Biocontrol potential of bacteria belonging to the Bacillus subtilis group against pests and diseases of agricultural interest through genome exploration.

The usage of microorganisms as biocontrol agents and biofertilizers has been recommended and recognized as an ecologically correct alternative to maintaining the productivity and safety of crops. Thus, the objectives of this work were to characterize twelve strains belonging to Invertebrate Bacteria Collection of Embrapa Genetic Resources and Biotechnology by molecular, morphological, and biochemical methods and to evaluate the pathogenicity of these strains against pests and diseases of agricultural interest. The morphological characteristic of the strains was performed according to the principles of Bergy's Manual of Systematic Bacteriology. The genomes of the 12 strains were sequenced in Macrogen, Inc. (Seoul, Korea) using the HiSeq2000 and GS-FLX Plus high-performance platforms. In the determination of antibiotic sensibility profiles, disc-diffusion methods (Cefar Diagnótica Ltda) were adopted©. Selective bioassays were carried out with insects of the Lepidoptera (Spodoptera frugiperda, Helicoverpa armigera, and Chrysodeixis includens), Coleoptera (Anthonomus grandis), Diptera (Aedes aegypti) and Hemiptera (Euschistus heros) orders, and with the nematode Caenorhabditis elegans. In addition, the antagonistic action of the phytopathogens Fusarium oxysporum f. sp. vasinfectum and Sclerotinia sclerotiorum against the strains under study, and in vitro assays of phosphate solubilization were also performed. Sequencing of the complete genome of the 12 strains determined that all of them belonged to the Bacillus subtilis sensu lato group. In the strains genome were detected genic clusters responsible for encoding secondary metabolites such as surfactin, iturin, fengycins/plipastatin, bacillomycin, bacillisin, and siderophores. Due to the production of these compounds, there was a survival reduction of the Lepidoptera order insects and a reduction in the phytopathogens mycelial growth. These results show that the species of group B. subtilis s.l. can become promising microbiological alternatives to pest and disease control.

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae.

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

Differentially methylated regions identified in bovine embryos are not observed in adulthood.

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.

Overexpression of the GmEXPA1 gene reduces plant susceptibility to Meloidogyne incognita.

KEY MESSAGE: The overexpression of the soybean GmEXPA1 gene reduces plant susceptibility to M. incognita by the increase of root lignification. Plant expansins are enzymes that act in a pH-dependent manner in the plant cell wall loosening and are associated with improved tolerance or resistance to abiotic or biotic stresses. Plant-parasitic nematodes (PPN) can alter the expression profile of several expansin genes in infected root cells. Studies have shown that overexpression or downregulation of particular expansin genes can reduce plant susceptibility to PPNs. Root-knot nematodes (RKN) are obligate sedentary endoparasites of the genus Meloidogyne spp. of which M. incognita is one of the most reported species. Herein, using a transcriptome dataset and real-time PCR assays were identified an expansin A gene (GmEXPA1; Glyma.02G109100) that is upregulated in the soybean nematode-resistant genotype PI595099 compared to the susceptible cultivar BRS133 during plant parasitism by M. incognita. To understand the role of the GmEXPA1 gene during the interaction between soybean plant and M. incognita were generated stable A. thaliana and N. tabacum transgenic lines. Remarkably, both A. thaliana and N. tabacum transgenic lines overexpressing the GmEXPA1 gene showed reduced susceptibility to M. incognita. Furthermore, plant growth, biomass accumulation, and seed yield were not affected in these transgenic lines. Interestingly, significant upregulation of the NtACC oxidase and NtEFE26 genes, involved in ethylene biosynthesis, and NtCCR and Nt4CL genes, involved in lignin biosynthesis, was observed in roots of the N. tabacum transgenic lines, which also showed higher lignin content. These data suggested a possible link between GmEXPA1 gene expression and increased lignification of the root cell wall. Therefore, these data support that engineering of the GmEXPA1 gene in soybean offers a powerful biotechnology tool to assist in RKN management.

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae.

