Allele mining of eukaryotic translation initiation factor genes in Prunus for the identification of new sources of resistance to sharka.

Members of the eukaryotic translation initiation complex are co-opted in viral infection, leading to susceptibility in many crop species, including stone fruit trees (Prunus spp.). Therefore, modification of one of those eukaryotic translation initiation factors or changes in their gene expression may result in resistance. We searched the crop and wild Prunus germplasm from the Armeniaca and Amygdalus taxonomic sections for allelic variants in the eIF4E and eIFiso4E genes, to identify alleles potentially linked to resistance to Plum pox virus (PPV). Over one thousand stone fruit accessions (1397) were screened for variation in eIF4E and eIFiso4E transcript sequences which are in single copy within the diploid Prunus genome. We identified new alleles for both genes differing from haplotypes associated with PPV susceptible accessions. Overall, analyses showed that eIFiso4E is genetically more constrained since it displayed less polymorphism than eIF4E. We also demonstrated more variations at both loci in the related wild species than in crop species. As the eIFiso4E translation initiation factor was identified as indispensable for PPV infection, a selection of ten different eIFiso4E haplotypes along 13 accessions were tested by infection with PPV and eight of them displayed a range of reduced susceptibility to resistance, indicating new potential sources of resistance to sharka.

The complex evolutionary history of apricots: Species divergence, gene flow and multiple domestication events.

Domestication is an excellent model to study diversification and this evolutionary process can be different in perennial plants, such as fruit trees, compared to annual crops. Here, we inferred the history of wild apricot species divergence and of apricot domestication history across Eurasia, with a special focus on Central and Eastern Asia, based on microsatellite markers and approximate Bayesian computation. We significantly extended our previous sampling of apricots in Europe and Central Asia towards Eastern Asia, resulting in a total sample of 271 cultivated samples and 306 wild apricots across Eurasia, mainly Prunus armeniaca and Prunus sibirica, with some Prunus mume and Prunus mandshurica. We recovered wild Chinese species as genetically differentiated clusters, with P. sibirica being divided into two clusters, one possibly resulting from hybridization with P. armeniaca. Central Asia also appeared as a diversification centre of wild apricots. We further revealed at least three domestication events, without bottlenecks, that gave rise to European, Southern Central Asian and Chinese cultivated apricots, with ancient gene flow among them. The domestication event in China possibly resulted from ancient hybridization between wild populations from Central and Eastern Asia. We also detected extensive footprints of recent admixture in all groups of cultivated apricots. Our results thus show that apricot is an excellent model for studying speciation and domestication in long-lived perennial fruit trees.

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production.

BACKGROUND: In plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. Many S genes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due to Plum pox virus (PPV). RESULTS: For this purpose, hairpin-inducing silencing constructs based on Prunus persica orthologs were used to generate Prunus salicina (Japanese plum) 4E and 4G silenced plants by Agrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulated eIFiso4E transgenic Japanese plums were not regenerated in our conditions, eIFiso4G11-, but not the eIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka. CONCLUSIONS: Our results indicate that S gene function of the translation initiation complex isoform is conserved in Prunus species. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.

New insights into the history of domesticated and wild apricots and its contribution to Plum pox virus resistance.

Studying domesticated species and their wild relatives allows understanding of the mechanisms of population divergence and adaptation, and identifying valuable genetic resources. Apricot is an important fruit in the Northern hemisphere, where it is threatened by the Plum pox virus (PPV), causing the sharka disease. The histories of apricot domestication and of its resistance to sharka are however still poorly understood. We used 18 microsatellite markers to genotype a collection of 230 wild trees from Central Asia and 142 cultivated apricots as representatives of the worldwide cultivated apricot germplasm; we also performed experimental PPV inoculation tests. The genetic markers revealed highest levels of diversity in Central Asian and Chinese wild and cultivated apricots, confirming an origin in this region. In cultivated apricots, Chinese accessions were differentiated from more Western accessions, while cultivated apricots were differentiated from wild apricots. An approximate Bayesian approach indicated that apricots likely underwent two independent domestication events, with bottlenecks, from the same wild population. Central Asian native apricots exhibited genetic subdivision and high frequency of resistance to sharka. Altogether, our results contribute to the understanding of the domestication history of cultivated apricot and point to valuable genetic diversity in the extant genetic resources of wild apricots.

