Pamp triggered responses as the first layer of immunity reprogramming cells for defense in both mammals and plants
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Both mammals and plants live in a constant co-existence with a broad range of microbes. Some of these microbes have the potential to be pathogenic and will cause disease symptoms if they successfully invade the hosts. As an important early line of defense, mammals and plants have evolved perception systems to detect the presence of potentially invading microbes. In addition to the initial recognition, proper intracellular responses are needed to restrict microbial growth and prevent a full infection. This line of defense depends on the recognition of pathogen associated molecular patterns (PAMPs). PAMPs are conserved among and specific to microbes but not to the host. Common bacterial PAMPs recognized by both mammals and plants include lipopolysaccharides (LPS) and flagellin. PAMPs are recognized by pattern recognition receptors (PRRs). The recognition induces many intracellular changes such as ion influx, changes in protein phosphorylation including activation of mitogen-activated protein kinases (MAPK), and transcriptional reprogramming. Many general strategies of these responses are shared in both mammals and plants, but most of the specific proteins and events are unique between mammals and plants. In mammals, one set of PRRs, TLRs, recognize a broad range of PAMPs. PAMPinduced TLR signaling leads to transient rise of cytoplasmic Zn2+, MAPKs activation and the releasing of the central pro-inflammatory mammalian transcription factor NF-?B, which leads to the production of pro-inflammatory cytokines. Cytokines facilitate the eradication of invading pathogen and the establishment of adaptive immunity. Most PRRs in plants, such as the flagellin receptor FLAGELLIN SENSITIVE 2 (FLS2), are cell surface-located receptor-like kinases (RLKs). Like TLRs in mammals, RLKs signaling pathways are thought to utilize common signaling components. Intracellular changes induced by PAMP perception include rapid Ca2+ influx, MAPKs activation, PAMP responsive gene expression, and at later stages, the accumulation of a defense hormone salicylic acid (SA). PAMP perception has been demonstrated to play a role in limiting bacterial growth. Some or all of these changes lead to effective restricting of bacterial growth called PAMP triggered immunity (PTI). A significant feature of TLR signaling is that ubiquitination directed protein complex scaffolding and signal components degradation play an important role in signaling transduction. In addition, many TLRs signaling transduction including TLR4 signaling has a special requirement for metal zinc. Zinc is an essential element in immunity, the deficiency of which results in a depressed immune response and an increased susceptibility to infections. Although free zinc in the cytoplasm is maintained at extremely low concentrations, a transient pool of zinc has been observed in TLR1-, TLR2-, and TLR4 activation in monocytes and granulocytes, indicating a role of zinc in signaling transduction. Depletion of zinc using specific chelators has been shown to block downstream activation of MAPK and NF-?B upon LPS stimulation of macrophage cell lines and primary monocytes. However, the underlying steps in TLR pathway affected by zinc deficiency are poorly understood. In Chapter II, the effect of variable zinc availability on LPS-induced TLR4 signaling was systematically investigated in the macrophage RAW264.7 cell line. It was found that zinc deficiency blocked the phosphorylation of several downstream kinases including IKK, MKK, and MAPKs, which was restored by supplementation with zinc in a dose-dependent manner. Whereas zinc deficiency did not affect the phosphorylation or ubiquitination of the upstream kinase, IRAK1, and the subsequent degradation of this protein was dependent on zinc availability. Significantly, IRAK degradation was not affected by proteasome inhibitors. These results revealed a novel Zn- dependent but proteasome-independent IRAK1 degradation upon LPS stimulation for further TLR4 signal transduction. In plants, there are still many gaps in the understanding of PAMP-induced RLK signaling. For instance, the substrates of MAPKs that directly regulate PAMP responsive gene transcriptional reprogramming are largely unknown. In addition, the mechanisms by which intracellular signaling, such as MAPK activation and SA accumulation, contribute to PTI are still enigmatic. Recently, mkp1 (Ws) was reported to display enhanced early PAMP-triggered responses as well as enhanced resistance against bacterial infection in our lab. In addition, mkp1 resistance is completely dependent on the presence of a specific MAPK, MPK6, making this mutant a useful tool to investigate PAMP signaling and resistance. In chapter III, I further investigated whether resistance in mkp1 depends on SA by measuring SA content as well as using SA- deficient mutants in mkp1. Unexpectedly, I found a novel, SA-independent resistance in mkp1 mutant. Interestingly, I found mkp1 compromises bacterial virulence by manipulating effector delivery. MKP1 is a negative regulator of MPK3/6. However, activation of MPK3/6 alone is not sufficient to yield resistance in plant. RLKs in plants utilize both MAPKs and CDPKs for signal transduction. It was reported that MAPKs and CDPKs synergistically affect a subset of PAMP responsive gene expression. Notably, MKP1 has a unique domain architecture including two proposed calmodulin binding domains. These facts indicate that MKP1 may be involved in calcium signaling as well. To test this hypothesis, I made cpk5,6,11/mkp1 quadruple mutant. Bioassays revealed that the quadruple mutant is able to suppress some, but not all mkp1 phenotypes. These results support the hypothesis that the resistance in mkp1 is achieved by integrating both MAPK and calcium-related signaling. MPK3/6 are activated during various biotic and abiotic stresses. This poses the question of signal specificity, which may occur through specific MPK3/6 substrates. ATPHOS32 is a PAMP-activated MPK6 substrate conserved in many plant species. It is possible that through ATPHOS32, MPK6 is involved in PAMP signaling and/or in PTI. Because increased MPK6 signaling in mkp1 should involve increased phosphorylation of MPK6 substrate, mutants in MPK6 substrates such as PHOS32 may recapitulate the suppression some mkp1 phenotypes by mpk6. One difficulty in genetically investigating MAPKs substrates' functions is that many of them are multi-gene family members. To accelerate the testing process, I designed an artificial microRNA (amiRNA) that specifically silences all three members of PHOS32 family. Preliminary data suggests that the protein levels of all PHOS32 family members are decreased in amiR-phos32 lines. The amiR-phos32 lines were slightly more susceptible to Pto infection than WT. My preliminary data suggests that this amiRNA strategy successfully silenced the family of target protein(s), and implicated that PHOS32 acts as a weak positive regulator in defense responses. This work established the precedence for pursuing an amiRNA strategy to deal with gene function redundancy for both investigating mkp1 phenotypes and, more broadly, PAMP signalling. Taken together, MAP kinase phosphatase 1 (MKP1) mutant mkp1 (Ws) has been used as an useful tool to investigate how intracellular signaling contributes to PTI. My results suggest that the integration of multiple signal pathways such as MAPKs- and calcium-pathways, and the manipulating effector delivery into host cells contribute to resistance. In addition, amiRNA has been demonstrated as a robust tool to knock down expression level of gene family, which has broader implications in studying potential MPK3/6 substrates' functions.
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