Identification of genes differentially expressed in Pinus pinaster and Pinus pinea after infection with the pine wood nematode

Abstract

The purpose of this study was to compare the response to infestation by the pine wood nematode (PWN) Bursaphelenchus xylophilus between Pinus pinaster and P. pinea at an initial stage of the disease, 3 h after inoculation. The PWN is the causal agent of pine wilt disease and is destroying pine forests all over the world. In Portugal its main host is the maritime pine, P. pinaster, and its vector is the longhorn beetle Monochamus galloprovincialis. Interestingly, this disease does not seem to affect the species P. pinea and several factors could be behind this difference in susceptibility. With regards to the effects of the disease at a transcriptional level, the suppression subtractive hybridization (SSH) technique was utilized to identify Expressed Sequence Tags (EST) in P. pinaster and P. pinea when inoculated with PWN. EST were isolated, cloned, sequenced and identified using BlastN and BlastX, and indicated that at an initial stage of the disease there is activation of a tree defence response at a molecular level, mainly related to oxidative stress, production of lignin and ethylene and posttranscriptional regulation of nucleic acids. 58% of the isolated sequences are not yet described, which shows the lack of genomic information currently available for pine.

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

“…This gene had previously been identified in our laboratory to be involved in the response to PWD [16]. Additionally, the analysis confirmed the differential expression of the malic oxidoreductase (also an antioxidant enzyme) and ricin B-related lectin, that belong to a class of participants in the general defense against a multitude of plant pathogens [44].…”

“…Therefore, it is understandable why terpene metabolism related proteins, like (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMB-PP) reductase and thiolase like protein, both involved in terpenoid synthesis, were differentially expressed by infested P. pinaster (Figure 5A) [30,31]. Subsequently, as the water potential decreases, pine trees suffer severe oxidative stress and here, likewise other PWD-related studies [16,32], several oxidative-related genes were found, namely, a cytochrome c, found in the oxidation of phenolic elements in cell wall polymers under biotic stress, that has been associated with nematode infection in other studies [32] and an aldo/keto reductase, a member of NADPH-dependent oxidoreductases, that intervenes in the elimination of reactive oxygen species produced by plant cells after suffering from a great amount of stress [33]. …”

“…Particularly, FMN reductase has already been identified in previous studies in our lab as possibly related to B. xylophilus infection [16]. Also, a phox/Bem1 (PB1) domain was found to be more represented by infested P. pinea (Figure 5B) and this domain is usually found in signaling proteins including oxidases and cytosolic factors [47] and a 2-hydroxyacid dehydrogenase, that is associated with 3-phosphoglycerate dehydrogenase and may play a role in the oxidation-reduction process [48].…”

“…Other defense response genes detected in the transcriptome that could impose a physical and chemical barrier to nematode progression were the cell wall defense related SNARE protein [66], the abiotic stresses (like drought) related RING zinc finger protein [67], the lignin production related DAHP synthetase [16], and RNA recognition motif connected to protein modification [68]. Up-regulation of genes which constrict nematode progression via increased cell wall strenghtening were also detected in PWN-resistant P. thumbergii [15].…”

“…Different technologies have given us some insight regarding the pine genome and its response to biotic and abiotic stresses. A few examples include: 1) single nucleotide polymorphism genotyped using GoldenGate assay, where a consensus map was created for maritime pine [12]; 2) microarray technology, that identified 2,445 differentially expressed genes that were responsive to severe drought stress in roots of loblolly pine [13]; 3) LongSAGE technique, that provided a total of 20,818 tags, from which 38 were differentially expressed in the resistant Japanese black pine and 25 in non-resistant pine [14]; 4) and suppression subtractive hybridization, showing the up-regulation of stress response and defense related genes by pine wood nematode infestation [15,16]. …”