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Background The Root-Knot Nematode (RKN), Meloidogyne arenaria , significantly reduces peanut grain quality and yield worldwide. Whilst the cultivated species has low levels of resistance to RKN and other pests and diseases, peanut wild relatives ( Arachis spp.) show rich genetic diversity and harbor high levels of resistance to many pathogens and environmental constraints. Comparative transcriptome analysis can be applied to identify candidate resistance genes. Results Transcriptome analysis during the early stages of RKN infection of two peanut wild relatives, the highly RKN resistant Arachis stenosperma and the moderately susceptible A. duranensis , revealed genes related to plant immunity with contrasting expression profiles. These included genes involved in hormone signaling and secondary metabolites production and also members of the NBS-LRR class of plant disease resistance (R) genes. From 345 NBS-LRRs identified in A.duranensis reference genome, 52 were differentially expressed between inoculated and control samples, with the majority occurring in physical clusters unevenly distributed on eight chromosomes with preferential tandem duplication. The majority of these NBS-LRR genes showed contrasting expression behaviour between A. duranensis and A. stenosperma , particularly at 6 days after nematode inoculation, coinciding with the onset of the Hypersensitive Response in the resistant species. The physical clustering of some of these NBS-LRR genes correlated with their expression patterns in the contrasting genotypes. Four NBS-LRR genes exclusively expressed in A. stenosperma are located within clusters on chromosome Aradu. A09, which harbors a QTL for RKN resistance, suggesting a functional role for their physical arrangement and their potential involvement in this defense response. Conclusion The identification of functional novel R genes in wild Arachis species responsible for triggering effective defense cascades can contribute to the crop genetic improvement and enhance peanut resilience to RKN.

Background Peanut ( Arachis hypogaea ) production is largely affected by a variety of abiotic and biotic stresses, including the root-knot nematode (RKN) Meloidogyne arenaria that causes yield losses worldwide. Transcriptome studies of wild Arachis species, which harbor resistance to a number of pests and diseases, disclosed several candidate genes for M. arenaria resistance. Peanut is recalcitrant to genetic transformation, so the use of Agrobacterium rhizogenes -derived hairy roots emerged as an alternative for in-root functional characterization of these candidate genes. Results The present report describes an ex vitro methodology for hairy root induction in detached leaves based on the well-known ability of peanut to produce roots spontaneously from its petiole, which can be maintained for extended periods under high-humidity conditions. Thirty days after infection with the A. rhizogenes ‘K599’ strain, 90% of the detached leaves developed transgenic hairy roots with 5 cm of length in average, which were then inoculated with M. arenaria . For improved results, plant transformation, and nematode inoculation parameters were adjusted, such as bacterial cell density and growth stage; moist chamber conditions and nematode inoculum concentration. Using this methodology, a candidate gene for nematode resistance, AdEXLB8, was successfully overexpressed in hairy roots of the nematode-susceptible peanut cultivar ‘Runner’, resulting in 98% reduction in the number of galls and egg masses compared to the control, 60 days after M. arenaria infection. Conclusions This methodology proved to be more practical and cost-effective for functional validation of peanut candidate genes than in vitro and composite plant approaches, as it requires less space, reduces analysis costs and displays high transformation efficiency. The reduction in the number of RKN galls and egg masses in peanut hairy roots overexpressing AdEXLB8 corroborated the use of this strategy for functional characterization of root expressing candidate genes. This approach could be applicable not only for peanut–nematode interaction studies but also to other peanut root diseases, such as those caused by fungi and bacteria, being also potentially extended to other crop species displaying similar petiole-rooting competence.

MicroRNAs (miRNAs) are key post-transcriptional regulators of plant development and stress responses, with many being conserved across diverse plant lineages. In this study, we investigated the expression profiles of miRNAs and their corresponding target genes in Arachis stenosperma, a wild peanut relative that exhibits robust resistance to root-knot nematodes (RKN). Small RNA sequencing of nematode-infected roots identified 107 miRNA loci, of which 93 corresponded to conserved miRNA families and 14 represented novel candidates, designated as miRNOVO. Among these, 18 miRNAs belonging to 11 conserved families were identified as differentially expressed (DEMs). Notably, miR399 and miR319 showed the highest upregulation (logFC = 4.25 and 4.20), while miR393 and miR477 were the most downregulated (logFC = −0.83 and −0.79). Integrated analysis of miRNA and transcriptome data revealed several regulatory interactions involving key defense-related genes. These included NLR genes targeted by miR393 and miR477, hormone signaling components such as the auxin response factor ARF8 targeted by miR167, and the growth regulator GRF2 targeted by miR396. Additionally, miR408 was predicted to target laccase3, a gene involved in the oxidation of phenolic compounds, lignin biosynthesis, copper homeostasis and defense responses. Remarkably, four immune receptor genes belonging to the nucleotide-binding site leucine-rich repeat (NLR) family displayed inverse expression patterns relative to their regulatory miRNAs, suggesting miRNA-mediated post-transcriptional control during the early stages of nematode infection. These findings reveal both conserved and species-specific miRNA–mRNA modules associated with nematode resistance in A. stenosperma, highlighting promising targets for developing RKN-tolerant peanut cultivars through miRNA-based strategies.

