Abstract
The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota.
Highlights
Advances in nucleic acid sequencing and proteomics technologies have revolutionized our ability to study the biology of plant hosts and their associated microbiota [1,2,3]
These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota
We describe validated protocols for the extraction of DNA, RNA, and proteins from the oak caulosphere for use in parallel host-microbiota analysis of Acute Oak Decline (AOD), a major threat to native British oak (Quercus robur and Quercus petraea) [6,7]. These procedures demonstrated the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry, and it is hoped they will be of value for scientists in the field
Summary
Advances in nucleic acid sequencing and proteomics technologies have revolutionized our ability to study the biology of plant hosts and their associated microbiota [1,2,3]. We describe validated protocols for the extraction of DNA, RNA, and proteins from the oak caulosphere (stem tissue) for use in parallel host-microbiota analysis of Acute Oak Decline (AOD), a major threat to native British oak (Quercus robur and Quercus petraea) [6,7] These procedures demonstrated the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry, and it is hoped they will be of value for scientists in the field. The procedures described here are cost effective, relatively uncomplicated, and avoid the use of hazardous chemicals like phenol and chloroform These protocols were used to extract proteins and nucleic acids for analysis of the diversity and function of the oak microbiome in our previous studies of Acute Oak Decline, a complex Decline disease affecting native oak in the United Kingdom [6,7]. The techniques are designed to allow for the multi-omic analysis of trees and their associated microbiota, making them effective for taking forward the expanding use of multi-omics and host-microbiota interactions in tree disease research
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