Engineering Resistance against Sclerotinia sclerotiorum Using a Truncated NLR (TNx) and a Defense-Priming Gene.

The association of both cell-surface PRRs (Pattern Recognition Receptors) and intracellular receptor NLRs (Nucleotide-Binding Leucine-Rich Repeat) in engineered plants have the potential to activate strong defenses against a broad range of pathogens. Here, we describe the identification, characterization, and in planta functional analysis of a novel truncated NLR (TNx) gene from the wild species Arachis stenosperma (AsTIR19), with a protein structure lacking the C-terminal LRR (Leucine Rich Repeat) domain involved in pathogen perception. Overexpression of AsTIR19 in tobacco plants led to a significant reduction in infection caused by Sclerotinia sclerotiorum, with a further reduction in pyramid lines containing an expansin-like B gene (AdEXLB8) potentially involved in defense priming. Transcription analysis of tobacco transgenic lines revealed induction of hormone defense pathways (SA; JA-ET) and PRs (Pathogenesis-Related proteins) production. The strong upregulation of the respiratory burst oxidase homolog D (RbohD) gene in the pyramid lines suggests its central role in mediating immune responses in plants co-expressing the two transgenes, with reactive oxygen species (ROS) production enhanced by AdEXLB8 cues leading to stronger defense response. Here, we demonstrate that the association of potential priming elicitors and truncated NLRs can produce a synergistic effect on fungal resistance, constituting a promising strategy for improved, non-specific resistance to plant pathogens.

Defining the combined stress response in wild Arachis.

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.

Brazilian Kayabi Indian accessions of peanut, Arachis hypogaea (Fabales, Fabaceae): origin, diversity and evolution.

Peanut is a crop of the Kayabi tribe, inhabiting the Xingu Indigenous Park, Brazil. Morphological analysis of Xingu accessions showed variation exceeding that described for cultivated peanuts. This raised questions as to the origin of the Xingu accessions: are they derived from different species, or is their diversity a result of different evolutionary and selection processes? To answer these questions, cytogenetic and genotyping analyses were conducted. The karyotypes of Xingu accessions analyzed are very similar to each other, to an A. hypogaea subsp. fastigiata accession and to the wild allotetraploid A. monticola. The accessions share the number and general morphology of the chromosomes; DAPI+ bands; 5S and 45S rDNA loci distribution and a high genomic affinity with A. duranensis and A. ipaënsis genomic probes. However, the number of CMA3+ bands differs from those determined for A. hypogaea and A. monticola, which are also different from each other. SNP genotyping grouped all Arachis allotetraploids into four taxonomic groups: Xingu accessions were closer to A. monticola and A. hypogaea subsp. hypogaea. Our data suggests that the morphological diversity within these accessions is not associated with a different origin and can be attributed to morphological plasticity and different selection by the Indian tribes.

Contrasting Effects of Wild Arachis Dehydrin Under Abiotic and Biotic Stresses.

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 (YnSKn, SKn, and Kn) subclasses sharing similar gene organization and protein structure. Two additional low conserved DHN Φ-segments specific to the legume SKn-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 SKn-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.

Comparative root transcriptome of wild Arachis reveals NBS-LRR genes related to nematode resistance.

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.

Early responses to dehydration in contrasting wild Arachis species.

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.

Comparative transcriptomic analysis indicates genes associated with local and systemic resistance to Colletotrichum graminicola in maize.

The hemibiotrophic fungus Colletotrichum graminicola may cause severe damage to maize, affecting normal development of the plant and decreasing grain yield. In this context, understanding plant defense pathways at the inoculation site and systemically in uninoculated tissues can help in the development of genetic engineering of resistance against this pathogen. Previous work has discussed the molecular basis of maize - C. graminicola interaction. However, many genes involved in defense have not yet been exploited for lack of annotation in public databases. Here, changes in global gene expression were studied in root, male and female inflorescences of maize under local and systemic fungal infection treatments, respectively. RNA-Seq with qPCR was used to indicate genes involved in plant defense. We found that systemic acquired resistance induction in female inflorescences mainly involves accumulation of salicylic acid (SA)-inducible defense genes (ZmNAC, ZmHSF, ZmWRKY, ZmbZIP and PR1) and potential genes involved in chromatin modification. Furthermore, transcripts involved in jasmonic acid (JA) and ethylene (ET) signaling pathways were also accumulated and may participate in plant immunity. Moreover, several genes were functionally re-annotated based on domain signature, indicating novel candidates to be tested in strategies involving gene knockout and overexpression in plants.

Ex vitro hairy root induction in detached peanut leaves for plant-nematode interaction studies.

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.

Genome-wide analysis of expansin superfamily in wild Arachis discloses a stress-responsive expansin-like B gene.

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.

Clavanin bacterial sepsis control using a novel methacrylate nanocarrier.

Controlling human pathogenic bacteria is a worldwide problem due to increasing bacterial resistance. This has prompted a number of studies investigating peptides isolated from marine animals as a possible alternative for control of human pathogen infections. Clavanins are antimicrobial peptides isolated from the marine tunicate Styela clava, showing 23 amino acid residues in length, cationic properties, and also high bactericidal activity. In spite of clear benefits from the use of peptides, currently 95% of peptide properties have limited pharmaceutical applicability, such as low solubility and short half-life in the circulatory system. Here, nanobiotechnology was used to encapsulate clavanin A in order to develop nanoantibiotics against bacterial sepsis. Clavanin was nanostructured using EUDRAGIT(®) L 100-55 and RS 30 D solution (3:1 w:w). Atomic force, scanning electron microscopy and dynamic light scattering showed nanoparticles ranging from 120 to 372 nm in diameter, with a zeta potential of -7.16 mV and a polydispersity index of 0.123. Encapsulation rate of 98% was assessed by reversed-phase chromatography. In vitro bioassays showed that the nanostructured clavanin was partially able to control development of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, nanostructures did not show hemolytic activity. In vivo sepsis bioassays were performed using C57BL6 mice strain inoculated with a polymicrobial suspension. Assays led to 100% survival rate under sub-lethal sepsis assays and 40% under lethal sepsis assays in the presence of nanoformulated clavanin A until the seventh day of the experiment. Data here reported indicated that nanostructured clavanin A form shows improved antimicrobial activity and has the potential to be used to treat polymicrobial infections.