Stress priming is an important strategy for enhancing plant defense capacity to deal with environmental challenges and involves reprogrammed transcriptional responses. Although ultraviolet (UV) light exposure is a widely adopted approach to elicit stress memory and tolerance in plants, the molecular mechanisms underlying UV-mediated plant priming tolerance are not fully understood. Here, we investigated the changes in the global transcriptome profile of wild Arachis stenosperma leaves in response to UV-C exposure. A total of 5751 differentially expressed genes (DEGs) were identified, with the majority associated with cell signaling, protein dynamics, hormonal and transcriptional regulation, and secondary metabolic pathways. The expression profiles of DEGs known as indicators of priming state, such as transcription factors, transcriptional regulators and protein kinases, were further characterized. A meta-analysis, followed by qRT-PCR validation, identified 18 metaDEGs as being commonly regulated in response to UV and other primary stresses. These genes are involved in secondary metabolism, basal immunity, cell wall structure and integrity, and may constitute important players in the general defense processes and establishment of a priming state in A. stenosperma. Our findings contribute to a better understanding of transcriptional dynamics involved in wild Arachis adaptation to stressful conditions of their natural habitats.

Agrobacterium rhizogenes -mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean ( Glycine max ). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes ( Meloidogyne incognita and M . javanica ). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M . incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene ( AdEXPA24 ) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene ( GmPG ). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean.

Nematodes and drought are major constraints in tropical agriculture and often occur simultaneously. Plant responses to these stresses are complex and require crosstalk between biotic and abiotic signaling pathways. In this study, we explored the transcriptome data of wild Arachis species subjected to drought (A-metaDEG) and the root-knot nematode Meloidogyne arenaria (B-metaDEG) via meta-analysis, to identify core-stress responsive genes to each individual and concurrent stresses in these species. Transcriptome analysis of a nematode/drought bioassay (cross-stress) showed that the set of stress responsive DEGs to concurrent stress is distinct from those resulting from overlapping A- and B-metaDEGs, indicating a specialized and unique response to combined stresses in wild Arachis . Whilst individual biotic and abiotic stresses elicit hormone-responsive genes, most notably in the jasmonic and abscisic acid pathways, combined stresses seem to trigger mainly the ethylene hormone pathway. The overexpression of a cross-stress tolerance candidate gene identified here, an endochitinase-encoding gene ( AsECHI ) from Arachis stenosperma , reduced up to 30% of M. incognita infection and increased post-drought recovery in Arabidopsis plants submitted to both stresses. The elucidation of the network of cross-stress responsive genes in Arachis contributes to better understanding the complex regulation of biotic and abiotic responses in plants facilitating more adequate crop breeding for combined stress tolerance.

Expansins are plant cell wall-loosening proteins involved in adaptive responses to environmental stimuli and various developmental processes. The first genome-wide analysis of the expansin superfamily in the Arachis genus identified 40 members in A. duranensis and 44 in A. ipaënsis , the wild progenitors of cultivated peanut ( A. hypogaea ). These expansins were further characterized regarding their subfamily classification, distribution along the genomes, duplication events, molecular structure, and phylogeny. A RNA-seq expression analysis in different Arachis species showed that the majority of these expansins are modulated in response to diverse stresses such as water deficit, root-knot nematode (RKN) infection, and UV exposure, with an expansin-like B gene ( AraEXLB8 ) displaying a highly distinct stress-responsive expression profile. Further analysis of the AraEXLB8 coding sequences showed high conservation across the Arachis genotypes, with eight haplotypes identified. The modulation of AraEXLB8 expression in response to the aforementioned stresses was confirmed by qRT-PCR analysis in distinct Arachis genotypes, whilst in situ hybridization revealed transcripts in different root tissues according to the stress imposed. The overexpression of AraEXLB8 in soybean ( Glycine max ) composite plants remarkably decreased the number of galls in transformed hairy roots inoculated with RKN. This study improves the current understanding of the molecular evolution, divergence, and gene expression of expansins in Arachis , and provides molecular and functional insights into the role of expansin-like B, the less-studied plant expansin subfamily.

