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Background Peanuts are a fundamental legume in human diet, yet they are exposed to simultaneous and interacting biotic and abiotic stresses. Root-knot nematodes (RKN) and reduced water availability constitute a significant threat to this crop. Resistance signatures to each of those stresses have been identified in wild relatives such as Arachis stenosperma and Arachis duranensis . However, plant response to multiple stresses is very complex, requiring the activation of the appropriate signalling pathways to respond to all or by prioritising the response to one stress factor. Despite the experimental data availability of wild Arachis spp. subjected to RKN and/or drought stresses, the global regulome and crosstalk among biotic and abiotic molecular mechanisms have not yet been fully elucidated. Results In this study, we applied HIVE, an integrative analytical framework, to study transcriptional responses in two wild Arachis species subjected to the root-knot nematode Meloidogyne arenaria and/or drought stress across six independent, unpaired experiments. We inferred a global regulome of biotic and abiotic response of two wild Arachis species from HIVE findings. The study of this gene regulatory network allowed the identification of novel regulatory mechanisms, specifically focusing on transcription factors and signaling pathways, potentially involved in M. arenaria and/or drought stresses response. Conclusions Our results demonstrate that HIVE outperformed conventional meta-analysis approaches enabling the identification of novel and promising candidate genes potentially responsible for triggering effective defence responses to multiple stresses.

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.

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.

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.

Wild peanut relatives (Arachis spp.) are genetically diverse and were selected throughout evolution to a range of environments constituting, therefore, an important source of allelic diversity for abiotic stress tolerance. In particular, A. duranensis and A. stenosperma, the parents of the reference Arachis A-genome genetic map, show contrasting transpiration behavior under limited water conditions. This study aimed to build a comprehensive gene expression profile of these two wild species under dehydration stress caused by the withdrawal of hydroponic nutrient solution. For this purpose, roots of both genotypes were collected at seven time-points during the early stages of dehydration and used to construct cDNA paired-end libraries. Physiological analyses indicated initial differences in gas exchange parameters between the drought-tolerant genotype of A. duranensis and the drought-sensitive genotype of A. stenosperma. High-quality Illumina reads were mapped against the A. duranensis reference genome and resulted in the identification of 1,235 and 799 Differentially Expressed Genes (DEGs) that responded to the stress treatment in roots of A. duranensis and A. stenosperma, respectively. Further analysis, including functional annotation and identification of biological pathways represented by these DEGs confirmed the distinct gene expression behavior of the two contrasting Arachis species genotypes under dehydration stress. Some species-exclusive and common DEGs were then selected for qRT-PCR analysis, which corroborated the in silico expression profiling. These included genes coding for regulators and effectors involved in drought tolerance responses, such as activation of osmosensing molecular cascades, control of hormone and osmolyte content, and protection of macromolecules. This dataset of transcripts induced during the dehydration process in two wild Arachis genotypes constitute new tools for the understanding of the distinct gene regulation processes in these closely related species but with contrasting drought responsiveness. In addition, our findings provide insights into the nature of drought tolerance in wild germoplasm, which might be explored as novel sources of diversity and useful wild alleles to develop climate-resilient crop varieties.

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 .

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.

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.

DUF538 proteins belong to a large group of uncharacterized protein families sharing the highly conserved Domain of Unknown Function (DUF). Attention has been given to DUF538 domain-containing proteins due to changes in their gene expression behavior and protein abundance during plant development and responses to stress. Putative roles attributed to DUF538 in plants under abiotic and biotic constraints include involvement in cell redox balance, chlorophyll breakdown and pectin degradation. Our previous transcriptome studies suggested that DUF538 is also involved in the resistance responses of wild Arachis species against the highly hazardous root-knot nematodes (RKNs). To clarify the role of the AsDUF538 gene from the wild peanut relative Arachis stenosperma in this interaction, we analyzed the effect of its overexpression on RKN infection in peanut and soybean hairy roots and Arabidopsis transgenic plants. AsDUF538 overexpression significantly reduced the infection in all three heterologous plant systems against their respective RKN counterparts. The distribution of AsDUF538 transcripts in RKN-infected Arachis roots and the effects of AsDUF538 overexpression on hormonal pathways and redox system in transgenic Arabidopsis were also evaluated. This is the first time that a DUF538 gene is functionally validated in transgenic plants and the earliest report on its role in plant defense against RKNs.

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.