An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Pyramiding dsRNAs increases phytonematode tolerance in cotton plants.

MAIN CONCLUSION: Host-derived suppression of nematode essential genes decreases reproduction of Meloidogyne incognita in cotton. Root-knot nematodes (RKN) represent one of the most damaging plant-parasitic nematode genera worldwide. RNAi-mediated suppression of essential nematode genes provides a novel biotechnological strategy for the development of sustainable pest-control methods. Here, we used a Host Induced Gene Silencing (HIGS) approach by stacking dsRNA sequences into a T-DNA construct to target three essential RKN genes: cysteine protease (Mi-cpl), isocitrate lyase (Mi-icl), and splicing factor (Mi-sf), called dsMinc1, driven by the pUceS8.3 constitutive soybean promoter. Transgenic dsMinc1-T4 plants infected with Meloidogyne incognita showed a significant reduction in gall formation (57-64%) and egg masses production (58-67%), as well as in the estimated reproduction factor (60-78%), compared with the susceptible non-transgenic cultivar. Galls of the RNAi lines are smaller than the wild-type (WT) plants, whose root systems exhibited multiple well-developed root swellings. Transcript levels of the three RKN-targeted genes decreased 13- to 40-fold in nematodes from transgenic cotton galls, compared with those from control WT galls. Finally, the development of non-feeding males in transgenic plants was 2-6 times higher than in WT plants, indicating a stressful environment for nematode development after RKN gene silencing. Data strongly support that HIGS of essential RKN genes is an effective strategy to improve cotton plant tolerance. This study presents the first application of dsRNA sequences to target multiple genes to promote M. incognita tolerance in cotton without phenotypic penalty in transgenic plants.

Identification and characterization of the GmRD26 soybean promoter in response to abiotic stresses: potential tool for biotechnological application.

BACKGROUND: Drought is one of the most harmful abiotic stresses for plants, leading to reduced productivity of several economically important crops and, consequently, considerable losses in the agricultural sector. When plants are exposed to stressful conditions, such as drought and high salinity, they modulate the expression of genes that lead to developmental, biochemical, and physiological changes, which help to overcome the deleterious effects of adverse circumstances. Thus, the search for new specific gene promoter sequences has proved to be a powerful biotechnological strategy to control the expression of key genes involved in water deprivation or multiple stress responses. RESULTS: This study aimed to identify and characterize the GmRD26 promoter (pGmRD26), which is involved in the regulation of plant responses to drought stress. The expression profile of the GmRD26 gene was investigated by qRT-PCR under normal and stress conditions in Williams 82, BR16 and Embrapa48 soybean-cultivars. Our data confirm that GmRD26 is induced under water deficit with different induction folds between analyzed cultivars, which display different genetic background and physiological behaviour under drought. The characterization of the GmRD26 promoter was performed under simulated stress conditions with abscisic acid (ABA), polyethylene glycol (PEG) and drought (air dry) on A. thaliana plants containing the complete construct of pGmRD26::GUS (2.054 bp) and two promoter modules, pGmRD26A::GUS (909 pb) and pGmRD26B::GUS (435 bp), controlling the expression of the ß-glucuronidase (uidA) gene. Analysis of GUS activity has demonstrated that pGmRD26 and pGmRD26A induce strong reporter gene expression, as the pAtRD29 positive control promoter under ABA and PEG treatment. CONCLUSIONS: The full-length promoter pGmRD26 and the pGmRD26A module provides an improved uidA transcription capacity when compared with the other promoter module, especially in response to polyethylene glycol and drought treatments. These data indicate that pGmRD26A may become a promising biotechnological asset with potential use in the development of modified drought-tolerant plants or other plants designed for stress responses.

Transcriptome Profiling of Wild Arachis from Water-Limited Environments Uncovers Drought Tolerance Candidate Genes.

Peanut (Arachis hypogaea L.) is an important legume cultivated mostly in drought-prone areas where its productivity can be limited by water scarcity. The development of more drought-tolerant varieties is, therefore, a priority for peanut breeding programs worldwide. In contrast to cultivated peanut, wild relatives have a broader genetic diversity and constitute a rich source of resistance/tolerance alleles to biotic and abiotic stresses. The present study takes advantage of this diversity to identify drought-responsive genes by analyzing the expression profile of two wild species, Arachis duranensis and Arachis magna (AA and BB genomes, respectively), in response to progressive water deficit in soil. Data analysis from leaves and roots of A. duranensis (454 sequencing) and A. magna (suppression subtractive hybridization (SSH)) stressed and control complementary DNA (cDNA) libraries revealed several differentially expressed genes in silico, and 44 of them were selected for further validation by quantitative RT-PCR (qRT-PCR). This allowed the identification of drought-responsive candidate genes, such as Expansin, Nitrilase, NAC, and bZIP transcription factors, displaying significant levels of differential expression during stress imposition in both species. This is the first report on identification of differentially expressed genes under drought stress and recovery in wild Arachis species. The generated transcriptome data, besides being a valuable resource for gene discovery, will allow the characterization of new alleles and development of molecular markers associated with drought responses in peanut. These together constitute important tools for the peanut breeding program and also contribute to a better comprehension of gene modulation in response to water deficit and rehydration.

