FY 2019

High-throughput screening for genes to improve performance of crop plant microbiomes using functional metagenomics

PIs: Nathan Crook, Ph.D., Assistant Professor, Chemical and Biomolecular Engineering, and Manuel Kleiner, Ph.D., Assistant Professor, Plant and Microbial Biology.

Plant-associated microbiomes are critical for productivity and resilience against stress and disease in agricultural crops. Therefore, there has been recent interest in the development of methods to promote the colonization of plants with beneficial microbes or microbiomes, most often through seed treatments. However, a major emerging problem with this approach is that beneficial microbes are unable to persist under field conditions and get outcompeted by resident microbes very quickly. While recent work has uncovered colonization-enhancing genes for beneficial gut microbes and predicted colonization-related genes in plant commensal microbes, no persistence-enhancing genes are known for plant commensals. The goal of this proposal is to develop and apply functional metagenomics for large-scale screening of millions of metagenomic fragments for genes that confer desirable traits to plant-associated microorganisms. For this we will, (1) optimize transformation protocols for bacterial colonizers of plant roots and (2) identify genes which confer dominance to transformable plant root colonizers.


FY 2018

Development and evaluation of metaproteomics methods for root-associated microbes

PIs: Manuel Kleiner, Ph.D., Assistant Professor, Plant and Microbial Biology, and Shuijin Hu, Ph.D., Professor, Entomology and Plath Pathology. Postdoc: Fernando Salvato, Ph.D.

Root-associated microorganisms play important roles in plant growth and health. A large number of these microorganisms have been identified, however, the functional basis of the interactions between these microbes and plants is, in most cases, not understood. We will develop high-resolution mass spectrometry methods in the realm of metaproteomics to study the metabolism, physiology and interactions of root-associated microorganisms. The core approach that we will employ to develop plant-microbe metaproteomics into a robust and reproducible tool is the use of two plant species with defined, fully characterized microbial communities to systematically develop and validate the methods. This project will lay the groundwork for root and soil microbiota metaproteomics by (1) developing and validating protein extraction and cleanup methods for root-associated microbes; (2) optimizing the LC-MS/MS workflow to achieve a high metaproteome coverage; and (3) applying the developed workflows to two model systems for plant-microbe interactions to demonstrate the application of the method.

Are seed banks repositories of beneficial plant associated microbes?

PIs: Robert Dunn, Ph.D., Professor, Applied Ecology, Ignazio Carbone, Ph.D., Professor, Center for Integrated Fungal Research, Department of Entomology and Plant Pathology, Amy Grunden, Ph.D., Professor, Plant and Microbial Biology, and Christine Hawkes (beginning at NCSU in 2018). Postdoc: Anne Madden, Ph.D.

Around the world many varieties of seeds are saved in seed collections. The Svalbard seed collection alone now holds nearly a million varieties of crop seeds, to say nothing of the varieties of non crops present in collections. Such collections offer a potentially enormous value not only in terms of the plant genetic diversity they represent, but also in terms of the varieties of fungi and bacteria inside the seeds. Seed collections have ignored the bacteria and fungi in seeds, except in as much as they have tried to kill pathogens. We have little idea how many varieties of useful bacteria and fungi may be in such seeds but it too could be millions. Our goal in our project is to characterise the beneficial fungi and bacteria present in the major domesticated linages of grains, as well as in the wild relatives of such grains. If grains seeds in collections still hold many useful bacteria and fungi, changes need to be made in seed collections in order to find ways to preserve those microbes. At the same time, we to (and will begin the process here) develop a pipeline to study and use such microbes in agriculture as well as in other contexts such as food preservation.


 FY 2017

Effects of hybridization on the maize metagenome

PIs: Dan Bowman, Ph.D., Professor, Crop and Soil Science, and Jim Holland, Ph.D., USDA Professor, Crop and Soil Science. Postdoc: Maggie Wagner, Ph.d., NSF Plant Genome Postdoctoral Fellow

Hybrid vigor, the phenomenon in which hybridization between inbred genotypes results in phenotypically superior offspring, is a subject of immense economic value and research interest. In maize, many traits exhibit heterosis, where mean trait values in the F1 exceed those of either parent; however, the effect of hybridization on maize microbiome composition and diversity (which are known to be heritable) has never been tested. The proposed research will compare the rhizosphere and foliar microbiomes of inbred maize lines and their hybrid offspring, and explore the consequences for metagenome content and plant growth. Because crosses between inbred lines are a crucial mechanism in crop breeding, understanding the relationship between hybridization, metagenome content, and microbiome function is an important step toward incorporating microbiome science into breeding programs.


