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.

Systemic responses in a tolerant olive (Olea europaea L.) cultivar upon root colonization by the vascular pathogen Verticillium dahliae.

Verticillium wilt of olive (VWO) is caused by the vascular pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant cultivars; however, our knowledge on VWO tolerance/resistance genetics is very limited. A transcriptomic analysis was conducted to (i) identify systemic defense responses induced/repressed in aerial tissues of the tolerant cultivar Frantoio upon root colonization by V. dahliae, and (ii) determine the expression pattern of selected defense genes in olive cultivars showing differential susceptibility to VWO. Two suppression subtractive hybridization cDNA libraries, enriched in up-regulated (FU) and down-regulated (FD) genes respectively, were generated from "Frantoio" aerial tissues. Results showed that broad systemic transcriptomic changes are taking place during V. dahliae-"Frantoio" interaction. A total of 585 FU and 381 FD unigenes were identified, many of them involved in defense response to (a)biotic stresses. Selected genes were then used to validate libraries and evaluate their temporal expression pattern in "Frantoio." Four defense genes were analyzed in cultivars Changlot Real (tolerant) and Picual (susceptible). An association between GRAS1 and DRR2 gene expression patterns and susceptibility to VWO was observed, suggesting that these transcripts could be further evaluated as markers of the tolerance level of olive cultivars to V. dahliae.

Complete genome sequence of Pseudomonas fluorescens strain PICF7, an indigenous root endophyte from olive (Olea europaea L.) and effective biocontrol agent against Verticillium dahliae.

Pseudomonas fluorescens strain PICF7 is a native endophyte of olive roots. Previous studies have shown this motile, Gram-negative, non-sporulating bacterium is an effective biocontrol agent against the soil-borne fungus Verticillium dahliae, the causal agent of one of the most devastating diseases for olive (Olea europaea L.) cultivation. Here, we announce and describe the complete genome sequence of Pseudomonas fluorescens strain PICF7 consisting of a circular chromosome of 6,136,735 bp that encodes 5,567 protein-coding genes and 88 RNA-only encoding genes. Genome analysis revealed genes predicting factors such as secretion systems, siderophores, detoxifying compounds or volatile components. Further analysis of the genome sequence of PICF7 will help in gaining insights into biocontrol and endophytism.

Endophytic colonization and biocontrol performance of Pseudomonas fluorescens†PICF7 in olive (Olea europaea L.) are determined neither by pyoverdine production nor swimming motility.

Pseudomonas fluorescens PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild-type PICF7, discarding these traits as relevant for its endophytic lifestyle.

The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots.

Pseudomonas fluorescens PICF7, a native olive root endophyte and effective biocontrol agent (BCA) against Verticillium wilt of olive, is able to trigger a broad range of defense responses in root tissues of this woody plant. In order to elucidate whether strain PICF7 also induces systemic defense responses in above-ground organs, aerial tissues of olive plants grown under non-gnotobiotic conditions were collected at different time points after root bacterization with this endophytic BCA. A suppression subtractive hybridization (SSH) cDNA library, enriched in up-regulated genes, was generated. This strategy enabled the identification of 376 ESTs (99 contigs and 277 singlets), many of them related to response to different stresses. Five ESTs, involved in defense responses, were selected to carry out time-course quantitative real-time PCR (qRT-PCR) experiments aiming to: (1) validate the induction of these genes, and (2) shed light on their expression pattern along time (from 1 to 15 days). Induction of olive genes potentially coding for lipoxygenase 2, catalase, 1-aminocyclopropane-1-carboxylate oxidase, and phenylananine ammonia-lyase was thus confirmed at some time points. Computational analysis also revealed that different transcription factors were up-regulated in olive aerial tissues (i.e., JERF, bHLH, WRKY), as previously reported for roots. Results confirmed that root colonization by this endophytic bacterium does not only trigger defense responses in this organ but also mounts a wide array of systemic defense responses in distant tissues (stems, leaves). This sheds light on how olive plants respond to the "non-hostile" colonization by a bacterial endophyte and how induced defense response can contribute to the biocontrol activity of strain PICF7.

Genetic responses induced in olive roots upon colonization by the biocontrol endophytic bacterium Pseudomonas fluorescens PICF7.

Knowledge on the genetic basis underlying interactions between beneficial bacteria and woody plants is still very limited, and totally absent in the case of olive. We aimed to elucidate genetic responses taking place during the colonization of olive roots by the native endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive. Roots of olive plants grown under non-gnotobiotic conditions were collected at different time points after PICF7 inoculation. A Suppression Subtractive Hybridization cDNA library enriched in induced genes was generated. Quantitative real time PCR (qRT-PCR) analysis validated the induction of selected olive genes. Computational analysis of 445 olive ESTs showed that plant defence and response to different stresses represented nearly 45% of genes induced in PICF7-colonized olive roots. Moreover, quantitative real-time PCR (qRT-PCR) analysis confirmed induction of lipoxygenase, phenylpropanoid, terpenoids and plant hormones biosynthesis transcripts. Different classes of transcription factors (i.e., bHLH, WRKYs, GRAS1) were also induced. This work highlights for the first time the ability of an endophytic Pseudomonas spp. strain to mount a wide array of defence responses in an economically-relevant woody crop such as olive, helping to explain its biocontrol activity.

Root hairs play a key role in the endophytic colonization of olive roots by Pseudomonas spp. with biocontrol activity.

The use of indigenous bacterial root endophytes with biocontrol activity against soil-borne phytopathogens is an environmentally-friendly and ecologically-efficient action within an integrated disease management framework. The earliest steps of olive root colonization by Pseudomonas fluorescens PICF7 and Pseudomonas putida PICP2, effective biocontrol agents (BCAs) against Verticillium wilt of olive (Olea europaea L.) caused by the fungus Verticillium dahliae Kleb., are here described. A gnotobiotic study system using in vitro propagated olive plants, differential fluorescent-protein tagging of bacteria, and confocal laser scanning microscopy analysis have been successfully used to examine olive roots-Pseudomonas spp. interactions at the single-cell level. In vivo simultaneous visualization of PICF7 and PICP2 cells on/in root tissues enabled to discard competition between the two bacterial strains during root colonization. Results demonstrated that both BCAs are able to endophytically colonized olive root tissues. Moreover, results suggest a pivotal role of root hairs in root colonization by both biocontrol Pseudomonas spp. However, colonization of root hairs appeared to be a highly specific event, and only a very low number of root hairs were effectively colonized by introduced bacteria. Strains PICF7 and PICP2 can simultaneously colonize the same root hair, demonstrating that early colonization of a given root hair by one strain did not hinder subsequent attachment and penetration by the other. Since many environmental factors can affect the number, anatomy, development, and physiology of root hairs, colonization competence and biocontrol effectiveness of BCAs may be greatly influenced by root hair's fitness. Finally, the in vitro study system here reported has shown to be a suitable tool to investigate colonization processes of woody plant roots by microorganisms with biocontrol potential.