Development of molecular markers for resistance to pine wilt disease in Pinus pinaster

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Characterization of microRNAs implicated in maritime pine resistance to pinewood nematode

Publication . Mendes, André Vieira; Miguel, Célia Maria Romba Rodrigues; Castro, Dora Cristina Vicente Batista Lyon de

Pine wilt disease (PWD), caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus, is a serious threat to coniferous forests worldwide. Post-transcriptional regulation of gene expression by microRNAs (miRNAs) plays important roles during plant-pathogen interactions. Recently, a few miRNAs have been identified as differentially expressed between PWD resistant and susceptible maritime pine (Pinus pinaster Ait.) plants. The aim of this work was to further characterize the expression patterns of four of these miRNAs during early stages of PWN infection and evaluate how they relate with maritime pine responses. P. pinaster 4-year-old plants from half-sib families previously characterized regarding their predicted survival mean to PWN, were inoculated and symptom development was recorded. Stem samples were collected before (0 hours) and at 24, 48 and 72 hours post inoculation. After RNA extraction, reverse transcription quantitative PCR (RT-qPCR) data was obtained for quantification of miRNA expression in susceptible, resistant and control plant stem samples. First, reference miRNAs for normalization of expression data were identified using well-established algorithms. Then, a step toward functional characterization of the potentially defense-related P. pinaster miR166h, miR951f, miR3627m and Novel_110 was given by comparing their expression patterns along time between resistant and susceptible plants. The results show that miR11532_4 and miR396_48 are good reference miRNAs, being more appropriate than currently used reference genes. Expression analysis suggests the potential contribution of miR166h and miR951f upregulation to inefficient activation of susceptible plants’ immunity, as well as the possible involvement of Novel_110 in cell wall reinforcement of resistant genotypes. Furthermore, the potential regulation of oxidoreductase activity by miR3627m is discussed. Overall, valuable information on post-transcriptional regulation of maritime pine response to PWD was obtained, although further functional characterization of these miRNAs is needed. Ultimately, this knowledge might be helpful for breeding programs that aim at developing resistant P. pinaster plants.

2022Master thesis

Open access

SNP Detection in Pinus pinaster Transcriptome and Association with Resistance to Pinewood Nematode

Publication . Modesto, Inês; Inácio, Vera; Novikova, Polina; Carrasquinho, Isabel; Van de Peer, Yves; Miguel, Célia M.

Pinewood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of pine wilt disease (PWD), which severely affects Pinus pinaster stands in southwestern Europe. Despite the high susceptibility of P. pinaster, individuals of selected half-sib families have shown genetic variability in survival after PWN inoculation, indicating that breeding for resistance can be a valuable strategy to control PWD. In this work, RNA-seq data from susceptible and resistant plants inoculated with PWN were used for SNP discovery and analysis. A total of 186,506 SNPs were identified, of which 31 were highly differentiated between resistant and susceptible plants, including SNPs in genes involved in cell wall lignification, a process previously linked to PWN resistance. Fifteen of these SNPs were selected for validation through Sanger sequencing and 14 were validated. To evaluate SNP-phenotype associations, 40 half-sib plants were genotyped for six validated SNPs. Associations with phenotype after PWN inoculation were found for two SNPs in two different genes (MEE12 and PCMP-E91), as well as two haplotypes of HIPP41, although significance was not maintained following Bonferroni correction. SNPs here detected may be useful for the development of molecular markers for PWD resistance and should be further investigated in future association studies

2022Journal article

Open access

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

Publication . Modesto, Inês; Mendes, André; Carrasquinho, Isabel; Miguel, Célia M.

Pine wilt disease (PWD) is a severe environmental problem in Eastern Asia andWestern 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 RNAinterference 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

2022Journal article

Open access