Banana (Musa spp.), which is one of the world's most popular and most traded fruits, is highly susceptible to pests and diseases. Pseudocercospora musae, responsible for Sigatoka leaf spot disease, is a principal fungal pathogen of Musa spp., resulting in serious economic damage to cultivars in the Cavendish subgroup. The aim of this study was to characterize genetic components of the early immune response to P. musae in Musa acuminata subsp. burmannicoides, var. Calcutta 4, a resistant wild diploid. Leaf RNA samples were extracted from Calcutta 4 three days after inoculation with fungal conidiospores, with paired-end sequencing conducted in inoculated and non-inoculated controls using lllumina HiSeq 4000 technology. Following mapping to the reference M. acuminata ssp. malaccensis var. Pahang genome, differentially expressed genes (DEGs) were identified and expression representation analyzed on the basis of gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes orthology and MapMan pathway analysis. Sequence data mapped to 29,757 gene transcript models in the reference Musa genome. A total of 1073 DEGs were identified in pathogen-inoculated cDNA libraries, in comparison to non-inoculated controls, with 32% overexpressed. GO enrichment analysis revealed common assignment to terms that included chitin binding, chitinase activity, pattern binding, oxidoreductase activity and transcription factor (TF) activity. Allocation to KEGG pathways revealed DEGs associated with environmental information processing, signaling, biosynthesis of secondary metabolites, and metabolism of terpenoids and polyketides. With 144 up-regulated DEGs potentially involved in biotic stress response pathways, including genes involved in cell wall reinforcement, PTI responses, TF regulation, phytohormone signaling and secondary metabolism, data demonstrated diverse early-stage defense responses to P. musae. With increased understanding of the defense responses occurring during the incompatible interaction in resistant Calcutta 4, these data are appropriate for the development of effective disease management approaches based on genetic improvement through introgression of candidate genes in superior cultivars.

The early response of oil palm (Elaeis guineensis Jacq.) plants to water deprivation: Expression analysis of miRNAs and their putative target genes, and similarities with the response to salinity stress.

Oil palm (Elaeis guineensis Jacq.) is a oilseed crop of great economic importance drastically affected by abiotic stresses. MicroRNAs (miRNAs) play crucial roles in transcription and post-transcription regulation of gene expression, being essential molecules in the response of plants to abiotic stress. To better understand the molecular mechanisms behind the response of young oil palm plants to drought stress, this study reports on the prediction and characterization of miRNAs and their putative target genes in the apical leaf of plants subjected to 14 days of water deprivation. Then, the data from this study were compared to the data from a similar study that focused on salinity stress. Both, the drought-and salt-responsive miRNAs and their putative target genes underwent correlation analysis to identify similarities and dissimilarities among them. Among the 81 identified miRNAs, 29 are specific for oil palm, including two (egu-miR28ds and egu-miR29ds) new ones - described for the first time. As for the expression profile, 62 miRNAs were significantly differentially expressed under drought stress, being five up-regulated (miR396e, miR159b, miR529b, egu-miR19sds, and egu-miR29ds) and 57 down-regulated. Transcription factors, such as MYBs, HOXs, and NF-Ys, were predicted as putative miRNA-target genes in oil palm under water deprivation; making them the most predominant group of such genes. Finally, the correlation analysis study revealed a group of putative target genes with similar behavior under salt and drought stresses. Those genes that are upregulated by these two abiotic stresses encode lncRNAs and proteins linked to stress tolerance, stress memory, modulation of ROS signaling, and defense response regulation to abiotic and biotic stresses. In summary, this study provides molecular evidence for the possible involvement of miRNAs in the drought stress response in oil palm. Besides, it shows that, at the molecular level, there are many similarities in the response of young oil palm plants to these two abiotic stresses.

Overexpression of a soybean Globin (GmGlb1-1) gene reduces plant susceptibility to Meloidogyne incognita.

MAIN CONCLUSION: The overexpression of the GmGlb1-1 gene reduces plant susceptibility to Meloidogyne incognita. Non-symbiotic globin class #1 (Glb1) genes are expressed in different plant organs, have a high affinity for oxygen, and are related to nitric oxide (NO) turnover. Previous studies showed that soybean Glb1 genes are upregulated in soybean plants under flooding conditions. Herein, the GmGlb1-1 gene was identified in soybean as being upregulated in the nematode-resistant genotype PI595099 compared to the nematode-susceptible cultivar BRS133 during plant parasitism by Meloidogyne incognita. The Arabidopsis thaliana and Nicotiana tabacum transgenic lines overexpressing the GmGlb1-1 gene showed reduced susceptibility to M. incognita. Consistently, gall morphology data indicated that pJ2 nematodes that infected the transgenic lines showed developmental alterations and delayed parasitism progress. Although no significant changes in biomass and seed yield were detected, the transgenic lines showed an elongated, etiolation-like growth under well-irrigation, and also developed more axillary roots under flooding conditions. In addition, transgenic lines showed upregulation of some important genes involved in plant defense response to oxidative stress. In agreement, higher hydrogen peroxide accumulation and reduced activity of reactive oxygen species (ROS) detoxification enzymes were also observed in these transgenic lines. Thus, based on our data and previous studies, it was hypothesized that constitutive overexpression of the GmGlb1-1 gene can interfere in the dynamics of ROS production and NO scavenging, enhancing the acquired systemic acclimation to biotic and abiotic stresses, and improving the cellular homeostasis. Therefore, these collective data suggest that ectopic or nematode-induced overexpression, or enhanced expression of the GmGlb1-1 gene using CRISPR/dCas9 offers great potential for application in commercial soybean cultivars aiming to reduce plant susceptibility to M. incognita.

