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.)

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.

When seeking solutions to today's elevated atmospheric CO2 levels, it is critical that we include data from the past, because atmospheric CO2 concentrations have fluctuated throughout Earth history. In fact, CO2 levels have been consistently higher in the past������������������often significantly higher, at times perhaps as much as 6x pre-industrial values. The biological response of life on Earth to these global conditions, from their onset to their cessation, is recorded in the rock record. Intriguingly, Konservat Lagerst��������tte (e.g., sedimentary deposits that preserve fossils in extraordinary detail) occur more frequently in the distant past (i.e., deep time) than in more recent depositional environments. Could these be linked? We hypothesize that ancient microorganisms responded to pre-Cenozoic high atmospheric CO2 by sequestering carbon through very rapid precipitation of carbonate minerals in terrestrial, as well as marine settings. This increase in microbial precipitation of carbonates, sometimes as concretions, created conditions favorable to the stabilization of normally labile tissues and the exclusion of exogenous, degradative influences. These factors very likely contributed to exceptional preservation of fossil remains, including persistence of non-biomineralized (i.e., ����������������soft���������������) tissues. Although microbes have been invoked as agents of preservation as well as destruction, because they act to ����������������seal��������������� sediments surrounding bone to form a relatively closed system, to date, the effect of contemporaneous atmospheric CO2 levels on microbial carbonate precipitation, and its implications for preservation, have not been explored. The convergence research we propose would enable us to design and implement empirical studies that directly test this idea, and characterize the microbial influence in depositional environments producing exceptionally preserved fossils. Thus, we ask the following: 1) Did the elevated CO2 in Mesozoic atmospheres play a role in microbially mediated exceptional preservation? 2) If this can be demonstrated through actualistic experiments and fossil studies, could this mechanism of fossil preservation also shed light on microbial sequestration of atmospheric CO2 in terrestrial environments? 3) Furthermore, can this understanding of microbially mediated CO2 sequestration be harnessed for development of robust, scalable carbon-capture systems? To test these hypotheses, we propose a two-pronged approach. We will conduct empirical tests that involve growing known microbially induced carbonate precipitation (MCIP) strains, as well as microbial communities from relevant environments, under conditions of Mesozoic proxy atmospheres. We will compare the rate and degree of precipitation in organisms grown in enriched CO2 with those of the same strains grown in ambient atmospheres, to characterize the effects of elevated CO2 on precipitation rates. Then, we will examine: 1) the sediments surrounding exceptionally preserved fossils, 2) the composition of concretions that contain fossil material, 3) the morphological and molecular preservation of the fossils themselves, and 4) biomarkers associated with microbes in these fossil materials, using a combination of chemical and molecular techniques. Our interdisciplinary team will work synergistically to examine the role of microbes in both fostering and impeding exceptional preservation, the relationship of exceptional preservation to elevated atmospheric CO2, and potential microbial pathways that can be exploited to accomplish terrestrial carbon sequestration. Such pathways are rarely considered in the dialogue regarding potential solutions to anthropogenic carbon release, but may present a viable, cost-effective mitigation measure

One of the grand challenges facing humanity is to secure sufficient and healthy food for the increasing world population. This requires maintaining sustainable cultivation of crop plants under changing climate conditions. Plant roots and soil microbes have been associated since the emergence of plants on land. Nevertheless, the mechanisms that coevolved to control and regulate microbiota associations with healthy plants are largely unexplored. The photosynthetically active green leaf tissues supply assimilated carbon to roots for development and also to feed its associated microbes. To maintain balanced growth, plants have to integrate this underground demand and regulate the rate of photosynthetic CO2 fixation, and sugar allocation needs to be coordinated between root and shoot. Research on plants and their naturally associated microorganisms is therefore in a prime position to provide new perspectives and concepts for understanding plant function, plant performance and plant growth under limited input conditions with a reduced environmental footprint and could also define breeding targets and develop microbial interventions. InRoot aims to: 1. Disentangle the effects of climate and soil type from the impact of root-microbe interactions through transplantation experiments and exploit natural variation to identify the plant genetic components responsible for adaptation to the local microbiota. 2. Identify key bacterial taxa governing the establishment of host-driven microbial networks in the rhizosphere by analysing the microbe-microbe and microbe-host interactions established in tailored synthetic communities (SynComs) with direct consequences on host performance. 3. Define the plant genetic components that control infection of plant roots by ubiquitous and host-specific endophytes using advanced genetic screens and new methods for quantifying root cellular responses to microbes 4. Understand molecular mechanisms integrating root-microbe interactions into whole-plant physiology by investigating systemic physiological responses induced by SynComs using whole plant phenotyping. 5. Predict plant performance as a function of plant and microbiota genotypes by building multiscale models based on genotype, phenotype, and mechanistic data thereby providing knowledge for application. InRoot perspective: Provide knowledge and tools for science-based development of new crop varieties and associated microbial interventions that will improve productivity, reduce the need for fertilizers and pesticides, and alleviate negative environmental impact.