Simultaneous silencing of juvenile hormone metabolism genes through RNAi interrupts metamorphosis in the cotton boll weevil.

The cotton boll weevil (CBW) (Anthonomus grandis) is one of the major insect pests of cotton in Brazil. Currently, CBW control is mainly achieved by insecticide application, which is costly and insufficient to ensure effective crop protection. RNA interference (RNAi) has been used in gene function analysis and the development of insect control methods. However, some insect species respond poorly to RNAi, limiting the widespread application of this approach. Therefore, nanoparticles have been explored as an option to increase RNAi efficiency in recalcitrant insects. Herein, we investigated the potential of chitosan-tripolyphosphate (CS-TPP) and polyethylenimine (PEI) nanoparticles as a dsRNA carrier system to improve RNAi efficiency in the CBW. Different formulations of the nanoparticles with dsRNAs targeting genes associated with juvenile hormone metabolism, such as juvenile hormone diol kinase (JHDK), juvenile hormone epoxide hydrolase (JHEH), and methyl farnesoate hydrolase (MFE), were tested. The formulations were delivered to CBW larvae through injection (0.05-2 µg), and the expression of the target genes was evaluated using RT-qPCR. PEI nanoparticles increased targeted gene silencing compared with naked dsRNAs (up to 80%), whereas CS-TPP-dsRNA nanoparticles decreased gene silencing (0%-20%) or led to the same level of gene silencing as the naked dsRNAs (up to 50%). We next evaluated the effects of targeting a single gene or simultaneously targeting two genes via the injection of naked dsRNAs or dsRNAs complexed with PEI (500 ng) on CBW survival and phenotypes. Overall, the gene expression analysis showed that the treatments with PEI targeting either a single gene or multiple genes induced greater gene silencing than naked dsRNA (∼60%). In addition, the injection of dsJHEH/JHDK, either naked or complexed with PEI, significantly affected CBW survival (18% for PEI nanoparticles and 47% for naked dsRNA) and metamorphosis. Phenotypic alterations, such as uncompleted pupation or malformed pupae, suggested that JHEH and JHDK are involved in developmental regulation. Moreover, CBW larvae treated with dsJHEH/JHDK + PEI (1,000 ng/g) exhibited significantly lower survival rate (55%) than those that were fed the same combination of naked dsRNAs (30%). Our findings demonstrated that PEI nanoparticles can be used as an effective tool for evaluating the biological role of target genes in the CBW as they increase the RNAi response.

Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection.

BACKGROUND: Cultivated peanut (Arachis hypogaea) is one of the most widely grown grain legumes in the world, being valued for its high protein and unsaturated oil contents. Worldwide, the major constraints to peanut production are drought and fungal diseases. Wild Arachis species, which are exclusively South American in origin, have high genetic diversity and have been selected during evolution in a range of environments and biotic stresses, constituting a rich source of allele diversity. Arachis stenosperma harbors resistances to a number of pests, including fungal diseases, whilst A. duranensis has shown improved tolerance to water limited stress. In this study, these species were used for the creation of an extensive databank of wild Arachis transcripts under stress which will constitute a rich source for gene discovery and molecular markers development. RESULTS: Transcriptome analysis of cDNA collections from A. stenosperma challenged with Cercosporidium personatum (Berk. and M.A. Curtis) Deighton, and A. duranensis submitted to gradual water limited stress was conducted using 454 GS FLX Titanium generating a total of 7.4 x 10(5) raw sequence reads covering 211 Mbp of both genomes. High quality reads were assembled to 7,723 contigs for A. stenosperma and 12,792 for A. duranensis and functional annotation indicated that 95% of the contigs in both species could be appointed to GO annotation categories. A number of transcription factors families and defense related genes were identified in both species. Additionally, the expression of five A. stenosperma Resistance Gene Analogs (RGAs) and four retrotransposon (FIDEL-related) sequences were analyzed by qRT-PCR. This data set was used to design a total of 2,325 EST-SSRs, of which a subset of 584 amplified in both species and 214 were shown to be polymorphic using ePCR. CONCLUSIONS: This study comprises one of the largest unigene dataset for wild Arachis species and will help to elucidate genes involved in responses to biological processes such as fungal diseases and water limited stress. Moreover, it will also facilitate basic and applied research on the genetics of peanut through the development of new molecular markers and the study of adaptive variation across the genus.

Integrated Omic Approaches Reveal Molecular Mechanisms of Tolerance during Soybean and Meloidogyne incognita Interactions.

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.

Increasing Anthonomus grandis susceptibility to Metarhizium anisopliae through RNAi-induced AgraRelish knockdown: a perspective to combine biocontrol and biotechnology.

