Pathogen-associated molecular pattern (PAMP) recognition occurs by plasma membrane located receptors that induce among other processes nuclear gene expression. The plant FLS2-BAK1 receptor complex binds the bacterial PAMP, flg22 and induces a series of defense responses. The resulting signal transduction events occur through the activation of two MAPK signaling cascades, which trigger a rapid and strong activation of MPK3, MPK4 and MPK6. Cellular responses to pathogens are regulated by the activated MAPKs, which lead to the eventual phosphorylation of cytoplasmic and nuclear substrates. These MAPK substrates in turn respond to phosphorylation by reprogramming the expression of defense genes. A large scale phosphoproteomics screen of nuclear proteins in wild type and mpk mutant plants in response to flg22 revealed several novel putative targets of MAP kinases. This thesis is aimed at identifying the role of two of these chromatin associated proteins in plant immunity and their signaling mechanisms. The chromatin associated proteins we chose to study here are LITTLE NUCLEI/CROWDED NUCLEI (LINC/CRWN), LINC1 and the AT-HOOK MOTIF CONTANING NUCLEAR LOCALIZED 13 (AHL13) proteins. We demonstrate that these two chromatin associated proteins play a positive regulatory role in jasmonic acid signaling and immunity. Knock out mutants for both genes exhibit impairment in early and late innate immune reposes to both PAMP and hemibiotrophic pathogen strains. We also demonstrate that these mutants are compromised in regulating the expression of genes involved in jasmonic acid (JA) signaling and responses and genes involved in the biosynthesis both the indole and aliphatic glucosinolate (GS) pathways. Moreover, Pst DC3000 hrcC triggers JA and JAIle accumulation in these mutants, whereas salicylic acid (SA) levels are unchanged. We were also able to identify and validate two novel MAPK targeted phosphosites in AHL13 that affect the protein stability of AHL13 and we establish its role as a MPK6 substrate that affects jasmonic acid biosynthesis and PTI responses. Together this work identifies two novel signaling components involved in the regulation of jasmonic acid homeostasis and immunity.

Mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine protein kinases which link upstream receptors to their downstream targets. These targets can be localized in the cytoplasm or the nucleus. Pathogens produce pathogen-associated molecular patterns (PAMPs) that are known to trigger the activation of MAPK cascades. In plants, MAPK signaling cascades regulate development and cellular processes such as stress responses, immunity, and apoptosis by means of the phosphorylation of specific targets. Phosphoproteomics analysis of PAMP-induced Arabidopsis plants led to the identification of several putative MAPK targets. USP1 (Universal Stress Protein A) (At1g11360) and WLIM2A (At2g39900) are two potential phosphorylation targets of MAPKs, and are the focus of this thesis. So far, little is known about their role in plant immunity. CRISPR-Cas9 generated knockout usp1 mutant lines enhanced resistance to infection by Pst DC3000, usp1 mutant showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. Although the physiological role of USP1 has been established, the biochemical and molecular functions are unknown. We discovered a new role for USP1, demonstrating that it functions as a molecular chaperone and is involved in thermal priming. Overall, these data show that phosphoprotein USP1 plays an important role in orchestrating plant immunity. CRISPR-Cas9 generated knockout wlim2a mutant showed susceptibility to infection by Pst DC3000. wlim2a mutants showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated in wlim2a mutants. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. wlim2a mutants show enhanced fungal infection by Botrytis cinerea. Overall, the data shows that WLIM2A phosphorylation is important during plant immunity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.