Tolerance of olive (Olea europaea) cv Frantoio to Verticillium dahliae relies on both basal and pathogen-induced differential transcriptomic responses.

Verticillium wilt of olive (VWO) is one of the most serious biotic constraints for this tree crop. Our knowledge of the genetics of the tolerance/resistance to this disease is very limited. Here we show that tolerance of the cv Frantoio relies on both basal and early pathogen-induced differential transcriptomic responses. A comparative transcriptomic analysis (RNA-seq) was conducted in root tissues of cvs Frantoio (VWO-tolerant) and Picual (VWO-susceptible). RNA samples originated from roots of inoculated olive plants during the early infection stages by Verticillium dahliae (highly virulent, defoliating pathotype). A huge number of differentially expressed genes (DEGs) were found between 'Frantoio' and 'Picual' (27 312 unigenes) in the absence of the pathogen. Upon infection with V. dahliae, 'Picual' and 'Frantoio' plants responded differently too. In the early infection stages, four clusters of DEGs could be identified according to their time-course expression patterns. Among others, a pathogenesis-related protein of the Bet v I family and a dirigent-like protein involved in lignification, and several BAK1, NHL1, reactive oxygen species stress response and BAM unigenes showed noticeable differences between cultivars. Tolerance of 'Frantoio' plants to VWO is a consequence of a complex and multifaceted process which involves many plant traits.

Gene Expression Pattern in Olive Tree Organs (Olea europaea L.).

The olive tree (Olea europaea L.) was one of the first plant species in history to be domesticated. Throughout olive domestication, gene expression has undergone drastic changes that may affect tissue/organ-specific genes. This is an RNA-seq study of the transcriptomic activity of different tissues/organs from adult olive tree cv. "Picual" under field conditions. This analysis unveiled 53,456 genes with expression in at least one tissue, 32,030 of which were expressed in all organs and 19,575 were found to be potential housekeeping genes. In addition, the specific expression pattern in each plant part was studied. The flower was clearly the organ with the most exclusively expressed genes, 3529, many of which were involved in reproduction. Many of these organ-specific genes are generally involved in regulatory activities and have a nuclear protein localization, except for leaves, where there are also many genes with a plastid localization. This was also observed in stems to a lesser extent. Moreover, pathogen defense and immunity pathways were highly represented in roots. These data show a complex pattern of gene expression in different organs, and provide relevant data about housekeeping and organ-specific genes in cultivated olive.

Transposon activation is a major driver in the genome evolution of cultivated olive trees (Olea europaea L.).

The primary domestication of olive (Olea europaea L.) in the Levant dates back to the Neolithic period, around 6,000-5,500 BC, as some archeological remains attest. Cultivated olive trees are reproduced clonally, with sexual crosses being the sporadic events that drive the development of new varieties. In order to determine the genomic changes which have occurred in a modern olive cultivar, the genome of the Picual cultivar, one of the most popular olive varieties, was sequenced. Additional 40 cultivated and 10 wild accessions were re-sequenced to elucidate the evolution of the olive genome during the domestication process. It was found that the genome of the 'Picual' cultivar contains 79,667 gene models, of which 78,079 were protein-coding genes and 1,588 were tRNA. Population analyses support two independent events in olive domestication, including an early possible genetic bottleneck. Despite genetic bottlenecks, cultivated accessions showed a high genetic diversity driven by the activation of transposable elements (TE). A high TE gene expression was observed in presently cultivated olives, which suggests a current activity of TEs in domesticated olives. Several TEs families were expanded in the last 5,000 or 6,000 years and produced insertions near genes that may have been involved in selected traits during domestication as reproduction, photosynthesis, seed development, and oil production. Therefore, a great genetic variability has been found in cultivated olive as a result of a significant activation of TEs during the domestication process.

The Transcriptome of Verticillium dahliae Responds Differentially Depending on the Disease Susceptibility Level of the Olive (Olea europaea L.) Cultivar.

