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.

Epigenetic Regulation of Verticillium dahliae Virulence: Does DNA Methylation Level Play A Role?

Verticillium dahliae is the etiological agent of Verticillium wilt of olive. The virulence of Defoliating V. dahliae isolates usually displays differences and high plasticity. This work studied whether an epigenetic mechanism was involved in this plasticity. An inverse correlation between virulence and DNA methylation of protein-coding genes was found. A set of 831 genes was selected for their highly consistent inverse methylation profile and virulence in the five studied isolates. Of these genes, ATP-synthesis was highly represented, which indicates that the more virulent D isolates are, the more energy requirements they may have. Furthermore, there were numerous genes in the protein biosynthesis process: genes coding for the chromatin structure, which suggests that epigenetic changes may also affect chromatin condensation; many transmembrane transporter genes, which is consistent with denser compounds, traffic through membranes in more virulent isolates; a fucose-specific lectin that may play a role in the attachment to plant cell walls during the host infection process; and pathogenic cutinases that facilitate plant invasion and sporulation genes for rapid spreading alongside plants. Our findings support the notion that differences in the virulence of the Defoliating V. dahliae isolates may be controlled, at least to some extent, by an epigenetic mechanism.

Genome Wild Analysis and Molecular Understanding of the Aquaporin Diversity in Olive Trees (Olea Europaea L.).

Cellular aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life, playing important roles in the uptake of water and many solutes across the membranes. In olive trees, AQP diversity, protein features and their biological functions are still largely unknown. This study focuses on the structure and functional and evolution diversity of AQP subfamilies in two olive trees, the wild species Olea europaea var. sylvestris (OeuAQPs) and the domesticated species Olea europaea cv. Picual (OleurAQPs), and describes their involvement in different physiological processes of early plantlet development and in biotic and abiotic stress tolerance in the domesticated species. A scan of genomes from the wild and domesticated olive species revealed the presence of 52 and 79 genes encoding full-length AQP sequences, respectively. Cross-genera phylogenetic analysis with orthologous clustered OleaAQPs into five established subfamilies: PIP, TIP, NIP, SIP, and XIP. Subsequently, gene structures, protein motifs, substrate specificities and cellular localizations of the full length OleaAQPs were predicted. Functional prediction based on the NPA motif, ar/R selectivity filter, Froger's and specificity-determining positions suggested differences in substrate specificities of Olea AQPs. Expression analysis of the OleurAQP genes indicates that some genes are tissue-specific, whereas few others show differential expressions at different developmental stages and in response to various biotic and abiotic stresses. The current study presents the first detailed genome-wide analysis of the AQP gene family in olive trees and it provides valuable information for further functional analysis to infer the role of AQP in the adaptation of olive trees in diverse environmental conditions in order to help the genetic improvement of domesticated olive trees.

Corrigendum: Study protocol for FIBROKIT: a new tool for fibromyalgia diagnosis and patient follow-up.

[This corrects the article DOI: 10.3389/fneur.2023.1286539.].

Study protocol for FIBROKIT: a new tool for fibromyalgia diagnosis and patient follow-up.

Fibromyalgia (FM) is a complex disease that is characterized by chronic musculoskeletal pain and has great economic impact. FM prevalence is about 2% to 4% worldwide, affecting mainly middle-aged women, and its complex pathophysiology complicates diagnosis, treatment and the findings of solid biomarkers. Previous studies have suggested an association between the disease and oxidative stress, mitochondrial metabolism, intestinal microbiota and inflammation, providing sufficient data to support the multifactorial origin of FM. Hence, the objective of this randomized, prospective, low-interventional, double-blinded and placebo-controlled clinical trial is the development of a specific panel of FM biomarkers and the evaluation of their response to a six-month nutritional intervention based on the Mediterranean diet supplemented with extra virgin olive oil (EVOO). For this purpose, the experimental design implies the recruitment of a large cohort of female Spanish patients. Middle-aged women who meet the diagnostic criteria for FM according to the American College of Rheumatology (ACR) will be eligible, along with age-matched healthy women. Both groups will be randomly divided into placebo (olive oil, OO) and treatment groups (extra virgin olive oil, EVOO), and will provide samples at the beginning (T0), after 3 months of nutritional intervention (T1), at the end of the nutritional intervention in 6 months (T2), and 6 months after the end of nutritional intervention (TF), being enrolled for 1 year. Data will be collected through health questionnaires, and whole blood and stool samples will be taken and analyzed. Blood will be used for western-blotting and proteomic analysis of mitochondrial homeostasis and plasma proteome, while stool will undergo metagenomic analysis, respectively. This study represents the first low-interventional investigation with more than 200 participants focused on exploring the association of oxidative stress, mitochondrial metabolism, intestinal microbiota and related pathways with a nutritional intervention in the context of FM. As a result, the outcomes of this study will significantly contribute to the development of a comprehensive and robust panel of diagnostic biomarkers, and will shed some light on their modulation with non-pharmacological therapies such as nutrition. Clinical trial registration: https://clinicaltrials.gov/study/NCT05921409, identifier: NCT05921409.

