Garoutte, A. 2016. Identifying the activities of rhizosphere microbial communities using metatranscriptomics. Dissertation, Michigan State University, East Lansing, Michigan.
Soil microbial communities carry out many functions, most of which are beneficial to the planet as well as to humans. Soil microbial communities control the biogeochemical cycling rates of key elements such as carbon, nitrogen, sulfur and phosphorous and can also aid in plant growth and disease defense. Microbial ecologists have studied the functional activities of microbial communities for decades often using laboratory incubations. Metagenomics has allowed the identification of the microbes and the potentially functional genes in an environmental sample, but does not allow an assessment of activity. Direct observation of microbial community activity in the field is the desired strategy to build the foundational knowledge required to assess, predict and potentially manage soil microbial community activity.
In this dissertation I combine the use of metagenomics with metatranscriptomics to identify functional activity of the microbial community in the soil and rhizosphere of candidate biofuel crops. First, I assessed the efficiency of a novel method of rRNA removal, called the duplex specific nuclease normalization, to remove the dominating rRNA from samples of total RNA, to allow greater sequence coverage of mRNA. While this method did result in about 17% non-rRNA, it did not provide a major gain in sequencing depth. I also established best practices for computational metatranscriptomic analysis, especially the importance of assembling short reads into longer contigs to improve annotation accuracy.
Second, I examined the activity of the rhizosphere microbial community of switchgrass, a candidate biofuel crop, using a combination of metagenomics, metatranscriptomics and metaproteomics. I defined a minimum core of microbial community functions of both metagenomic and metatranscriptomic sequences to focus the analysis on the most common sequences that were expressed. Beyond the expected housekeeping functions, the ecologically important functions related to biogeochemical cycling expressed were glycoside hydrolases, ligninolytic enzymes, ammonia assimilation, phosphate metabolism and functions related to plant-microbe interactions were production of auxin, trehalose and ACC-deaminase. Ecologically important genes had lower abundance than housekeeping functions indicating that ecologically important genes may represent keystone functions.
I also examined the effect of two plants, switchgrass and corn, on the presence and activity of microbial community functions at various distances from living roots using metagenomics and metatranscriptomics. The metagenomic data was able to differentiate between microbial communities associated with the two different crops and differentiate communities in direct contact with the roots versus those not in direct contact. The metatranscriptomic data was unable to differentiate between bulk and rhizosphere samples indicating others factors are stronger determinants of community transcription.
I show that direct observation of the activity of microbial communities associated with biofuel crops in field collected samples is possible through metatranscriptomics and aided by metagenomics and metaproteomics. These data allow the detection of microbial activities related to biogeochemical cycling and plant microbe interactions as well as reveal differences in genetic potential across different soil treatments.
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