Manuel Kleiner

In maize and many other plants, F1 hybrids perform better than their inbred parent lines - a phenomenon known as heterosis or hybrid vigor. The causes of heterosis have been investigated for over a century but are still poorly understood. Our preliminary data suggest a novel mechanism in which growth in sterile conditions reduces or eliminates heterosis for root size- a pattern that we term Microbe-Dependent Heterosis (MDH). Potential explanations for MDH include (1) superior resistance of hybrids to weakly pathogenic soil biota, or (2) immune over-reactions by inbred maize in response to innocuous soil biota. The proposed experiments will help to distinguish between these possibilities by exploring the genetic, ecological, and molecular causes of MDH. First, we will test a wide range of individual microbial strainsas well as naturally-occurring soil biota for the ability to induce MDH. Second, we will map the genetic architecture of MDH to identify genomic loci whose effect on heterosis is dependent on the microbial environment, and test for a genetic correlation with loci underlying resistance to a variety of pathogenic microbes in the field. Third, we will investigate the molecular mechanisms of MDH by measuring gene and protein expression of both hybrid and inbred plants as well as the microbes inside their roots. The results of these experiments will clarify the microbial features and patterns of plant immune activity that result in MDH. This work will be led by Drs. Maggie Wagner (U. Kansas), Peter Balint-Kurti (USDA-ARS), and Manuel Kleiner (North Carolina State U.)

Research has shown that microbial communities substantively impact plant health and resilience to both abiotic and biotic environmental stresses. To address rising global food security concerns, it is critical to gain a more comprehensive and integrated understanding of plant-microbe systems. Current technologies generate large quantities of data, but there is an urgent need to develop a more comprehensive systems understanding of the metabolic pathways that lead to observed results. Our central hypothesis is that microbial interactions and metabolite exchanges with key microbial species drive plant health (maize) and maintain robustness in the face of stress. Our goal in addressing this hypothesis is to elucidate the interactions and metabolite exchanges taking place by integrating theoretical modeling with experimental data to build a comprehensive metabolic model of the maize rhizosphere. We will employ a systems approach to guide our inquiries into plant-microbe interactions. Genome-scale metabolic models will be constructed, and metabolic pathways will be analyzed with elementary flux mode analysis, a pathway analysis technique which allows a comprehensive, unbiased examination of all possible metabolic pathways in a network. Multiple layers of experimental data will be incorporated as constraints to restrict the community model to a physiologically relevant solution space. Expected outcomes from the proposed project include the development of an experimentally validated community-level metabolic model to describe plant-microbe interactions and the effect of nutrient limitation. This will improve our understanding of maize response to nutrient stress conditions and the role that microbial communities play. Moreover, this project will provide new knowledge about the interplay between different levels of cellular expression by collecting extensive corresponding data sets on a single system to better understand the patterns between different levels of cellular expression and activity. Ultimately, the methods and concepts will be translatable to other crop systems and will contribute to development of microbial strategies for improving growth and managing susceptibility to pests and pathogens.

In maize and many other plants, F1 hybrids perform better than their inbred parent lines - a phenomenon known as heterosis. The causes of heterosis have been investigated for over a century but are still poorly understood. Our preliminary data suggest a novel mechanism, not previously reported, in which growth in sterile conditions reduces or eliminates heterosis for root size- a pattern that we term Microbe-Dependent Heterosis (MDH). The causes of MDH are unclear; potential explanations include (1) superior resistance of hybrids to weakly pathogenic soil biota, or (2) immune over-reactions by inbred maize in response to innocuous soil biota. The proposed experiments will help to distinguish between these possibilities by exploring the genetic, ecological, and molecular causes of MDH. First, we will test a wide range of individual microbial strains as well as naturally-occurring soil biota for the ability to induce MDH. Second, we will map the genetic architecture of MDH to identify genomic loci whose effect on heterosis is dependent on the microbial environment, and test for a genetic correlation with loci underlying resistance to a variety of pathogenic microbes in the field. Third, we will investigate the molecular mechanisms of MDH by measuring gene and protein expression of both hybrid and inbred plants as well as the microbes inside their roots. The results of these experiments will clarify the microbial features and patterns of plant immune activity that result in MDH.

Our Overall Goal is to identify genes and proteins that are causal to functional interactions within the microbiome and between the microbiome and the plant that impact drought tolerance in maize. We propose to develop new experimental resources and metaproteomics methods for studying plant microbiome function, and use them to identify molecular mechanisms underlying plant-microbiome interactions that confer improved drought resilience in maize. First, we will isolate and sequence a collection of root-associated bacteria from across a natural precipitation gradient in Kansas, and screen them for beneficial effects on drought-stressed maize. Second, we will develop and optimize metaproteomics methods for the study of plant-associated microbiomes. Finally, we will deploy these new resources in a tractable, fully controlled, simplified maize-microbiome experimental system to investigate causal relationships between plant and microbial genes, proteins, and enhanced drought resistance in maize. The resulting research tools and mechanistic knowledge would bring us closer to the goal of using beneficial microbes to protect maize yields under water-limited conditions without further depleting our natural freshwater resources. This project thus specifically addresses the Program Area Priority ����������������Agricultural Microbiomes in Plant Systems and Natural Resources���������������, A1402.

Our Vision is to provide a science-based platform for new agricultural practices enabling plant producers to manage their production ecosystems in a resource-efficient way with limited environmental footprint based on an in-depth understanding of key ecological functions in the soilplant interphase (rhizosphere). Our Motivation is to address the major research gaps in deciphering the complexity of microbemicrobe and microbe-plant interactions in the rhizosphere, and thereby provide new conceptual understanding on how these interactions influence plant performance. This motivation is timely due to recent developments in methodology and will enable us to provide the knowledge-base for unlocking the potential of the soil rhizobiota (microbes living on in the rhizosphere) as the key to development of sustainable and resilient plant production systems. Our Focus is to identify and quantify main determinants of microbial interactions and networks in the rhizosphere leading toward a resilient ecological unit, and thus reveal the importance and potential of microbial interactions and functions in the rhizosphere. The proposed research will take advantage of a multi-faceted, integrative and cross-disciplinary approach, which is fundamental for 1) achieving a deep understanding of the chemical and biological factors that control microbe-microbe and plantmicrobe interactions and functions under natural soil conditions, 2) establishing improved predictive models for microbial interactions in soil and 3) exploiting the microbial potential in plant-soil production systems for the benefit of plant growth and resilience. INTERACT will decode these important, yet often transient, microbial interactions in the complex soil matrix, in relation to soil biogeochemical status, water stress as well as pathogen attack, and the impact of these interactions on plant performance. We will challenge the currently accepted view among scientists that plants are the primary drivers for rhizobiome assembly. Hence, we will determine whether in fact soil microbes, largely through chemical communication and signaling, play a greater role in rhizobiome development and function than has been previously appreciated. INTERACT will provide critical insight into the rhizosphere ecology, as a basis for actively influencing the assembly of effective rhizosphere communities to support plant health and productivity, either through biotechnological or agronomic approaches.