Biology subjects

Dinesh-Kumar, S. P.

Publications and source records attributed to Dinesh-Kumar, S. P..

5 recordsLinked to original sources

bioRxiv2025

High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB

Genome editing has revolutionized plant biology research. However, efficient and straightforward delivery of editing reagents remains a major challenge. Viral delivery systems can address these issues, but CRISPR-Cas nucleases are often too large for viral vectors. Recently, smaller editors like TnpBs have been identified, but wild-type TnpBs are significantly less active than commonly used Cas9 nucleases. Here, we optimized a tobacco rattle virus (TRV)-based system to deliver newly discovered, highly active engineered ISDra2 TnpB variants. Our results demonstrate that the eTnpBc variant delivered via TRV enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci, significantly higher than that of wild-type ISDra2 TnpB. Additionally, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here are expected to accelerate broader adoption of eTnpBc for transformation- and transgene-free genome editing in plants.

plant biology↗

bioRxiv2024

Directed evolution of a plant immune receptor for broad spectrum recognition of pathogen effectors

Rapid development of immune receptors that protect crops from emerging pathogens is a critical challenge 1,2. While novel immune receptors that recognize previously undetected pathogen effectors could provide protection against a wider range of pathogens, engineering such receptors has been constrained by the low throughput and speed of in planta testing. We established yeast surface display as a high throughput platform to recapitulate plant immune receptor-ligand interactions and evolve new binding capabilities. Using this directed evolution platform, we engineered the ligand binding domain of the rice immune receptor Pik-1 to recognize diverse effectors from the fast-evolving fungal pathogen Magnaporthe oryzae. Our approach yielded Pik-1 ligand binding domains with affinity for variants of the M. oryzae effector Avr-Pik that previously escaped detection by known rice alleles of Pik-1, with in planta assays confirming functional recognition of these effectors. Additional rounds of mutagenesis and selection led to a Pik-1 domain that binds all tested Avr-Pik variants as well as the evolutionarily divergent effector AvrPiz-t. These results demonstrate the potential of directed evolution to engineer immune receptors with new-to-nature recognition of a wide range of pathogen-derived ligands and accelerate development of broad spectrum resistance in crops.

plant biology↗

bioRxiv2022

The Sw-5b NLR immune receptor induces earlier transcriptional changes in response to thrips-mediated inoculation of Tomato spotted wilt orthotospovirus compared to mechanical inoculation

The nucleotide-binding leucine-rich repeat (NLR) class of immune receptor, Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the NSm of TSWV, we know very little about the downstream events that lead to resistance. Here, we investigated the early transcriptomic changes that occur in response to both mechanical and thrips-mediated inoculation of TSWV using near-isogenic resistant and susceptible tomato lines. Interestingly, the Sw-5b induces earlier transcriptional changes in response to thrips-mediated inoculation compared to mechanical inoculation of TSWV. A subset of the differentially expressed genes (DEGs) observed at 12 and 24 hours post thrips-mediated inoculation of TSWV was only present at 72 hours post mechanical inoculation. Although some DEGs were shared between thrips and mechanical inoculation at 72 hours postinfection, 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, and transcription factors such as the ethylene response factor 1 and the calmodulin-binding protein 60 were induced. Whereas, in response to mechanical inoculation, fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, and transcription factors such as ERF017 and WRKY75 were differentially expressed. Our findings reveal novel insights into Sw-5b responses specific to the method of TSWV inoculation. Given that TSWV is transmitted in nature primarily by the thrips, the DEGs we have identified provide a foundation for understanding the mechanistic roles of these genes in the Sw-5b-mediated resistance.

plant biology↗

bioRxiv2022

High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus

Delivery of gene editing components such as the Cas nuclease and single guide RNAs (sgRNAs) into plant cells is commonly accomplished by agrobacterium-mediated transformation. Although Arabidopsis is easy to transform, generation of biallelic edited plants requires screening a large number of plants in subsequent generations. Here, we describe optimization of the Tobacco rattle virus (TRV) for in planta delivery of sgRNAs fused to a tRNAIleu that induces efficient multiplex somatic and biallelic heritable editing in Arabidopsis. Inclusion of tRNAIleu enhances the systemic movement of TRV and the mutant phenotype is visible in the initial TRV::sgRNA-tRNAIleu infected Arabidopsis, which allows for the uncovering of lethal phenotypes. Mutant progeny are recovered in the next generation (M1) at frequencies ranging from 30-60%, with 100% mutant recovery in the following (M2) generation. TRV::tRNAIleu system described here allows generation of biallelic edited plants in a single generation and is amenable for large-scale high throughput CRISPR screens.

plant biology↗

bioRxiv2021

Rapid screening of pest resistance genes in maize using a sugarcane mosaic virus vector

Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production world-wide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although several additional transgenes have confirmed insecticidal activity in other plants, 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 transgenes for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defense signaling 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) spider and scorpion toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth on maize was reduced by transient SCMV-mediated overexpression of genes encoding these proteins. Additionally, experiments with some 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 can be exploited as a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.

plant biology↗