Molecular Defense Response of Pine Trees (Pinus spp.) to the Parasitic Nematode Bursaphelenchus xylophilus.

Pine wilt disease (PWD) is a severe environmental problem in Eastern Asia and Western Europe, devastating large forest areas and causing significant economic losses. This disease is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, a parasitic migratory nematode that infects the stem of conifer trees. Here we review what is currently known about the molecular defense response in pine trees after infection with PWN, focusing on common responses in different species. By giving particular emphasis to resistance mechanisms reported for selected varieties and families, we identified shared genes and pathways associated with resistance, including the activation of oxidative stress response, cell wall lignification, and biosynthesis of terpenoids and phenylpropanoids. The role of post-transcriptional regulation by small RNAs in pine response to PWN infection is also discussed, as well as the possible implementation of innovative RNA-interference technologies, with a focus on trans-kingdom small RNAs. Finally, the defense response induced by elicitors applied to pine plants before PWN infection to prompt resistance is reviewed. Perspectives about the impact of these findings and future research approaches are discussed.

Primary Metabolite Adjustments Associated With Pinewood Nematode Resistance in Pinus pinaster.

The pinewood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of the pine wilt disease (PWD) and represents one of the major threats to conifer forests. The detection of the PWN in Portugal, associated with Pinus pinaster, increased the concern of its spread to European forests. Despite its susceptibility to PWD, genetic variability found among P. pinaster populations has been associated with heritable PWD resistance. Understanding the mechanisms underlying tree resistance constitutes a valuable resource for breeding programs toward more resilient forest plantations. This study investigated changes in anatomy, chlorophyll a fluorescence (ChlF), and primary metabolism in susceptible and resistant P. pinaster half-sib plants, after PWN inoculation. Susceptible plants showed a general shutdown of central metabolism, osmolyte accumulation, photosynthetic inhibition, and a decrease in the plant water status. The ChlF transient rise (OJIP curve) revealed the appearance of L- and K-bands, indicators of environmental stress. In contrast, resistant plants revealed a regulated defense response and were able to restrict PWN migration and cellular damage. Furthermore, the accumulation of γ-aminobutyric acid (GABA) and succinate suggested a role of these metabolites in PWD resistance and the possible activation of the GABA shunt. Altogether, these results provide new insights to the role of primary metabolism in PWD resistance and in the selection of resistant phenotypes for disease mitigation.

Insights Into the Mechanisms Implicated in Pinus pinaster Resistance to Pinewood Nematode.

Pine wilt disease (PWD), caused by the plant-parasitic nematode Bursaphelenchus xylophilus, has become a severe environmental problem in the Iberian Peninsula with devastating effects in Pinus pinaster forests. Despite the high levels of this species' susceptibility, previous studies reported heritable resistance in P. pinaster trees. Understanding the basis of this resistance can be of extreme relevance for future programs aiming at reducing the disease impact on P. pinaster forests. In this study, we highlighted the mechanisms possibly involved in P. pinaster resistance to PWD, by comparing the transcriptional changes between resistant and susceptible plants after infection. Our analysis revealed a higher number of differentially expressed genes (DEGs) in resistant plants (1,916) when compared with susceptible plants (1,226). Resistance to PWN is mediated by the induction of the jasmonic acid (JA) defense pathway, secondary metabolism pathways, lignin synthesis, oxidative stress response genes, and resistance genes. Quantification of the acetyl bromide-soluble lignin confirmed a significant increase of cell wall lignification of stem tissues around the inoculation zone in resistant plants. In addition to less lignified cell walls, susceptibility to the pine wood nematode seems associated with the activation of the salicylic acid (SA) defense pathway at 72 hpi, as revealed by the higher SA levels in the tissues of susceptible plants. Cell wall reinforcement and hormone signaling mechanisms seem therefore essential for a resistance response.

Pinus pinaster Early Hormonal Defence Responses to Pinewood Nematode (Bursaphelenchus xylophilus) Infection.

The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phytohormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most methodologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection.

Characterization of the cork formation and production transcriptome in Quercus cerris × suber hybrids.

Cork oak is the main cork-producing species worldwide, and plays a significant economic, ecological and social role in the Mediterranean countries, in particular in Portugal and Spain. The ability to produce cork is limited to a few species, hence it must involve specific regulation mechanisms that are unique to these species. However, to date, these mechanisms remain largely understudied, especially with approaches involving the use of high-throughput sequencing technology. In this study, the transcriptome of cork-producing and non-cork-producing Quercus cerris × suber hybrids was analyzed in order to elucidate the differences between the two groups of trees displaying contrasting phenotypes for cork production. The results revealed the presence of a significant number of genes exclusively associated with cork production, in the trees that developed cork. Moreover, several gene ontology subcategories, such as cell wall biogenesis, lipid metabolic processes, metal ion binding and apoplast/cell wall, were only detected in the trees with cork production. These results indicate the existence, at the transcriptome level, of mechanisms that seem to be unique and necessary for cork production, which is an advancement in our knowledge regarding the genetic regulation behind cork formation and production.

The draft genome sequence of cork oak.

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.