Among biotic constraints affecting olive trees cultivation worldwide, the soil-borne fungus Verticillium dahliae is considered one of the most serious threats. Olive cultivars display differential susceptibility to the disease, but our knowledge on the pathogen's responses when infecting varieties differing in susceptibility is scarce. A comparative transcriptomic analysis (RNA-seq) was conducted in olive cultivars Picual (susceptible) and Frantoio (tolerant). RNA samples originated from roots during the first two weeks after inoculation with V. dahliae defoliating (D) pathotype. Verticillium dahliae mRNA amount was overwhelmingly higher in roots of the susceptible cultivar, indicating that proliferation of pathogen biomass is favored in 'Picual'. A significant larger number of V. dahliae unigenes (11 fold) were only induced in this cultivar. Seven clusters of differentially expressed genes (DEG) were identified according to time-course expression patterns. Unigenes potentially coding for niche-adaptation, pathogenicity, virulence and microsclerotia development were induced in 'Picual', while in 'Frantoio' expression remained negligible or null. Verticillium dahliae D pathotype transcriptome responses are qualitatively and quantitatively different, and depend on cultivar susceptibility level. The much larger V. dahliae biomass found in 'Picual' roots is a consequence of both host and pathogen DEG explaining, to a large extent, the higher aggressiveness exerted over this cultivar.

Transcriptomic Analysis of L. Roots during the Early Infection Process.

Olive cultivation is affected by a wide range of biotic constraints. Verticillium wilt of olive is one of the most devastating diseases affecting this woody crop, inflicting major economic losses in many areas, particularly within the Mediterranean Basin. Little is known about gene-expression changes during plant infection by of woody plants such as olive. A complete RNA-seq transcriptomic analysis of olive tree roots was made. Trinity assembler proved to be the best option to assemble the olive and transcriptomes. The olive transcriptome (Oleup) consisted of 68,259 unigenes (254,252 isoforms/transcripts), and the transcriptome (Vedah) consisted of 37,425 unigenes (52,119 isoforms/transcripts). Most unigenes of the Oleup transcriptome corresponded to cellular processes (12,339), metabolic processes (10,974), single-organism processes (7263), and responses to stimuli (5114). As for the Vedah transcriptome, most unigenes correspond to metabolic processes (25,372), cellular processes (23,718), localization (6385), and biological regulation (4801). Differential gene-expression analysis of both transcriptomes was made at 2 and 7 d post-infection. The induced genes of both organisms during the plant-pathogen interaction were clustered in six subclusters, depending on the expression patterns during the infection. Subclusters A to C correspond to plant genes, and subcluster D to F correspond to genes. A relevant finding was that the differentially expressed gene (DEGs) included in subclusters B and C were highly enriched in proteolysis as well as protein-folding and biosynthesis genes. In addition, a reactive oxygen species (ROS) defense was induced first in the pathogen and later in the plant roots.

Early and delayed long-term transcriptional changes and short-term transient responses during cold acclimation in olive leaves.

Low temperature severely affects plant growth and development. To overcome this constraint, several plant species from regions having a cool season have evolved an adaptive response, called cold acclimation. We have studied this response in olive tree (Olea europaea L.) cv. Picual. Biochemical stress markers and cold-stress symptoms were detected after the first 24 h as sagging leaves. After 5 days, the plants were found to have completely recovered. Control and cold-stressed plants were sequenced by Illumina HiSeq 1000 paired-end technique. We also assembled a new olive transcriptome comprising 157,799 unigenes and found 6,309 unigenes differentially expressed in response to cold. Three types of response that led to cold acclimation were found: short-term transient response, early long-term response, and late long-term response. These subsets of unigenes were related to different biological processes. Early responses involved many cold-stress-responsive genes coding for, among many other things, C-repeat binding factor transcription factors, fatty acid desaturases, wax synthesis, and oligosaccharide metabolism. After long-term exposure to cold, a large proportion of gene down-regulation was found, including photosynthesis and plant growth genes. Up-regulated genes after long-term cold exposure were related to organelle fusion, nucleus organization, and DNA integration, including retrotransposons.