Peter Balint-Kurti

"Project is in support of PSI" Plant disease resistance proteins of the nucleotide binding leucine-rich repeat (NLR) type are activated and induce a strong defense response known as effector-triggered immunity or ETI, upon recognition of specific pathogen-derived effector proteins. The effectiveness of this system depends on its inactivity when the cognate pathogen is not present, rapid induction when a pathogen is recognized followed by a rapid suppression after induction. The ubiquitin-proteasome pathway, mediated by the sequential actions of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating) and E3 (ubiquitin ligase) enzymes is a major protein modification process found in all eukaryotes. Our preliminary data indicates that maize ZmCER9 E3-ligase mediates degradation of the Rp1-D disease resistance protein specifically after its activation. We have further evidence that CER9 may act in a similar way to degrade other plant resistance proteins once activated. This appears to be a previously undescribed mechanism that mediates the deactivation of the defense response after activation. Based on its homology, ZmCER9 appears to be a component of the endoplasmic reticulum associated degradation (ERAD), a fundamental eukaryotic quality-control system that degrades incorrectly folded proteins. In plants this pathway has been relatively poorly characterized and there are no known substrates of the branch of the pathway mediated by CER9. Activated Rp1-D may represent the first known substrate of this branch of the ERAD pathway in plants. We hypothesize that ERAD-Mediated Degradation of Activated NLRs (EMDAN) is a general mechanism for the deactivation of ETI in plants. We propose to use a range of molecular, genomics and cell biology techniques to characterize the role of CER9, ERAD and related pathways involved in ubiquitin/proteasome associated processes in controlling ETI in maize and Arabidopsis.

Quantitative disease resistance (QDR) is the most important form of resistance used in maize and by crops in general. Prior work by our research team and many others has shown that QDR is based on a large variety of genes and mechanisms, most of which are still poorly understood (if at all). We have identified and characterized a number of QTL and genes associated with resistance to multiple maize diseases. We have also developed and characterized two large maize populations that are ideal for the genetic dissection of quantitative traits, QDR in particular. This proposal is aimed at exploiting these these resources and data to extend our knowledge of QDR, focusing on (a) underlying mechanisms of QDR and (b) QDR associations with and effects on other traits. we will focus on four fungal diseases that are among the most important diseases of maize in the US and worldwide; the foliar blights, southern leaf blight (SLB) and northern leaf blight (NLB) and the ear rots Fusarium ear rot (FER) and Gibberella ear rot (GER). Both ear rots additionally produce mycotoxins that harm livestock and human health and cause seedling blights that lead to significant crop losses.

We seek to understand the genetic basis of non-race-specific resistance to fusiform rust disease caused by Cronartium quercuum f. sp. fusiforme (Cqf) in Pinus taeda, an economically critical pine species. In previous research, our group mapped two major resistance QTL with high genetic resolution in the genome of a P. taeda resistance donor. In a parallel bulked-segregant RNAseq experiment, we identified candidate resistance genes with SNP highly associated with resistance to Cqf. These genes were part of the nucleotide-binding leucine-rich repeat. Here, we will leverage our newly gained knowledge of the genetics of host resistance to generate a pine population segregating for the same two resistance QTL. To understand the genetics of avirulence in the pathogen, the pine population will then be challenged with a diverse basidiospore mixture of Cqf in an artificial inoculation experiment. Following symptom development, fungal strains capable of growing on each of four host resistance genotypes will be sampled directly from diseased tissue and sequenced. Following SNP discovery, the fungal genome will be scanned for the presence of selective sweeps that would indicate proximity to genes selected for virulence against one or the other QTL, such as effectors.

Planned Activity, Objectives, and Methods Most plant pathogens produce effectors, proteins that are introduced into the plant cell to facilitate the pathogenesis process. Plants carry nucleotide-binding leucine rich repeat (NLR) proteins which, upon recognition of specific effectors, trigger a defense response called effector-triggered immunity, usually including a hypersensitive response (HR), a rapid cell death at the point of infection. The maize Rp1-D gene encodes an NLR resistance protein that confers resistance to common rust disease conferred by the fungus Puccinia sorghi. We previously used genetics and molecular biological approaches to identify several host proteins responsible for controlling the activity of Rp1-D21, an auto-active derivative of Rp1- D. In this project, we will use complementary approaches, including bioinformatics, functional genomics, cell biology, and spectroscopy techniques, to identify and analyze the molecular components of the interaction deriving from P. sorghi as well as other important host-derived components. We will identify effectors associated with the control of host cell death and suppression of the host defense response. We will define how these effectors influence important physiological changes in host cells, such as changes in pH, reactive oxygen species production, and calcium flux, and will characterize their subcellular localizations. We will also examine the maize HR with respect to these same physiological changes and the organelle dynamics in the cell. We will examine in particular the formation of stromules, narrow stromafilled tubes that extend from plastids, often connected to other subcellular compartments, including the nucleus, that are believed to facilitate the exchange of signaling components between the plastids and nucleus during HR. Finally, we will characterize the physical interactions of all the host- and pathogenderived components that interact with Rp1-D and are likely to constitute components of the Rp1-D signaling complex, the ���������������resistosome������������������. The proposal explicitly addresses the focus of the PBI program to ����������������support ��������������� fundamental ��������������� research on the mechanisms and principles that mediate the interaction of plants with their biotic partners���������������. Intellectual merit Despite significant progress, there remains much to learn about NLR-mediated resistance. This is particularly true in monocots. This project employs state-of-the-art biochemical and cell biology techniques to augment and extend our understanding of the control of the defense response mediated by Rp1-D focusing on pathogen derived components. This will result in an understanding of the control of the NLRmediated response that is unique in maize and among the most detailed in any plant species. Broader impacts The broader impacts of this proposal are twofold. The proposed research will elucidate a pivotal defense mechanism in maize which is both a model species for plant quantitative genetics and the number one crop in the U.S. Our results will be of direct relevance to efforts to genetically improve this important crop. Since the HR is a general defense response found in all multicellular plants, our findings will be relevant to improving other important crop species, particularly other grasses. The second impact is through the planned outreach activities with the NCSU Science House. All outreach activities will educate the public on genetics, plant breeding, biotechnology, and associated societal implications. They build on existing successful programs that have developed several instructional modules for teachers and students.