Metabarcoding versus mapping unassembled shotgun reads for identification of prey consumed by arthropod epigeal predators.

BACKGROUND: A central challenge of DNA gut content analysis is to identify prey in a highly degraded DNA community. In this study, we evaluated prey detection using metabarcoding and a method of mapping unassembled shotgun reads (Lazaro). RESULTS: In a mock prey community, metabarcoding did not detect any prey, probably owing to primer choice and/or preferential predator DNA amplification, while Lazaro detected prey with accuracy 43-71%. Gut content analysis of field-collected arthropod epigeal predators (3 ants, 1 dermapteran, and 1 carabid) from agricultural habitats in Brazil (27 samples, 46-273 individuals per sample) revealed that 64% of the prey species detections by either method were not confirmed by melting curve analysis and 87% of the true prey were detected in common. We hypothesized that Lazaro would detect fewer true- and false-positive and more false-negative prey with greater taxonomic resolution than metabarcoding but found that the methods were similar in sensitivity, specificity, false discovery rate, false omission rate, and accuracy. There was a positive correlation between the relative prey DNA concentration in the samples and the number of prey reads detected by Lazaro, while this was inconsistent for metabarcoding. CONCLUSIONS: Metabarcoding and Lazaro had similar, but partially complementary, detection of prey in arthropod predator guts. However, while Lazaro was almost 2× more expensive, the number of reads was related to the amount of prey DNA, suggesting that Lazaro may provide quantitative prey information while metabarcoding did not.

Expression analysis of miRNAs and their putative target genes confirm a preponderant role of transcription factors in the early response of oil palm plants to salinity stress.

BACKGROUND: Several mechanisms regulating gene expression contribute to restore and reestablish cellular homeostasis so that plants can adapt and survive in adverse situations. MicroRNAs (miRNAs) play roles important in the transcriptional and post-transcriptional regulation of gene expression, emerging as a regulatory molecule key in the responses to plant stress, such as cold, heat, drought, and salt. This work is a comprehensive and large-scale miRNA analysis performed to characterize the miRNA population present in oil palm (Elaeis guineensis Jacq.) exposed to a high level of salt stress, to identify miRNA-putative target genes in the oil palm genome, and to perform an in silico comparison of the expression profile of the miRNAs and their putative target genes. RESULTS: A group of 79 miRNAs was found in oil palm, been 52 known miRNAs and 27 new ones. The known miRNAs found belonged to 28 families. Those miRNAs led to 229 distinct miRNA-putative target genes identified in the genome of oil palm. miRNAs and putative target genes differentially expressed under salinity stress were then selected for functional annotation analysis. The regulation of transcription, DNA-templated, and the oxidation-reduction process were the biological processes with the highest number of hits to the putative target genes, while protein binding and DNA binding were the molecular functions with the highest number of hits. Finally, the nucleus was the cellular component with the highest number of hits. The functional annotation of the putative target genes differentially expressed under salinity stress showed several ones coding for transcription factors which have already proven able to result in tolerance to salinity stress by overexpression or knockout in other plant species. CONCLUSIONS: Our findings provide new insights into the early response of young oil palm plants to salinity stress and confirm an expected preponderant role of transcription factors - such as NF-YA3, HOX32, and GRF1 - in this response. Besides, it points out potential salt-responsive miRNAs and miRNA-putative target genes that one can utilize to develop oil palm plants tolerant to salinity stress.

Dissecting protein domain variability in the core RNA interference machinery of five insect orders.

RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (K) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.

Transcriptome Profiling-Based Analysis of Carbohydrate-Active Enzymes in Aspergillus terreus Involved in Plant Biomass Degradation.

