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.

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. 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. 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.

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.

Plant dehydrins (DNHs) belong to the LEA (Late Embryogenesis Abundant) protein family and are involved in responses to multiple abiotic stresses. DHNs are classified into five subclasses according to the organization of three conserved motifs (K-; Y-; and S-segments). In the present study, the DHN protein family was characterized by molecular phylogeny, exon/intron organization, protein structure, and tissue-specificity expression in eight Fabaceae species. We identified 20 DHN genes, encompassing three (Y_nSK_n, SK_n, and K_n) subclasses sharing similar gene organization and protein structure. Two additional low conserved DHN Φ-segments specific to the legume SK_n-type of proteins were also found. The in silico expression patterns of DHN genes in four legume species (Arachis duranensis, A. ipaënsis, Glycine max, and Medicago truncatula) revealed that their tissue-specific regulation is associated with the presence or absence of the Y-segment. Indeed, DHN genes containing a Y-segment are mainly expressed in seeds, whereas those without the Y-segment are ubiquitously expressed. Further qRT-PCR analysis revealed that, amongst stress responsive dehydrins, a SK_n-type DHN gene from A. duranensis (AdDHN1) showed opposite response to biotic and abiotic stress with a positive regulation under water deficit and negative regulation upon nematode infection. Furthermore, transgenic Arabidopsis lines overexpressing (OE) AdDHN1 displayed improved tolerance to multiple abiotic stresses (freezing and drought) but increased susceptibility to the biotrophic root-knot nematode (RKN) Meloidogyne incognita. This contradictory role of AdDHN1 in responses to abiotic and biotic stresses was further investigated by qRT-PCR analysis of transgenic plants using a set of stress-responsive genes involved in the abscisic acid (ABA) and jasmonic acid (JA) signaling pathways and suggested an involvement of DHN overexpression in these stress-signaling pathways.

Using long-read sequencing, we assembled and unzipped the polyploid genomes of Meloidogyne incognita, M. javanica and M. arenaria, three of the most devastating plant-parasitic nematodes. We found the canonical nematode telomeric repeat to be missing in these and other Meloidogyne genomes. In addition, we find no evidence for the enzyme telomerase or for orthologs of C. elegans telomere-associated proteins, suggesting alternative lengthening of telomeres. Instead, analyzing our assembled genomes, we identify species-specific composite repeats enriched mostly at one extremity of contigs. These repeats are G-rich, oriented, and transcribed, similarly to canonical telomeric repeats. We confirm them as telomeric using fluorescent in situ hybridization. These repeats are mostly found at one single end of chromosomes in these species. The discovery of unusual and specific complex telomeric repeats opens a plethora of perspectives and highlights the evolutionary diversity of telomeres despite their central roles in senescence, aging, and chromosome integrity.

The root-knot nematode (RKN), Meloidogyne incognita, is a devastating soybean pathogen worldwide. The use of resistant cultivars is the most effective method to prevent economic losses caused by RKNs. To elucidate the mechanisms involved in resistance to RKN, we determined the proteome and transcriptome profiles from roots of susceptible (BRS133) and highly tolerant (PI 595099) Glycine max genotypes 4, 12, and 30 days after RKN infestation. After in silico analysis, we described major defense molecules and mechanisms considered constitutive responses to nematode infestation, such as mTOR, PI3K-Akt, relaxin, and thermogenesis. The integrated data allowed us to identify protein families and metabolic pathways exclusively regulated in tolerant soybean genotypes. Among them, we highlighted the phenylpropanoid pathway as an early, robust, and systemic defense process capable of controlling M. incognita reproduction. Associated with this metabolic pathway, 29 differentially expressed genes encoding 11 different enzymes were identified, mainly from the flavonoid and derivative pathways. Based on differential expression in transcriptomic and proteomic data, as well as in the expression profile by RT–qPCR, and previous studies, we selected and overexpressed the GmPR10 gene in transgenic tobacco to assess its protective effect against M. incognita. Transgenic plants of the T₂ generation showed up to 58% reduction in the M. incognita reproduction factor. Finally, data suggest that GmPR10 overexpression can be effective against the plant parasitic nematode M. incognita, but its mechanism of action remains unclear. These findings will help develop new engineered soybean genotypes with higher performance in response to RKN infections.