The purpose of this project is to develop a handheld Mueller matrix polarimeter that can be deployed to measure leaves in transmission. Leaves from different corn varieties will be quantified using both this handheld unit and our laboratory unit (an imaging Mueller Matrix polarimeter). These data will be compared to ground truth from e.g., enzymatic, colorimetric, 1D-NMR, and Mass-spectrometry based analyses, to correlate polarimetry measurements to metabolic concentration. Additionally, we will investigate polarization in reflection using a hyperspectral imaging polarimeter to quantify polarization������������������s ability to correct for bidirectional reflectance effects from canopy level measurements.

More than a third of crop yields are currently lost due to abiotic and biotic stressors such as drought, pests, and disease. These stressors are expected to worsen in a warmer, drier future, resulting in crop yields further declining ~25%; however, breeding is only expected to rescue 7-15% of that loss [1]. The plant microbiome is a new avenue of plant management that may help fill this gap. All plants have fungi living inside their leaves (����������������foliar fungal endophytes���������������). This is an ancient and intimate relationship in which the fungi affect plant physiology, biotic and abiotic stress tolerance, and productivity. For example, some foliar fungi prevent or delay onset of major yield-limiting diseases caused by pathogens such as Fusarium head blight [2]. Foliar endophytes also reduce plant water loss by up to half and delay wilting by several weeks [3, 4]. Endophyte effects on plants occur via diverse genes and metabolites, including genes involved in stress responses and plant defense [5]. Genes and metabolites also predict how interactions in fungal consortia affect host stress responses, which is important for developing field inoculations [6]. Because newly emergent leaves lack fungi, endophytes are also an attractive target for manipulation (particularly compared to soils, where competition with the existing microbial community inhibits microbial additives). We propose to address the role of endophytic ����������������mycobiomes��������������� in stress tolerance of five North Carolina food, fiber, and fuel crops (corn, hemp, soybean, switchgrass, wheat), and to develop tools that can push this field beyond its current limits. Our major objectives (Fig. 1) are to: 1. Identify key microbiome scales to optimally manage endophytes 2. Determine fungal mechanisms via greenhouse tests, modeling, and genetic engineering 3. Build tools for field detection of endophytes 4. Understand the regulatory environment and engage diverse stakeholders Results of these objectives will allow us to make significant progress in both understanding the basic biology of plant-fungal interactions and managing those interactions in real-world settings. Our extension efforts will also bring these ideas to the broader community. Finally, we will also be well positioned to pursue several future research endeavors supported by federal granting agencies.

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.

We generally assess levels of foliar disease in corn by observation in the field using a visual scale. While this method is robust and gives reproducible data, it does not provide qualitative data such as lesion size or shape, nor does it give us good data on speed of disease progression or on timing of initial symptoms. We will rate a set of 50 genetically similar lines each of which has a different allele conferring resistance to the foliar disease southern leaf blight. We will use a variety of approaches to rate disease , including hyperspectral imaging and digital imaging. We will also rate them conventionally at very frequent intervals. Finally, we will measure yield. This work will help to develop methods for the early detection of foliar disease in the field. It will also characterize disease progression and the relationship between symptoms and yield loss. This knowledge will aid in the development of predictive models to guide farmers in the decision of whether and when to apply fungicides. The characterization of different mechanisms used by different resistance genes will guide breeders in the combination of resistance genes to produce more optimally-robust disease resistant lines.

Arbuscular mycorrhiza (AM) are soil-borne fungi that form intimate symbiotic associations with the roots of most land plants. AM fungi act as extensions of the root system, increasing the root ����������������uptake area��������������� more than 1000-fold and considerably improving the plant's ability to acquire water and nutrients from the soil. AM fungi therefore substantially improve the host plant������������������s access to macronutrients including phosphorous (P), potassium (K) and nitrogen (N), and micronutrients. This proposal describes a project to understand the genetics controlling the association between corn and AM fungi. If successful, this will facilitate the increased used of AM fungi in corn cultivation and help reduce the expensive and ecologically-damaging use of fertilizers.

We generally assess levels of foliar disease in corn by observation in the field using a visual scale. While this method is robust and gives reproducible data, it does not provide qualitative data such as lesion size or shape, nor does it give us good data on speed of disease progression or on timing of initial symptoms. We will rate a set of 50 genetically similar lines each of which has a different allele conferring resistance to the foliar disease southern leaf blight. We will use a variety of approaches to rate disease , including hyperspectral imaging and digital imaging. We will also rate them conventionally at very frequent intervals. Finally, we will measure yield. This work will help to develop methods for the early detection of foliar disease in the field. It will also characterize disease progression and the relationship between symptoms and yield loss. This knowledge will aid in the development of predictive models to guide farmers in the decision of whether and when to apply fungicides. The characterization of different mechanisms used by different resistance genes will guide breeders in the combination of resistance genes to produce more optimally-robust disease resistant lines.