An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that ...promote cell-to-cell mobility. Mutant progeny are recovered in the next generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci.
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.
The identification of untranslated regions, introns, and coding regions within an organism remains challenging. We developed a quantitative sequencing-based method called RNA-Seq for mapping ...transcribed regions, in which complementary DNA fragments are subjected to high-throughput sequencing and mapped to the genome. We applied RNA-Seq to generate a high-resolution transcriptome map of the yeast genome and demonstrated that most (74.5%) of the nonrepetitive sequence of the yeast genome is transcribed. We confirmed many known and predicted introns and demonstrated that others are not actively used. Alternative initiation codons and upstream open reading frames also were identified for many yeast genes. We also found unexpected 3'-end heterogeneity and the presence of many overlapping genes. These results indicate that the yeast transcriptome is more complex than previously appreciated.
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.
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.
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•The immune receptor EFR phosphorylates BIK1 at S89/T90 in a unique extended loop•BIK1 S89/T90 are required for EFR-mediated defense responses•Defense phytohormones JA and SA are elevated in BIK1 phosphomimetic mutant plants•BIK1 localizes to the nucleus and targets WRKY transcription factors to regulate JA/SA
Receptor-like cytoplasmic kinases (RLCKs) function at the plasma membrane during plant pattern recognition receptor (PRR)-mediated immunity. Lal et al. show that the RLCK BIK1 is uniquely phosphorylated by the PRR EFR and localizes to the nucleus where it interacts with WRKY transcription factors to regulate defense hormones during plant immunity.
Abstract Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. ...Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.
A recently developed technique called RNA Sequencing (RNA-Seq) uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution than is available with Sanger ...sequencing- and microarray-based methods. In the RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA of interest are directly sequenced using next-generation sequencing technologies. The reads obtained from this can then be aligned to a reference genome in order to construct a whole-genome transcriptome map. RNA-Seq has been used successfully to precisely quantify transcript levels, confirm or revise previously annotated 5' and 3' ends of genes, and map exon/intron boundaries. This unit describes protocols for performing RNA-Seq using the Illumina sequencing platform.
Summary
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.
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