Microbiota and Mitochondrial Sex-Dependent Imbalance in Fibromyalgia: A Pilot Descriptive Study.

Fibromyalgia is a widespread chronic condition characterized by pain and fatigue. Among the long list of physiological disturbances linked to this syndrome, mitochondrial imbalance and oxidative stress stand out. Recently, the crosstalk between mitochondria and intestinal microbiota has caught the attention of biomedical researchers, who have found connections between this axis and several inflammatory and pain-related conditions. Hence, this pilot descriptive study focused on characterizing the mitochondrial mass/mitophagy ratio and total antioxidant capacity in PBMCs, as well as some microbiota components in feces, from a Peruvian cohort of 19 females and 7 males with FM. Through Western blotting, electrochemical oxidation, ELISA, and real-time qPCR, we determined VDAC1 and MALPLC3B protein levels; total antioxidant capacity; secretory immunoglobulin A (sIgA) levels; and Firmicutes/Bacteroidetes, Bacteroides/Prevotella, and Roseburia/Eubacterium ratios; as well as Ruminococcus spp., Pseudomonas spp., and Akkermansia muciniphila levels, respectively. We found statistically significant differences in Ruminococcus spp. and Pseudomonas spp. levels between females and males, as well as a marked polarization in mitochondrial mass in both groups. Taken together, our results point to a mitochondrial imbalance in FM patients, as well as a sex-dependent difference in intestinal microbiota composition.

Corrigendum to "Coupling the endophytic microbiome with the host transcriptome in olive roots" [Comput. Struct. Biotechnol. J. 19 (2021) 4777-4789].

[This corrects the article DOI: 10.1016/j.csbj.2021.08.035.].

Identification of Genetic Markers and Genes Putatively Involved in Determining Olive Fruit Weight.

The fruit size of a cultivated olive tree is consistently larger than its corresponding wild relatives because fruit size is one of the main traits associated with olive tree domestication. Additionally, large fruit size is one of the main objectives of modern olive breeding programs. However, as the long juvenile period is one main hindrance in classic breeding approaches, obtaining genetic markers associated with this trait is a highly desirable tool. For this reason, GWAS analysis of both genetic markers and the genes associated with fruit size determination, measured as fruit weight, was herein carried out in 50 genotypes, of which 40 corresponded to cultivated and 10 to wild olive trees. As a result, 113 genetic markers were identified, which showed a very high statistically significant correlation with fruit weight variability, p < 10−10. These genetic markers corresponded to 39 clusters of genes in linkage disequilibrium. The analysis of a segregating progeny of the cross of "Frantoio" and "Picual" cultivars allowed us to confirm 10 of the 18 analyzed clusters. The annotation of the genes in each cluster and the expression pattern of the samples taken throughout fruit development by RNAseq enabled us to suggest that some studied genes are involved in olive fruit weight determination.

Coupling the endophytic microbiome with the host transcriptome in olive roots.

