Transcriptomic analyses of Pinus contorta responses to Grosmannia clavigera under contrasting levels of nitrogen availability

• Author / Creator

  Normington, Louisa Charlotte

•  In western Canada alone, the current mountain pine beetle (MPB) outbreak has devastated nearly 19 million hectares of pine forest, negatively impacting communities, industries and ecosystems. Reforestation efforts can benefit from a deeper understanding of the impact of nitrogen (N)-based fertilization on pine trees under attack by bark beetles and their microbial cohorts. To this end, we employed transcriptomic tools to explore how N availability affects the molecular response of lodgepole pine to the pathogenic MPB fungal associate Grosmannia clavigera. 
  
   The first objective was to find the optimal approach to the de novo assembly of a lodgepole pine reference transcriptome in preparation for RNA-Seq. We hypothesized that the CLC Genomics Workbench would produce a faster assembly, but that Trans-ABySS, given its multiple k-mer de Bruijn graph algorithm, would produce a higher quality assembly. Following our findings, we suggest using Trans-ABySS for constructing reference transcriptomes using Illumina next-generation sequence data from non-model species.
  
   The second objective was to conduct a controlled environment experiment to evaluate the effects of N availability on the responses of lodgepole pine seedlings to G. clavigera infection. We hypothesized that higher N availability would result in an increase in foliar N concentration in the lodgepole pine seedlings. In addition, we hypothesized that fertilization with 1 mM or 10 mM NH4NO3 would impact lodgepole pine defense against G. clavigera, as measured by differences in lesion development. We demonstrated that increased fertilization significantly impacted foliar N content, resulting in an increase in foliar N concentration. Furthermore, the higher concentration fertilizer seemed to elicit a stronger defense response through the creation of a longer lesion, possibly in response to N-stimulated fungal growth. 
  
   The third objective was to use RNA-Seq to identify the defense response patterns of lodgepole pine seedlings fertilized with either low (0.3 mM NH4NO3) or high (10 mM NH4NO3) levels of N followed by inoculation with G. clavigera in growth chamber conditions. This portion of the thesis project produced a comprehensive lodgepole pine master transcriptome with accompanying annotations. Differential expression analysis was accompanied by data mining of significantly differentially expressed transcripts to uncover patterns of gene expression influenced by N availability, in accordance with the fourth objective, followed by a network analysis approach to identify genes that were co-expressed with key transcription factors. We hypothesized that N availability would: (1) affect known components of lodgepole pine defense against G. clavigera, such as monoterpene synthesis, (2) modulate expression of genes thought to be important in mediating G. clavigera-elicited responses in lodgepole pine, and (3) alter the ratio of N-based to carbon (C)-based defense-related genes that were up-regulated in response to G. clavigera inoculation, with a greater proportion of N-based defense genes up-regulated in response to higher N availability.
  
   Quantification of monoterpene levels in volatile organic compounds (VOCs), foliage and phloem determined that levels of a relatively small number of monoterpenes were significantly impacted by N fertilization and G. clavigera inoculation, including the VOC α-pinene. Significantly differentially expressed defense-associated genes, such as terpene synthase and chitinase genes, displayed a greater fold change induction in the 0.3 mM NH4NO3-treated seedlings compared with the 10 mM NH4NO3-treated seedlings. It is possible that more resources were allocated to growth in 10 mM NH4NO3 treated seedlings resulting in a lower intensity defense response, though without the appropriate growth measurements, we can only speculate. A greater proportion of N-based defenses were up-regulated compared with the up-regulated C-based defenses in the high N-treated tissues compared with the low N-treated tissues. Finally, network analysis identified JAZ and WRKY transcription factors as hub genes that represented potential regulators of lodgepole pine’s molecular response to G. clavigera. 
  
   The analyses carried out in this study suggest an intersection between N use and defense in lodgepole pine seedlings challenged by G. clavigera and grown under low and high N applications in growth chamber conditions.
  

• Subjects / Keywords

  • lodgepole pine
  • mountain pine beetle
  • nitrogen fertilization
  • Grosmannia clavigera
  • plant defense
  • transcriptomics

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-mwta-xy19

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.