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.