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

Abstract

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.

Figure 1. Gene Ontology (GO) terms distribution of 341 unigenes induced in olive (Olea europaea) roots colonized by Pseudomonas fluorescens PICF7.

Expressed sequences tags (ESTs) were categorized using the ‘Blast2GO’ software according to the terms of the three main GO vocabularies: ‘biological processes’ (a), ‘molecular functions’ (b) and ‘cellular components’ (c). The category ‘Other’ in the main GO vocabulary term ‘biological processes’ clusters 56% of the transcripts analyzed and refers to very low represented categories related to development, homeostasis, photosynthesis and cellular differentiation. In the main GO vocabulary term ‘molecular functions’, the category ‘Other’ clusters 42.5% of ESTs with similarity to proteins with putative or unknown function (see text for details).

Figure 2. Kyoto Encyclopaedia of Genes and Genomes (KEGG) for the phenylpropanoid biosynthesis pathway.

Several genes involved in this pathway have been found to be induced in olive roots upon Pseudomonas fluorescens PICF7 colonization. The caffeoyl-CoA O-methyltransferase (2.1.1.104) (yellow rectangles), the cinnamyl-alcohol deydrogenase (1.1.1.95) (orange rectangles) and the peroxidase (1.11.1.7) (red rectangles) enzymes are mapped (see text for details and also Table 2).

Figure 3. A proposed schematic representation of different defence responses induced in olive root tissues by the beneficial endophyte Pseudomonas fluorescens PICF7.

An ideal cross section from rhizosphere soil (left) to root cortex (right) is shown. PICF7 cells can effectively colonize the root surface, the intercellular spaces of subepidermal and cortical zones (red ellipses), and the interior of root hairs (orange ellipses) , . As yet unidentified signals/metabolites produced by bacteria (represented by curved blue lines) could activate the defence responses here reported (see main text for details). The interaction between P. fluorescens PICF7 and olive also induces the expression of different transcription factors involved in systemic acquired resistance (SAR) and induced systemic resistance (ISR), such as bHLH, ARF2 or GRAS1, and WRKYs (i.e., WRKY 5) known to be implicated in the crosstalk between both plant systemic defence response pathways. Genes whose expression has been validated by qRT-PCR analysis are indicated by pink solid circles. C-O-MT: Caffeoyl-O-methyltransferase; PAL: Phenylalanine ammonia-lyase; CAD: Cinnamyl-alcohol dehydrogenase; ACL: Acetone cyanohydrin lyase; MDH: Malate dehydrogenase.