Given the global abundance of plant biomass residues, potential exists in biorefinery-based applications with lignocellulolytic fungi. Frequently isolated from agricultural cellulosic materials, Aspergillus terreus is a fungus efficient in secretion of commercial enzymes such as cellulases, xylanases and phytases. In the context of biomass saccharification, lignocellulolytic enzyme secretion was analyzed in a strain of A. terreus following liquid culture with sugarcane bagasse (SB) (1% w/v) and soybean hulls (SH) (1% w/v) as sole carbon source, in comparison to glucose (G) (1% w/v). Analysis of the fungal secretome revealed a maximum of 1.017 UI.mL-1 xylanases after growth in minimal medium with SB, and 1.019 UI.mL-1 after incubation with SH as carbon source. The fungal transcriptome was characterized on SB and SH, with gene expression examined in comparison to equivalent growth on G as carbon source. Over 8000 genes were identified, including numerous encoding enzymes and transcription factors involved in the degradation of the plant cell wall, with significant expression modulation according to carbon source. Eighty-nine carbohydrate-active enzyme (CAZyme)-encoding genes were identified following growth on SB, of which 77 were differentially expressed. These comprised 78% glycoside hydrolases, 8% carbohydrate esterases, 2.5% polysaccharide lyases, and 11.5% auxiliary activities. Analysis of the glycoside hydrolase family revealed significant up-regulation for genes encoding 25 different GH family proteins, with predominance for families GH3, 5, 7, 10, and 43. For SH, from a total of 91 CAZyme-encoding genes, 83 were also significantly up-regulated in comparison to G. These comprised 80% glycoside hydrolases, 7% carbohydrate esterases, 5% polysaccharide lyases, 7% auxiliary activities (AA), and 1% glycosyltransferases. Similarly, within the glycoside hydrolases, significant up-regulation was observed for genes encoding 26 different GH family proteins, with predominance again for families GH3, 5, 10, 31, and 43. A. terreus is a promising species for production of enzymes involved in the degradation of plant biomass. Given that this fungus is also able to produce thermophilic enzymes, this first global analysis of the transcriptome following cultivation on lignocellulosic carbon sources offers considerable potential for the application of candidate genes in biorefinery applications.

MiDaf16-like and MiSkn1-like gene families are reliable targets to develop biotechnological tools for the control and management of Meloidogyne incognita.

Meloidogyne incognita is a plant-parasitic root-knot nematode (RKN, PPN) responsible for causing damage to several crops worldwide. In Caenorhabditis elegans, the DAF-16 and SKN-1 transcription factors (TFs) orchestrate aging, longevity, and defense responses to several stresses. Here, we report that MiDaf16-like1 and MiSkn1-like1, which are orthologous to DAF-16 and SKN-1 in C. elegans, and some of their targets, are modulated in M. incognita J2 during oxidative stress or plant parasitism. We used RNAi technology for the stable production of siRNAs in planta to downregulate the MiDaf16-like1 and MiSkn1-like1 genes of M. incognita during host plant parasitism. Arabidopsis thaliana and Nicotiana tabacum overexpressing a hairpin-derived dsRNA targeting these genes individually (single-gene silencing) or simultaneously (double-gene silencing) were generated. T2 plants were challenged with M. incognita and the number of eggs, galls, and J2, and the nematode reproduction factor (NRF) were evaluated. Our data indicate that MiDaf16-like1, MiSkn1-like1 and some genes from their networks are modulated in M. incognita J2 during oxidative stress or plant parasitism. Transgenic A. thaliana and N. tabacum plants with single- or double-gene silencing showed significant reductions in the numbers of eggs, J2, and galls, and in NRF. Additionally, the double-gene silencing plants had the highest resistance level. Gene expression assays confirmed the downregulation of the MiDaf16-like1 and MiSkn1-like1 TFs and defense genes in their networks during nematode parasitism in the transgenic plants. All these findings demonstrate that these two TFs are potential targets for the development of biotechnological tools for nematode control and management in economically important crops.

Spring Is Coming: Genetic Analyses of the Bud Break Date Locus Reveal Candidate Genes From the Cold Perception Pathway to Dormancy Release in Apple (Malus × domestica Borkh.).

