In silico structural-functional characterization of three differentially expressed resistance gene analogs identified in Dalbergia sissoo against dieback disease reveals their role in immune response regulation.

Plant immunity includes enemy recognition, signal transduction, and defensive response against pathogens. We experimented to identify the genes that contribute resistance against dieback disease to Dalbergia sissoo, an economically important timber tree. In this study, we investigated the role of three differentially expressed genes identified in the dieback-induced transcriptome in Dalbergia sissoo. The transcriptome was probed using DOP-rtPCR analysis. The identified RGAs were characterized in silico as the contributors of disease resistance that switch on under dieback stress. Their predicted fingerprints revealed involvement in stress response. Ds-DbRCaG-02-Rga.a, Ds-DbRCaG-04-Rga.b, and Ds-DbRCaG-06-Rga.c showed structural homology with the Transthyretin-52 domain, EAL associated YkuI_C domain, and Src homology-3 domain respectively, which are the attributes of signaling proteins possessing a role in regulating immune responses in plants. Based on in-silico structural and functional characterization, they were predicted to have a role in immune response regulation in D. sissoo.

Mutation introduced in DDTFR10/A gene of ethylene response element-binding protein (EREBP) family through CRISPR/Cas9 genome editing confers increased Fusarium wilt tolerance in tomato.

We investigated the role of the DDTFR10/A gene of the ethylene response element-binding protein (EREBP) family through the CRISPR/Cas9 genome editing approach. The associated role of this gene in tomato fruit ripening was known. The involvement of ripening-regulatory proteins in plant defense has been documented; therefore, to find the involvement of the DDTFR10/A gene in host susceptibility, we introduced the mutation in DDTFR10/A gene through CRISPR/cas9 in the genome of the tomato plant. The 50% biallelic and 50% homozygous mutations were observed in the T0 generation. The CRISPR/Cas9 edited plants showed 40% reduced symptoms of Fusarium wilt compared to control plants (non-edited). The DDTFR10/A gene expression in tomato plants was evaluated against biotic (Fusarium wilt) and abiotic (salinity) stresses, and the upregulated expression of this gene was found under both challenges. However, a comparative increase in DDTFR10/A gene expression was observed in tomato plants upon inoculation with Fusarium oxysporum f. sp. lycopersici. The phenotypic assay performed on edited tomato plants demonstrated the role of the DDTFR10/A gene in contributing toward susceptibility against Fusarium wilt. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01273-6.

Identification of resistance gene analogs of the NBS-LRR family through transcriptome probing and in silico prediction of the expressome of Dalbergia sissoo under dieback disease stress.

Dalbergia sissoo is an important timber tree, and dieback disease poses a dire threat to it toward extinction. The genomic record of D. sissoo is not available yet on any database; that is why it is challenging to probe the genetic elements involved in stress resistance. Hence, we attempted to unlock the genetics involved in dieback resistance through probing the NBS-LRR family, linked with mostly disease resistance in plants. We analyzed the transcriptome of D. sissoo under dieback challenge through DOP-rtPCR analysis using degenerate primers from conserved regions of NBS domain-encoded gene sequences. The differentially expressed gene sequences were sequenced and in silico characterized for predicting the expressome that contributes resistance to D. sissoo against dieback. The molecular and bioinformatic analyses predicted the presence of motifs including ATP/GTP-binding site motif A (P-loop NTPase domain), GLPL domain, casein kinase II phosphorylation site, and N-myristoylation site that are the attributes of proteins encoded by disease resistance genes. The physicochemical characteristics of identified resistance gene analogs, subcellular localization, predicted protein fingerprints, in silico functional annotation, and predicted protein structure proved their role in disease and stress resistance.

Functional inhibition of the StERF3 gene by dual targeting through CRISPR/Cas9 enhances resistance to the late blight disease in Solanum tuberosum L.

BACKGROUND: Disease-resistant cultivars are the best solution to get their maximum yield potential and avoid fungicide application. There is no doubt about the contribution, and use of R genes (resistance genes) in resistance development in plants, while S genes (susceptibility genes) also hold a strong position in pathogenesis by resistance repression, and their loss of function contributes to enhanced resistance. Hence, we attempted to knock out the function of the StERF3 gene in potatoes through CRISPR/Cas9-based genome editing and investigated the CRISPR/Cas9 approach as strategic control against late blight disease in potato plants. METHODS AND RESULTS: The StERF3 gene was edited in late blight susceptible cv. Lady Rosetta. Full allelic edited plants were identified through DnpI, and N1aIV mediated restriction digestion and then further analyzed through Indel Detection by Amplicon Analysis. Sequence analysis of targeted plants for indel identification showed full allelic editing. The detached leaf assay of full allelic edited plants demonstrated the role of the StERF3 gene in susceptibility to late blight in potatoes. In planta disease assay also showed reduced, slowed, and delayed disease progression in StERF3-loss-of-function mutants compared to wild-type (control) plants. Less fungal biomass was quantified in knockouts through Real-time qPCR that supported less susceptibility of edited plants to late blight. Besides, relatively high expression of pathogens-related genes, StPR1, and StNPR1, were also observed in StERF3-loss-of-function mutants compared to the corresponding control. CONCLUSION: The results showed the functional inhibition of StERF3 genes using the CRISPR/Cas9 approach. The functional knockouts (StERF3 gene-edited potato plants) revealed enhanced resistance against Phytophthora infestans, thereby demonstrating the best strategic control for late blight disease in potato plants.