Fusarium oxysporum causes devastating vascular wilt diseases in numerous crop species, resulting in substantial yield losses. The Arabidopsis thaliana - F. oxysporum f.sp. conglutinans (FOC) model system enables the identification of meaningful genotype–phenotype correlations and was applied in this study to evaluate the effects of overexpressing an NLR gene ( AsTIR19 ) from Arachis stenosperma against pathogen infection. AsTIR19 overexpression (OE) lines exhibited enhanced resistance to FOC without any discernible phenotype penalties. To elucidate the underlying resistance mechanisms mediated by AsTIR19 overexpression, we conducted whole transcriptome sequencing of an AsTIR19-OE line and non-transgenic wild-type (WT) plants inoculated and non-inoculated with FOC using Illumina HiSeq4000. Comparative analysis revealed 778 differentially expressed genes (DEGs) attributed to transgene overexpression, while fungal inoculation induced 434 DEGs in the OE line, with many falling into defense-related Gene Ontology (GO) categories. GO and KEGG enrichment analysis showed that DEGs were enriched in the phenylpropanoid and flavonoid pathways in the OE plants. This comprehensive transcriptomic analysis underscores how AsTIR19 overexpression reprograms transcriptional networks, modulating the expression of stress-responsive genes across diverse metabolic pathways. These findings provide valuable insights into the molecular mechanisms underlying the role of this NLR gene under stress conditions, highlighting its potential to enhance resistance to Fusarium oxysporum .

Root-knot nematodes (RKNs, genus Meloidogyne) affect a large number of crops causing severe yield losses worldwide, more specifically in tropical and sub-tropical regions. Several plant species display high resistance levels to Meloidogyne, but a general view of the plant immune molecular responses underlying resistance to RKNs is still lacking. Combining comparative genomics with differential gene expression analysis may allow the identification of widely conserved plant genes involved in RKN resistance. To identify genes that are evolutionary conserved across plant species, we used OrthoFinder to compared the predicted proteome of 22 plant species, including important crops, spanning 214 Myr of plant evolution. Overall, we identified 35,238 protein orthogroups, of which 6,132 were evolutionarily conserved and universal to all the 22 plant species (PLAnts Common Orthogroups—PLACO). To identify host genes responsive to RKN infection, we analyzed the RNA-seq transcriptome data from RKN-resistant genotypes of a peanut wild relative (Arachis stenosperma), coffee (Coffea arabica L.), soybean (Glycine max L.), and African rice (Oryza glaberrima Steud.) challenged by Meloidogyne spp. using EdgeR and DESeq tools, and we found 2,597 (O. glaberrima), 743 (C. arabica), 665 (A. stenosperma), and 653 (G. max) differentially expressed genes (DEGs) during the resistance response to the nematode. DEGs’ classification into the previously characterized 35,238 protein orthogroups allowed identifying 17 orthogroups containing at least one DEG of each resistant Arachis, coffee, soybean, and rice genotype analyzed. Orthogroups contain 364 DEGs related to signaling, secondary metabolite production, cell wall-related functions, peptide transport, transcription regulation, and plant defense, thus revealing evolutionarily conserved RKN-responsive genes. Interestingly, the 17 DEGs-containing orthogroups (belonging to the PLACO) were also universal to the 22 plant species studied, suggesting that these core genes may be involved in ancestrally conserved immune responses triggered by RKN infection. The comparative genomic approach that we used here represents a promising predictive tool for the identification of other core plant defense-related genes of broad interest that are involved in different plant–pathogen interactions.

Genus Arachis comprises 82 species distributed into nine taxonomic sections. Most Arachis species are wild and those from Arachis section have been evaluated for many traits, since they can be used in peanut breeding. Most of the remaining species have been neglected and understudied. Recently, resveratrol content and expression of a resveratrol synthase gene were analyzed in wild Arachis species. Our aim was to expand the knowledge about resveratrol in Arachis , analyzing species from five sections and evaluating the expression of a resveratrol synthase (RS) gene responsive to ultraviolet light (UV) along the time. In a first experiment, the resveratrol content after UV induction was analyzed on detached leaves of 12 species from five sections. Variation was observed among species and accessions of the same species. The highest contents were found in A. lignosa (843.9 μg/g) and A. triseminata (745.4 μg/g) . In a second experiment, RS expression and resveratrol content in four species and one synthetic amphidiploid were analyzed at 0, 7, 15 and 24 h pos induction (hpi) with UV. In most genotypes, the highest RS expression level was at 0 hpi, whereas the highest resveratrol content was at 15 hpi. Our results suggested that resveratrol is ubiquitously present in the genus Arachis with different capacities of synthesis among species and accessions in response to ultraviolet treatment. Presence of resveratrol in wild Arachis species adds new value to these genetic resources.