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The endosperm of cereal grains accumulates large amounts of storage compounds such as carbohydrates and proteins that are essential for seed germination. These compounds also constitute a principal source of human food, animal feed, and industrial raw material globally. However, the molecular mechanisms controlling the cell differentiation of cereal endosperm remain largely unknown. This dissertation has been focused on identification of the gene regulatory networks associated with the differentiation of basal endosperm transfer layer (BETL), the starchy endosperm (SE), and the aleurone (AL) of the maize (Zea mays) endosperm, which are the main cell types involved in nutrient uptake from the maternal tissue and the synthesis and deposition of storage compounds. As a first step toward characterizing the gene networks associated with cell differentiation of the maize endosperm, the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize kernel were profiled using RNA sequencing (RNA-Seq). Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types. Detailed analyses of a coexpression module strongly associated with BETL identified a regulatory module activated by the MYB-Related Protein-1 (MRP-1), a previously reported regulator of BETL differentiation. To understand the interplay between the endosperm cell differentiation programs and the storage program which is activated immediately after the main cell types become distinguishable, direct and indirect target genes of Opaque-2 (O2), which is a bZIP family transcription factor (TF) that has been known as a major regulator of the endosperm storage program, were identified using a chromatin immunoprecipitation followed

The Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants

This work encompasses studies on the development of biocontrol strategies to manage the disease lettuce drop, caused by the fungi Sclerotinia sclerotiorum and S. minor, using the mycoparasitic fungus Coniothyrium minitans, and to better understand interactions during mycoparasitism at the biochemical level. Results from field experiments revealed that two applications of C. minitans at manufacturer recommended rates significantly reduced the incidence of lettuce drop caused by S. sclerotiorum but not by S. minor. Applications of other biocontrol products tested did not significantly reduce disease incidence caused by either pathogen. Sclerotium population studies revealed that soil populations of S. sclerotiorum in lettuce production fields ranged from 0.08 to 2.9 sclerotia/100g of soil and were generally aggregated in their distribution. Continued field studies revealed that there was no significant effect of irrigation (sprinkler vs furrow) on either the impact of sclerotium density or the efficacy of C. minitans. Studies on the evaluation of different application rates of Contans against S. minor revealed that two applications of Contans at 5 X manufactures recommended rates significantly reduced the disease incidence. Examination of sclerotial exudates of Sclerotinia spp. revealed that crude exudates from both Sclerotinia spp. stimulated C. minitans spore germination and the stimulation was due to compounds within the polar fraction. Studies on the role of lectin-carbohydrate binding during fungal-mycoparasite interactions revealed that many plant lectins as well as crude proteins extracted from sclerotia of either Sclerotinia spp. induced agglutination of C. minitans spores in vitro. Spore germination of C. minitans stimulated by sclerotial exudates of either Sclerotinia spp. was significantly inhibited in the presence of the lectin Con A but not other plant lectins. In vitro studies on the directional growth of C. minitans preceding mycoparasitism revealed the

calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM Hþ-ATPase regulation.

An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contai

In plants, the regulatory networks that have evolved to generate the appropriate cellular responses to external and internal stimuli often include a transient increase in intracellular calcium (Ca²⁺). One ten member, plant-specific family of Ca²⁺ sensing elements, the calcineurin B-like (CBL) protein family, is thought to relay the Ca²⁺ signal to downstream targets when plants experience an abiotic stress. The purpose of this study was to uncover critical functions of four CBL proteins. CBL10 was chosen for its distinct amino acid sequence and unique genomic structure among the CBL proteins. CBL1 and CBL9, generated by segmental duplication, were chosen based on their high level of amino acid identity. CBL8 was chosen based on its sequence and genomic structure similarities to SOS3/CBL4, the founding member of the CBL family. An Arabidopsis CBL10 knock-out insertion, cbl10-1, was isolated and found to have reduced stamen elongation, leading to male sterility. The mutant also showed growth arrest in aerial portions of the plant and developed chlorosis in response to increasing salt concentrations. Alternative pre-mRNA splicing generated five CBL10.2 variants whose transcript levels were regulated by cold or salt treatment, suggesting that CBL10 is involved in normal development and plant growth during abiotic stress via tight regulation of transcript levels. Phylogenetic analysis suggests that CBL1 and CBL9 may compensate for each other in the regulation of essential functions in the single mutant backgrounds. To investigate the crucial functions of these genes, a cbl1 cbl9 double mutant was generated. When grown under drought conditions, the double mutant was less sensitive to ABA, lost more water and produced more shriveled seeds than wild-type plants suggesting that both CBL1 and CBL9 play key roles in relaying drought stress signals during vegetative and reproductive growth. Because phylogenetic analysis and amino acid sequence comparisons suggest that CBL8 may f

In Arabidopsis thaliana, the calcium binding protein Salt Overly Sensitive3 (SOS3) interacts with and activates the protein kinase SOS2, which in turn activates the plasma membrane Na1/H1 antiporter SOS1 to bring about sodium ion homeostasis and salt tolerance. Constitutively active alleles of SOS2 can be constructed in vitro by changing Thr168 to Asp in the activation loop of the kinase catalytic domain and/or by removing the autoinhibitory FISL motif from the C-terminal regulatory domain. We expressed various activated forms of SOS2 in Saccharomyces cerevisiae (yeast) and in A. thaliana and evaluated the salt tolerance of the transgenic organisms. Experiments in which the activated SOS2 alleles were coexpressed with SOS1 in S. cerevisiae showed that the kinase activity of SOS2 is partially sufficient for SOS1 activation in vivo, and higher kinase activity leads to greater SOS1 activation. Coexpression of SOS3 with SOS2 forms that retained the FISL motif resulted in more dramatic increases in salt tolerance. In planta assays showed that the Thr168-to-Asp–activated mutant SOS2 partially rescued the salt hypersensitivity in sos2 and sos3 mutant plants. By contrast, SOS2 lacking only the FISL domain suppressed the sos2 but not the sos3 mutation, whereas truncated forms in which the C terminus had been removed could not restore the growth of either sos2 or sos3 plants. Expression of some of the activated SOS2 proteins in wild-type A. thaliana conferred increased salt tolerance. These studies demonstrate that the protein kinase activity of SOS2 is partially sufficient for activation of SOS1 and for salt tolerance in vivo and in planta and that the kinase activity of SOS2 is limiting for plant salt tolerance. The results also reveal an essential in planta role for the SOS2 C-terminal regulatory domain in salt tolerance.