Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations

A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores

Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome‐inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti‐insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV‐mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV‐expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize

Perspectives on intracellular perception of plant viruses

The intracellular nucleotide-binding domain leucine-rich repeat (NLR) class of immune receptors plays an important role in plant viral defence. Plant NLRs recognize viruses through direct or indirect association of viral proteins, triggering a downstream defence response to prevent viral proliferation and movement within the plant. This review focuses on current knowledge of intracellular perception of viral pathogens, activation of NLRs and the downstream signalling components involved in plant viral defence

Perspectives on intracellular perception of plant viruses.

The intracellular nucleotide-binding domain leucine-rich repeat (NLR) class of immune receptors plays an important role in plant viral defence. Plant NLRs recognize viruses through direct or indirect association of viral proteins, triggering a downstream defence response to prevent viral proliferation and movement within the plant. This review focuses on current knowledge of intracellular perception of viral pathogens, activation of NLRs and the downstream signalling components involved in plant viral defence

Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays

<p>Abstract</p> <p>Background</p> <p>Mismatched oligonucleotides are widely used on microarrays to differentiate specific from nonspecific hybridization. While many experiments rely on such oligos, the hybridization behavior of various degrees of mismatch (MM) structure has not been extensively studied. Here, we present the results of two large-scale microarray experiments on <it>S. cerevisiae </it>and <it>H. sapiens </it>genomic DNA, to explore MM oligonucleotide behavior with real sample mixtures under tiling-array conditions.</p> <p>Results</p> <p>We examined all possible nucleotide substitutions at the central position of 36-nucleotide probes, and found that nonspecific binding by MM oligos depends upon the individual nucleotide substitutions they incorporate: C→A, C→G and T→A (yielding purine-purine mispairs) are most disruptive, whereas A→X were least disruptive. We also quantify a marked GC skew effect: substitutions raising probe GC content exhibit higher intensity (and vice versa). This skew is small in highly-expressed regions (± 0.5% of total intensity range) and large (± 2% or more) elsewhere. Multiple mismatches per oligo are largely additive in effect: each MM added in a distributed fashion causes an additional 21% intensity drop relative to PM, three-fold more disruptive than adding adjacent mispairs (7% drop per MM).</p> <p>Conclusion</p> <p>We investigate several parameters for oligonucleotide design, including the effects of each central nucleotide substitution on array signal intensity and of multiple MM per oligo. To avoid GC skew, individual substitutions should not alter probe GC content. RNA sample mixture complexity may increase the amount of nonspecific hybridization, magnify GC skew and boost the intensity of MM oligos at all levels.</p

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 successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16-24 h), and higher incubation temperature (37 °C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10 min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species

Proximity labeling: an emerging tool for probing in&nbsp;planta molecular interactions.

Protein-protein interaction&nbsp;(PPI) networks are key to nearly all aspects of cellular activity. Therefore, the identification of PPIs is important for understanding a specific biological process in an organism. Compared with conventional methods for probing PPIs, the recently described proximity labeling (PL) approach combined with mass spectrometry (MS)-based quantitative proteomics has emerged as a powerful approach for characterizing PPIs. However, the application of PL in planta remains in its infancy. Here, we summarize recent progress in PL and its potential utilization in plant biology. We specifically summarize advances in PL, including the development and comparison of different PL enzymes&nbsp;and the application of PL for deciphering various molecular interactions in different organisms with an emphasis on plant systems

Proximity labeling: an emerging tool for probing in&nbsp;planta molecular interactions.

Protein-protein interaction&nbsp;(PPI) networks are key to nearly all aspects of cellular activity. Therefore, the identification of PPIs is important for understanding a specific biological process in an organism. Compared with conventional methods for probing PPIs, the recently described proximity labeling (PL) approach combined with mass spectrometry (MS)-based quantitative proteomics has emerged as a powerful approach for characterizing PPIs. However, the application of PL in planta remains in its infancy. Here, we summarize recent progress in PL and its potential utilization in plant biology. We specifically summarize advances in PL, including the development and comparison of different PL enzymes&nbsp;and the application of PL for deciphering various molecular interactions in different organisms with an emphasis on plant systems

The Sw-5b NLR Immune Receptor Induces Early Transcriptional Changes in Response to Thrips and Mechanical Modes of Inoculation of Tomato spotted wilt orthotospovirus

The NLR (nucleotide-binding leucine-rich repeat) class immune receptor Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the TSWV movement protein NSm, we know very little about the downstream events that lead to resistance. Here, we investigated the Sw-5b–mediated early transcriptomic changes that occur in response to mechanical and thrips-mediated inoculation of TSWV, using near-isogenic tomato lines CNPH-LAM 147 (Sw5b+/+) and Santa Clara (Sw-5b−/−). We observed earlier Sw-5b–mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b responses specific to the method of TSWV inoculation. Although TSWV is transmitted in nature primarily by the thrips, Sw-5b responses to thrips inoculation have not been previously studied. Therefore, the DEGs we have identified in response to thrips-mediated inoculation provide a new foundation for understanding the mechanistic roles of these genes in the Sw-5b–mediated resistance. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores

Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome‐inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti‐insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV‐mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV‐expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.This article is published as Chung, Seung Ho, Mahdiyeh Bigham, Ryan R. Lappe, Barry Chan, Ugrappa Nagalakshmi, Steven A. Whitham, Savithramma P. Dinesh‐Kumar, and Georg Jander. "A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores." Plant Biotechnology Journal (2021). doi:10.1111/pbi.13585.</p