Chilling requirement (CR) for bud dormancy completion determines the time of bud break in apple (Malus × domestica Borkh.). The molecular control of bud dormancy is highly heritable, suggesting a strong genetic control of the trait. An available Infinium II SNP platform for genotyping containing 8,788 single nucleotide polymorphic markers was employed, and linkage maps were constructed in a F1 cross from the low CR M13/91 and the moderate CR cv. Fred Hough. These maps were used to identify quantitative trait loci (QTL) for bud break date as a trait related to dormancy release. A major QTL for bud break was detected at the beginning of linkage group 9 (LG9). This QTL remained stable during seven seasons in two different growing sites. To increase mapping efficiency in detecting contributing genes underlying this QTL, 182 additional SNP markers located at the locus for bud break were used. Combining linkage mapping and structural characterization of the region, the high proportion of the phenotypic variance in the trait explained by the QTL is related to the coincident positioning of Arabidopsis orthologs for ICE1, FLC, and PRE1 protein-coding genes. The proximity of these genes from the most explanatory markers of this QTL for bud break suggests potential genetic additive effects, reinforcing the hypothesis of inter-dependent mechanisms controlling dormancy induction and release in apple trees.

Analysis of the Transcriptome in Aspergillus tamarii During Enzymatic Degradation of Sugarcane Bagasse.

The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL-1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

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.

Systemic and sex-biased regulation of OBP expression under semiochemical stimuli.

Constitutive expression of Odorant-Binding Proteins (OBPs) in antennae and other body parts has been examined mainly to infer their involvement in insect olfaction, while their regulation in response to semiochemical stimuli has remained poorly known. Previous studies of semiochemical response were basically done using electrophysiology, which integrates the response of the set of OBPs present in an antenna or sensillum, without revealing the regulation of OBPs or which ones might be involved. In this study we used boll weevil as a model and mined its OBPs by RNA-Seq to study their simultaneous antennal expression by qPCR under controlled semiochemical stimuli with aggregation pheromone and plant volatiles. In the absence of a semiochemical stimulus, 23 of 24 OBPs were constitutively expressed in the antenna in both sexes. Semiochemicals changed systemically the expression of OBPs in both sexes. There were different patterns of up- and down-regulation in female antennae for each semiochemical stimulus, consistent with female chemical ecology. On the other hand, the only response in males was down-regulation of some OBPs. We suggest that these systemic changes in OBP expression might be related to enhancing detection of the semiochemical stimuli and/or priming the olfactory system to detect other environmental chemicals.

Gene expression analysis in Musa acuminata during compatible interactions with Meloidogyne incognita.

Background and Aims: Endoparasitic root-knot nematodes (RKNs) ( Meloidogyne spp.) cause considerable losses in banana ( Musa spp.), with Meloidogyne incognita a predominant species in Cavendish sub-group bananas. This study investigates the root transcriptome in Musa acuminata genotypes 4297-06 (AA) and Cavendish Grande Naine (CAV; AAA) during early compatible interactions with M. incognita . Methods: Roots were analysed by brightfield light microscopy over a 35 d period to examine nematode penetration and morphological cell transformation. RNA samples were extracted 3, 7 and 10 days after inoculation (DAI) with nematode J2 juveniles, and cDNA libraries were sequenced using lllumina HiSeq technology. Sequences were mapped to the M. acuminata ssp. malaccensis var. Pahang genome sequence, differentially expressed genes (DEGs) identified and transcript representation determined by gene set enrichment and pathway mapping. Key Results: Microscopic analysis revealed a life cycle of M. incognita completing in 24 d in CAV and 27 d in 4279-06. Comparable numbers of DEGs were up- and downregulated in each genotype, with potential involvement of many in early host defence responses involving reactive oxygen species and jasmonate/ethylene signalling. DEGs revealed concomitant auxin metabolism and cell wall modification processes likely to be involved in giant cell formation. Notable transcripts related to host defence included those coding for leucine-rich repeat receptor-like serine/threonine-protein kinases, peroxidases, thaumatin-like pathogenesis-related proteins, and DREB, ERF, MYB, NAC and WRKY transcription factors. Transcripts related to giant cell development included indole acetic acid-amido synthetase GH3.8 genes, involved in auxin metabolism, as well as genes encoding expansins and hydrolases, involved in cell wall modification. Conclusions: Expression analysis in M. acuminata during compatible interactions with RKNs provides insights into genes modulated during infection and giant cell formation. Increased understanding of both defence responses to limit parasitism during compatible interactions and effector-targeted host genes in this complex interaction will facilitate the development of genetic improvement measures for RKNs.

First Microsatellite Markers Developed from Cupuassu ESTs: Application in Diversity Analysis and Cross-Species Transferability to Cacao.

The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit´s pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta- and trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches' broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively.

Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.

Sugarcane giant borer transcriptome analysis and identification of genes related to digestion.

Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.