The connection between olive genetic responses to environmental and agro-climatic conditions and the composition, structure and functioning of host-associated, belowground microbiota has never been studied under the holobiont conceptual framework. Two groups of cultivars growing under the same environmental, pedological and agronomic conditions, and showing highest (AH) and lowest (AL) Actinophytocola relative abundances, were earlier identified. We aimed now to: i) compare the root transcriptome profiles of these two groups harboring significantly different relative abundances in the above-mentioned bacterial genus; ii) examine their rhizosphere and root-endosphere microbiota co-occurrence networks; and iii) connect the root host transcriptome pattern to the composition of the root microbial communities by correlation and co-occurrence network analyses. Significant differences in olive gene expression were found between the two groups. Co-occurrence networks of the root endosphere microbiota were clearly different as well. Pearson's correlation analysis enabled a first portray of the interaction occurring between the root host transcriptome and the endophytic community. To further identify keystone operational taxonomic units (OTUs) and genes, subsequent co-occurrence network analysis showed significant interactions between 32 differentially expressed genes (DEGs) and 19 OTUs. Overall, negative correlation was detected between all upregulated genes in the AH group and all OTUs except of Actinophytocola. While two groups of olive cultivars grown under the same conditions showed significantly different microbial profiles, the most remarkable finding was to unveil a strong correlation between these profiles and the differential gene expression pattern of each group. In conclusion, this study shows a holistic view of the plant-microbiome communication.

Hydroxytyrosol as a Promising Ally in the Treatment of Fibromyalgia.

Fibromyalgia (FM) is a chronic and highly disabling syndrome, which is still underdiagnosed, with controversial treatment. Although its aetiology is unknown, a number of studies have pointed to the involvement of altered mitochondrial metabolism, increased oxidative stress and inflammation. The intake of extra virgin olive oil, and particularly of one of its phenolic compounds, hydroxytyrosol (HT), has proven to be protective in terms of redox homeostatic balance and the reduction of inflammation. In this context, using a proteomic approach with nanoscale liquid chromatography coupled to tandem mass spectrometry, the present study analysed: (i) Changes in the proteome of dermal fibroblasts from a patient with FM versus a healthy control, and (ii) the effect of the treatment with a nutritional relevant dose of HT. Our results unveiled that fibroblast from FM show a differential expression in proteins involved in the turnover of extracellular matrix and oxidative metabolism that could explain the inflammatory status of these patients. Moreover, a number of these proteins results normalized by the treatment with HT. In conclusion, our results support that an HT-enriched diet could be highly beneficial in the management of FM.

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.

Insight into the biological pathways underlying fibromyalgia by a proteomic approach.

Fibromyalgia (FM) is a form of non-articular rheumatism difficult to diagnose and treat because its etiology remains still elusive. Proteomics makes possible the systematic analysis of hundreds of proteins in clinical samples. Consequently, it has become a key tool for finding altered molecular pathways in different diseases. In this context, the present study analyzes changes in the plasma proteome of patients with FM by nanoscale liquid chromatography coupled to tandem mass spectrometry. Deregulated proteins were studied using Ingenuity Pathways Analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes. Conventional analytical methods were used to validate selected proteins. We found a total of 33 proteins differentially expressed in patients with FM. Haptoglobin and fibrinogen showed the highest FM/control ratio. IPA analysis revealed that the top enriched canonical pathways were acute-phase response signaling, Liver-X Receptor/Retinoid-X Receptor activation, Farnesoid-X Receptor/Retinoid-X Receptor activation, and coagulation and complement systems. The importance of inflammation in FM was corroborated by the increase in erythrocyte sedimentation rate. In conclusion, our results support the existence of a plasma protein signature of FM that involves different biological pathways all of them related to inflammation, and point to haptoglobin and fibrinogen as plausible biomarker-candidates for future studies. SIGNIFICANCE: The etiology of fibromyalgia (FM) remains elusive making its diagnosis and treatment difficult. The characterization of the proteome signature of this syndrome will improve its understanding. However, to date proteomic analyses in FM are scarce. The goal of the present work is to analyse, for the first time, changes in plasma protein profiles of patients with FM in comparison to control subjects, using label free relative protein quantification by nanoscale liquid chromatography coupled to tandem mass spectrometry. Our data demonstrate the existence of a common protein signature in the plasma of patients with FM that could explain some of the symptoms associated to this syndrome. The analysis of the 33 proteins differentially expressed corroborates the crucial role of inflammation in the pathogenesis of this syndrome. The interplay of the complement and coagulation cascades contributes to the inflammatory process, while the activation of Liver-X Receptor/Retinoid-X Receptor and Farnesoid-X Receptor/Retinoid-X Receptor could attempt to alleviate it. Finally, we have identified two proteins, haptoglobin and fibrinogen, as potential biomarker-candidates of FM for future studies.