 5-bp deletion (green boxed) leads to a premature stop-codon (red boxed) in the R-allele. qRT-PCR primer positions were indicated by arrows (blue, forward R-allele-specific; red, forward S-allele-specific; black, reverse). (d) ParP-4 allele-specific PCR-genotyping in 4 PPV resistant and 5 PPV susceptible apricot cultivars (GO: ‘Goldrich’; HA: ‘Harlayne’; OR: ‘Orange Red’; ST: ‘Stella’; CA: ‘Canino’; KA: ‘Katy’; CU: ‘Currot’; GI: ‘Ginesta’; MI: ‘Mitger’)

Fig. 5

Maximum Likelihood phylogenetic tree of peach and apricot MATHd genes clustered in the PPVres locus. Confident positions (935 bases) from the alignment of CDS sequences were used. The Tamura 3-parameter model (T92) + G was used as the best-fitting evolutionary model. Bootstrapping support values of the nodes > 50 (using 500 replications) are indicated