Family-based linkage and association mapping reveals novel genes affecting Plum pox virus infection in Arabidopsis thaliana.

Sharka is a devastating viral disease caused by the Plum pox virus (PPV) in stone fruit trees and few sources of resistance are known in its natural hosts. Since any knowledge gained from Arabidopsis on plant virus susceptibility factors is likely to be transferable to crop species, Arabidopsis's natural variation was searched for host factors essential for PPV infection. To locate regions of the genome associated with susceptibility to PPV, linkage analysis was performed on six biparental populations as well as on multiparental lines. To refine quantitative trait locus (QTL) mapping, a genome-wide association analysis was carried out using 147 Arabidopsis accessions. Evidence was found for linkage on chromosomes 1, 3 and 5 with restriction of PPV long-distance movement. The most relevant signals occurred within a region at the bottom of chromosome 3, which comprises seven RTM3-like TRAF domain-containing genes. Since the resistance mechanism analyzed here is recessive and the rtm3 knockout mutant is susceptible to PPV infection, it suggests that other gene(s) present in the small identified region encompassing RTM3 are necessary for PPV long-distance movement. In consequence, we report here the occurrence of host factor(s) that are indispensable for virus long-distance movement.

PPV long-distance movement is occasionally permitted in resistant apricot hosts.

The interactions between Plum pox virus (PPV), a member of the Potyvirus genus, and Prunus host plants are, up to now, poorly understood. In the current paper, fluorescence stereomicroscopy, in situ hybridisation and immunogold detection were performed in order to evaluate the virus transport and cellular distribution. The behavior of PPV in several susceptible (cv. "Moniqui" and "Screara") and resistant apricot genotypes (cv. "Harlayne", "Henderson", "Harcot", "Goldrich", "Stella" and "Stark Early Orange") were compared. Viral RNA was detected by in situ hybridisation in stem tissues close to the inoculation point, irrespective of the resistance status of the variety. Systemic infection was evidenced by virus immunodetection and by fluorescence detection of a GFP-tagged PPV in distant leaf sections. The signal obtained by in situ hybridisation colocalised with the fluorescence produced by GFP-tagged PPV in the same plant material but did not colocalise with the signal obtained by immunostaining. Intensity of the PPV infection in susceptible apricot cultivars varied depending on genotypes. The behavior of PPV in systemic leaves was clearly distinct between susceptible and resistant cultivars. While PPV was spreading widely around the major and minor veins in susceptible leaves, in the resistant apricot genotypes it was restricted to isolated spots consisting of few cells embedded in the mesophyll tissue. In summary, differences in the ability of PPV to systemically infect susceptible and resistant apricot cultivars were evident but nevertheless, long-distance transport of PPV occured in resistant apricot scions.

Plum pox virus induces differential gene expression in the partially resistant stone fruit tree Prunus armeniaca cv. Goldrich.

We investigated the changes in the expression profiles of the partially resistant apricot (Prunus armeniaca L.) cultivar Goldrich following inoculation with Plum pox virus (PPV) using cDNA-amplification fragment length polymorphism (AFLP). Altered expression patterns were detected and twenty-one differentially expressed cDNA had homologies with genes in databases coding for proteins involved in metabolism, signal transduction, defense, stress and intra/intercellular connections. Seven of the modified expressed patterns were further investigated by semi-quantitative RT-PCR or Northern blotting. The expression patterns of five of these genes were confirmed in the partially resistant P. armeniaca cv. 'Goldrich' and assessed in a susceptible genotype. One of these cDNAs, coding for a putative class III chitinase, appeared to be repressed in infected plants of the partially resistant genotype and expressed in the susceptible one which could be related to the partially resistant phenotype. On the contrary, the expression patterns of the genes coding for a transketolase, a kinesin-like and an ankyrin-like protein, were clearly linked to the susceptible interaction. These candidate genes could play a role either in the compatible interaction leading to virus invasion or to the quantitative resistance of apricot to PPV.