Verticillium wilt resistant and susceptible olive cultivars express a very different basal set of genes in roots.

BACKGROUND: Olive orchards are threatened by a wide range of pathogens. Of these, Verticillium dahliae has been in the spotlight for its high incidence, the difficulty to control it and the few cultivars that has increased tolerance to the pathogen. Disease resistance not only depends on detection of pathogen invasion and induction of responses by the plant, but also on barriers to avoid the invasion and active resistance mechanisms constitutively expressed in the absence of the pathogen. In a previous work we found that two healthy non-infected plants from cultivars that differ in V. dahliae resistance such as 'Frantoio' (resistant) and 'Picual' (susceptible) had a different root morphology and gene expression pattern. In this work, we have addressed the issue of basal differences in the roots between Resistant and Susceptible cultivars. RESULTS: The gene expression pattern of roots from 29 olive cultivars with different degree of resistance/susceptibility to V. dahliae was analyzed by RNA-Seq. However, only the Highly Resistant and Extremely Susceptible cultivars showed significant differences in gene expression among various groups of cultivars. A set of 421 genes showing an inverse differential expression level between the Highly Resistant to Extremely Susceptible cultivars was found and analyzed. The main differences involved higher expression of a series of transcription factors and genes involved in processes of molecules importation to nucleus, plant defense genes and lower expression of root growth and development genes in Highly Resistant cultivars, while a reverse pattern in Moderately Susceptible and more pronounced in Extremely Susceptible cultivars were observed. CONCLUSION: According to the different gene expression patterns, it seems that the roots of the Extremely Susceptible cultivars focus more on growth and development, while some other functions, such as defense against pathogens, have a higher expression level in roots of Highly Resistant cultivars. Therefore, it seems that there are constitutive differences in the roots between Resistant and Susceptible cultivars, and that susceptible roots seem to provide a more suitable environment for the pathogen than the resistant ones.

Transcriptomic time-series analysis of early development in olive from germinated embryos to juvenile tree.

BACKGROUND: Despite its relevance, almost no studies account for the genetic control in the early stages of tree development, i.e. from germination on. This study seeks to make a quite complete transcriptome for olive development and to elucidate the dynamic regulation of the transcriptomic response during the early-juvenile period by RNAseq time-series expression analysis. The transcriptome was made from 342,049,597 paired-end reads of 101 bp in length. The assembled transcriptome contained 109,125 unigenes (N50 = 1490 bp, average length = 839). RESULTS: The time-series-expression analysis showed that, embryonic structures present at the first month after the induction of germination reached a more differentiated state in two-month-old seedlings. Once the plants were between three and four months old and reached a size around 6-7 nodes, the first developmental stages appeared to be complete and the developing seedling became a juvenile plant. In addition, an AGL-gene was rapidly downregulated during the induction of germination. The repression of this gene was very strong, as evidenced by the low levels of gene expression during plant development from the embryonic seedling to undetectable levels of expression in the adult tree. These results suggest that this gene may be involved in seed dormancy and could be a repressor of the germination. Also, an APL1-like olive gene was found to be expressed at high levels during flowering, and was also expressed during the cold incubation in the activation of embryo germination, suggesting a probable role in embryonic development. CONCLUSIONS: The early development from germination to the juvenile stage of olive seedlings occurred when plants reached a size around 6-7 nodes, and general changes of relevant groups of genes involved in development are described. An AGL-gene was proposed to be involved in germination repression. An APL1-like gene was found to have a probable role in embryonic development.

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.

Nitro-Fatty Acids in Plant Signaling: Nitro-Linolenic Acid Induces the Molecular Chaperone Network in Arabidopsis.

Nitro-fatty acids (NO2-FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO2-FAs are considered novel signaling mediators of cell function based on a proven antiinflammatory response. Nevertheless, the interaction of NO with fatty acids in plant systems has scarcely been studied. Here, we examine the endogenous occurrence of nitro-linolenic acid (NO2-Ln) in Arabidopsis and the modulation of NO2-Ln levels throughout this plant's development by mass spectrometry. The observed levels of this NO2-FA at picomolar concentrations suggested its role as a signaling effector of cell function. In fact, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO2-Ln was involved in plant defense response against different abiotic-stress conditions, mainly by inducing heat shock proteins and supporting a conserved mechanism of action in both animal and plant defense processes. Bioinformatics analysis revealed that NO2-Ln was also involved in the response to oxidative stress conditions, mainly depicted by H2O2, reactive oxygen species, and oxygen-containing compound responses, with a high induction of ascorbate peroxidase expression. Closely related to these results, NO2-Ln levels significantly rose under several abiotic-stress conditions such as wounding or exposure to salinity, cadmium, and low temperature, thus validating the outcomes found by RNA-seq technology. Jointly, to our knowledge, these are the first results showing the endogenous presence of NO2-Ln in Arabidopsis (Arabidopsis thaliana) and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations.

Transcriptomic Analyses on the Role of Nitric Oxide in Plant Disease Resistance.

Nitric oxide (NO) is a gaseous molecule having key roles in many physiological processes such as germination, growth, development and senescence. It has been also shown the important role of NO as a signaling molecule in the response to a wide variety of stress situations, including both biotic and abiotic stress conditions. In the last few years, a growing number of studies have focused on NO-cell targets by several approaches such as transcriptomic and proteomic analyses. This review is centered on offering an update about the principal medium- and large-scale transcriptomic analyses performed with several NO donors including microarray, cDNA-amplification fragment length polymorphism (AFLP) and high throughput sequencing (RNA-seq technology) approaches mainly focused on the role of this reactive nitrogen species in relation to plant disease resistance. Different putative NO-responsive genes have been identified in different plant tissues and plant species by application of several NO donors suggesting the implication of NO-responsive genes with plant adaptive responses to biotic stress processes. Finally, it is also provided an overview about common transcription factor-binding sites of NO-responsive genes and the need to further analyze the different NO-targets by other omics studies.

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.

Transcriptomic profiling of linolenic acid-responsive genes in ROS signaling from RNA-seq data in Arabidopsis.

Linolenic acid (Ln) released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA). The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signaling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC) with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2-fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs) and Allene oxide cyclases (AOCs). In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding), such as WRKY, JAZ, MYC, and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signaling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1), methionine sulfoxide reductase (MSR) and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signaling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity) and especially in stresses mediated by ROS.