Extension and validation of maximum resolution amplicon bioinformatics methods to fungal marker-gene data

PIs: Benjamin Callahan, Ph.D., Assistant Professor, Population Health & Pathobiology and Ignazio Carbone, Ph.D., Professor, Center for Integrated Fungal Research, Department of Entomology and Plant Pathology

Fungal communities are crucial components of soil communities, and fungi are the most important microbial partners for plant health and function. High-throughput amplicon sequencing has become a powerful tool to characterize soil and plant-associated fungal communities, but the variable length of the ITS region presents different challenges that often interfere with methods developed primarily for the 16S rRNA gene. We propose to bring the state-of-the-art bioinformatics methods from the bacterial world into the fungal world by extending the DADA2 method for sequence variant inference to work on variable-length amplicons like those generated from the ITS gene region, and to validate those methods on fungal data. These new methods will allow marker-gene studies of fungal communities to resolve individual fungi more precisely, and to better discriminate between related fungi that may functionally differ.


Investigating the impact of bacterial endophytes on mycorrhizal fungi, rhizosphere microbiome and plant nutrient acquisition in switchgrass

PI: Shuijin Hu, Ph.D., Professor, Entomology & Plant Pathology. Collaborator: Chuansheng Mei, Ph.D., The Center for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research (IALR)

The rhizosphere microbiome, including saprophytic microbes and arbuscular mycorrhizal fungi (AMF), plays a major role in plant nutrient and water acquisition. Endophytes, bacterial or fungal symbionts living within the plant, are ubiquitous and can confer some beneficial effects on host plants through improving plant growth and tolerance to abiotic and biotic stresses. They can be particularly useful for forage and fuel crops such as switchgrass that will preferentially be planted on marginal lands where environmental stresses are routine. The primary objective of this study is to assess the impact of endophytic bacteria on AMF in roots, the rhizosphere microbiome (with a focus on N-cycling microbes), and plant nutrient acquisition, using switchgrass a model plant. Results from this study will help understand the drivers that control tripartite interactions among plants, endophytes in roots, and soil microbes in the context of plant C allocation for microbes in exchange for nutrient acquisition.


 FY 2016

Multiple Disease Resistance and the Maize Microbiome

PIs: Eric Davis, Ph.D.,William Neal Reynolds Distinguished Professor of Plant Pathology and Peter Balint-Kurti, Ph.D., Professor of Plant Pathology. Postdoc: Maggie Wagner, Ph.D., NSF Plant Genome Postdoctoral Fellow

Robust genetic resistance to multiple diseases is a key goal for crop breeding programs. However, non-pathogenic endophytic microbes also affect plant health and productivity. Do loci conferring broad-spectrum pathogen resistance have side-effects on the maize microbiome? Are there trade-offs between multiple disease resistance and accumulation of beneficial endophytes? The proposed research will improve our understanding of how host genotype influences the microbiome of maize roots and leaves; specifically, whether QTL associated with multiple disease resistance also alter microbiome diversity and composition. This study will reveal which microbial taxa within the endophyte community are sensitive to or robust to host genes conferring broad-spectrum resistance. In addition, this work will generate a collection of fungal strains isolated from maize leaves in the field, and will screen them for plant growth promotion in disease-resistant and disease-susceptible maize lines.


The Microbiome of Rice Seed and Seedlings

PI: Ralph Dean, Ph.D., William Neal Reynolds Distinguished Professor of Plant Pathology.

The manipulation of the plant microbiome has untapped potential for reducing chemical inputs, tolerating stressful environments, lowering disease and enhancing agricultural productivity. In this project, the microbiome of rice seeds and seedlings growing independently of natural soil will be characterized. Specifically, the role of genotype, source of seeds as well as seed age on the microbiome will be examined by pyrosequencing of 16S ribosomal (prokaryotes) and ITS region of the rRNA operon (fungi) as well as Sanger sequencing of isolated microbes. Sequence data will be examined for microbial diversity and determination of a core seed and seedling microbiome.