turboid-based-proximity-labeling-for-planta-identification-protein

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

quantification-callose-plant-tissues-enzyme-linked-immunosorbent

Quantification of Callose in Plant Tissues by Enzyme-Linked Immunosorbent Assay/ Immunofluorescence Spectrophotometry

The existing methods of callose quantification from plant tissues include epifluorescence microscopy, fluorescence spectrophotometry, immunofluorescence ...

University of California, Davis

Plant innate immune response against viruses utilizes intracellular Nucleotide Binding domain Leucine Rich Repeat (NLR) class of receptors. NLRs recognize different viral proteins termed elicitors and initiate diverse signaling processes that induce programmed cell death (PCD) in infected cells and restrict virus spread. In this review we describe the recent advances made in the study of plant NLRs that detect viruses. We describe some of the physical and functional interactions these NLRs undertake. We elaborate on the intra-molecular and homotypic association of NLRs that function in self-regulation and activation. Nuclear role for some viral NLRs is discussed as well as the emerging importance of the RNAi pathway in regulating the NLR family.

The conformational and subcellular compartmental dance of plant NLRs during viral recognition and defense signaling.

Inter-organellar communication is vital for successful innate immune responses that confer defense against pathogens. However, little is known about how chloroplasts, which are a major production site of pro-defense molecules, communicate and coordinate with other organelles during defense. Here we show that chloroplasts send out dynamic tubular extensions called stromules during innate immunity or exogenous application of the pro-defense signals, hydrogen peroxide (H2O2) and salicylic acid. Interestingly, numerous stromules surround nuclei during defense response, and these connections correlate with an accumulation of chloroplast-localized NRIP1 defense protein and H2O2 in the nucleus. Furthermore, silencing and knockout of chloroplast unusual positioning 1 (CHUP1) that encodes a chloroplast outer envelope protein constitutively induces stromules in the absence of pathogen infection and enhances programmed cell death. These results support a model in which stromules aid in the amplification and/or transport of pro-defense signals into the nucleus and other subcellular compartments during immunity.

Chloroplast Stromules Function during Innate Immunity.

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFP) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10), the two proteins reconstitute GFP fluorescence. We generated a number of  transgenic lines expressing sfGFP1-10 targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using  effectors AvrB and AvrRps4 in  and transgenic Arabidopsis plants. The versatile split sfGFP system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.

Spatiotemporal Monitoring of  Effectors via Type III Secretion Using Split Fluorescent Protein Fragments.

Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis.

Plants employ cell-surface pattern recognition receptors (PRRs) to detect pathogens. Although phytohormones produced during PRR signaling play an essential role in innate immunity, a direct link between PRR activation and hormone regulation is unknown. EFR is a PRR that recognizes bacterial EF-Tu and activates immune signaling. Here we report that EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1. The BIK1 structure revealed that the EFR-phosphorylated sites reside on a uniquely extended loop away from the BIK1 kinase core domain. Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections. In addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation. These findings demonstrate the mechanistic basis of signal transduction from PRR to phytohormones, mediated through a PRR-BIK1-WRKY axis.

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylation than BioID and BioID2 in plants. TurboID-based PL of N followed by quantitative proteomic analysis and genetic screening revealed multiple regulators of N-mediated immunity. Interestingly, a putative E3 ubiquitin ligase, UBR7, directly interacts with the TIR domain of N. UBR7 downregulation leads to an increased amount of N protein and enhanced TMV resistance. TMV-p50 effector disrupts the N-UBR7 interaction and relieves negative regulation of N. These findings demonstrate the utility of TurboID-based PL in plants and the N-interacting proteins we identified enhance our understanding of the mechanisms underlying NLR regulation.

ArticleTotal : 2	Year
Publication title Cited by 37	2020
Publication title	2026

Article	Year
The conformational and subcellular compartmental dance of plant NLRs during viral recognition and defense signaling. Current opinion in microbiology\| PubMed ID: 24906192	2014
Chloroplast Stromules Function during Innate Immunity. Developmental cell\| PubMed ID: 26120031	2015
Spatiotemporal Monitoring of Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. The Plant cell\| PubMed ID: 28619883	2017
Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis. Scientific reports\| PubMed ID: 28717181	2017
The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity. Cell host & microbe\| PubMed ID: 29649442	2018
TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity. Nature communications\| PubMed ID: 31324801	2019

Savithramma P. Dinesh-Kumar

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