Integrated product and process development methodologies for environmentally conscious electronic products

This research focuses on integrated product and process development (IPPD) methodologies for environmentally conscious electronic products. After a review of current research issues in the field of product and process development, a generic framework for IPPD is proposed which describes most of the concerned issues formally as constrained optimization problems. These problems may include such optimization objectives as cost, benefit, and environmental impact. Based on this framework, an IPPD methodology is proposed as a systems approach to competitive and environmentally conscious product and process development. A case study on personal computer development is performed illustrating how to apply the methodology meaningfully and efficiently. Eco-compass concept is then integrated into the methodology to evaluate environmental impact, and a case study on business telephone development is performed. To automate the design of products and processes, a solution methodology for IPPD based on logical representation of process relations is proposed with two illustrating product development examples. Finally, a timed IPPD methodology is introduced with increased modeling capability and decision accuracy. It considers the execution duration of processes and their time-varying characteristics. The timed methodology is applied to the life cycle development of flexible manufacturing systems (FMSs) and provides a new way to develop cost-effective, high-quality, and environmentally conscious FMSs

Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R–RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector’s authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY–domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY–domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a “reversibly closed” conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants

MOS11: A New Component in the mRNA Export Pathway

Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS)–mediated nuclear protein import, Nuclear Export Signal (NES)–dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107–160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1, 11 (mos11). Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly(A) mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol

Protein-protein interactions in the RPS4/RRS1 immune receptor complex

Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants..D., S.U.H., S.W.,H.G., and L.Hu were supported on European Research Council (ERC) Grant“Immunity by pair design”Project ID 669926 (to J.D.G.J.). Y.M. was supported onBiotechnology and Biological Sciences Research Council (BBSRC) Grant BB/M008193/1 (to J.D.G.J.). S.U.H. was supported on Next-Generation BioGreen 21 Program (Project No. PJ01365301), Rural Development Administration, Republic of Korea. J.C. was supported by a Chinese Scholarship Council Postgraduate Fellowship. P.D. was supported by the European Union’s Horizon 2020 Research and Innovation Program under Marie Skłodowska-Curie Individual Fellowship (Project ID 656243) and a Future Leader Fellowship from BBSRC (Grant Agreement BB/R012172/1). P.N.M. was supported by a Marie Skłodowska-Curie Action Individual Fellowship (Project ID 656011

RRS1 promotes association of RPS4 and EDS1 in the nucleus.

<p>(A) In the presence of RRS1, the RPS4/EDS1 are predominantly localized to the nucleus. BiFC assays with the co-expression of <i>nVenus-RPS4</i>/<i>cCFP-EDS1</i>/<i>GUS-HF/mCherry</i> reveal reconstruction of YFP signal in the cytoplasmic aggregations and in the nucleus (arrows). In the presence of RRS1-HF, nVenus-RPS4/cCFP-EDS1 association revealed a YFP signal in the nucleus. Scale bar = 10 μm. (B) EDS1 associates with RPS4/RRS1. Upon transient co-delivery of <i>RPS4-HA</i> and <i>RRS1-HF</i> with <i>GFP-EDS1</i> or <i>GFP</i> in <i>N</i>. <i>benthamiana</i> leaves, samples were harvested at 2 dpi and total extracts were immunoprecipitated with anti-GFP beads. Specific protein-protein interactions were detected by immunoblotting with the indicated antibodies. All the experiments were repeated at least three times with similar results.</p

RPS4 homodimerization is dependent on RRS1.

<p>(A) BiFC assays using nVenus- and cCFP-tagged RPS4 reveal that RPS4 self-association in the nucleus is RRS1-dependent. The <i>nVenus-RPS4</i>, <i>cCFP-RPS4</i>, and <i>mCherry</i> were transiently co-expressed in the presence of <i>RRS1-HF</i> or <i>GUS-HF</i> in <i>N</i>. <i>benthamiana</i> leaves. At 2 dpi, the reconstruction YFP signal is observed with confocal microscope (Leica SP5). <i>mCherry</i> was used as a nuclear and cytoplasmic marker. Scale bar = 10 μm. (B) Co-immunoprecipitation (co-IP) assays reveal that RPS4 self-associates only in the presence of RRS1. <i>Agrobacterium</i>-mediated transient co-expression of <i>RRS1-GFP</i>/<i>RPS4-HF</i>/<i>RPS4-HA</i> or <i>GFP</i>/ <i>RPS4-HF</i>/<i>RPS4-HA</i> was performed in <i>N</i>. <i>benthamiana</i> leaves. Anti-FLAG co-IPs were performed with total protein extracts and probed with anti-GFP, -FLAG, and -HA antibodies. (C) Co-IPs show that RRS1 self-associates and forms a heteromeric complex with RPS4. Transient co-expression assays of <i>RRS1-GFP</i>/<i>RRS1-HF</i>, <i>RRS1-GFP</i>/<i>RPS4-HF</i> or <i>GFP</i>/<i>RRS1-HF</i> were performed in <i>N</i>. <i>benthamiana</i> leaves. Immunoblots show the presence of proteins in total extracts (input) and after immunoprecipitation with anti-GFP beads (IP-GFP). All the experiments were repeated at least three times with similar results.</p