BACKGROUND: The hemolymph and insect gut together have an essential role in the immune defense against microorganisms, including the production of antimicrobial peptides (AMP). AMPs are mainly induced by two specific signaling pathways, Toll and immune deficiency (IMD). Here, we characterize the expression profile of four genes from both pathways and describe the importance of AgraRelish in the immune defense of Anthonomus grandis against the entomopathogenic fungus Metarhizium anisopliae by RNA interference (RNAi). RESULTS: To characterize the pathway that is activated early during the A. grandis-M. anisopliae interaction, we assessed the expression profiles of AgraMyD88 and AgraDorsal (Toll pathway), AgraIMD and AgraRelish (IMD pathway), and several AMP genes. Interestingly, we found that IMD pathway genes are upregulated early, and Toll pathway genes are upregulated just 3 days after inoculation (DAI). Furthermore, nine AMPs were upregulated 24 h after fungus inoculation, including attacins, cecropins, coleoptericins, and defensins. AgraRelish knockdown resulted in a reduction in median lethal time (LT50 ) for M. anisopliae-treated insects of around 2 days compared to control treatments. In addition, AgraRelish remained knocked down at 3 DAI. Finally, we identified that AgraRelish knockdown increased fungal loads at 2 DAI compared to control treatments, possibly indicating a faster infection. CONCLUSIONS: Our data indicate the influence of the IMD pathway on the antifungal response in A. grandis. Combining biocontrol and RNAi could significantly improve cotton boll weevil management. Hence, AgraRelish is a potential target for the development of biotechnological tools aimed at improving the efficacy of M. anisopliae against A. grandis.

Comparative gut transcriptome analysis of Diatraea saccharalis in response to the dietary source.

The sugarcane borer (Diatraea saccharalis, Fabricius, 1794) is a devastating pest that causes millions of dollars of losses each year to sugarcane producers by reducing sugar and ethanol yields. The control of this pest is difficult due to its endophytic behavior and rapid development. Pest management through biotechnological approaches has emerged in recent years as an alternative to currently applied methods. Genetic information about the target pests is often required to perform biotechnology-based management. The genomic and transcriptomic data for D. saccharalis are very limited. Herein, we report a tissue-specific transcriptome of D. saccharalis larvae and a differential expression analysis highlighting the physiological characteristics of this pest in response to two different diets: sugarcane and an artificial diet. Sequencing was performed on the Illumina HiSeq 2000 platform, and a de novo assembly was generated. A total of 27,626 protein-coding unigenes were identified, among which 1,934 sequences were differentially expressed between treatments. Processes such as defence, digestion, detoxification, signaling, and transport were highly represented among the differentially expressed genes (DEGs). Furthermore, seven aminopeptidase genes were identified as candidates to encode receptors of Cry proteins, which are toxins of Bacillus thuringiensis used to control lepidopteran pests. Since plant-insect interactions have produced a considerable number of adaptive responses in hosts and herbivorous insects, the success of phytophagous insects relies on their ability to overcome challenges such as the response to plant defences and the intake of nutrients. In this study, we identified metabolic pathways and specific genes involved in these processes. Thus, our data strongly contribute to the knowledge advancement of insect transcripts, which can be a source of target genes for pest management.

Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution.

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter.

Vegetative desiccation tolerance of Tripogon spicatus (Poaceae) from the tropical semiarid region of northeastern Brazil.

The vegetative desiccation tolerance of Tripogon spicatus (Nees) Ekman was confirmed by its ability to recover the physiological functionality of intact plants previously subjected to extreme dehydration. Photosynthesis became undetectable when leaf relative water content (RWCleaf) achieved ~60%, whereas photochemical variables showed a partial decrease. Until the minimum RWCleaf of 6.41%, total chl decreased by 9%, and total carotenoids increased by 29%. Superoxide dismutase (SOD) activity decreased by 57%, on average, during dehydration, but catalase (CAT) and peroxidase (APX) activities showed no significant differences throughout the experiment. Malondialdehyde (MDA) content increased by 151%, total leaf and root amino acids decreased by 62% and 77%, respectively, whereas leaf and root proline decreased by 40% and 61%, respectively, until complete desiccation. After rehydration, leaves completely recovered turgidity and total chl contents. Carotenoids and MDA remained high, whereas SOD was 60% lower than the measured average measured before dehydration. With the exception of root amino acid contents, total amino acids and proline concentrations recovered completely. Gas exchange and photochemical variables remained substantially higher 4 days after rehydration, compared with the control. Besides increasing MDA, the overall physiological results showed that membrane functionality was preserved, leading to the vegetative desiccation tolerance of T. spicatus during the dehydration-rehydration cycle.

Root Transcriptome Analysis of Wild Peanut Reveals Candidate Genes for Nematode Resistance.

Wild peanut relatives (Arachis spp.) are genetically diverse and were adapted to a range of environments during the evolution course, constituting an important source of allele diversity for resistance to biotic and abiotic stresses. The wild diploid A. stenosperma harbors high levels of resistance to a variety of pathogens, including the root-knot nematode (RKN) Meloidogyne arenaria, through the onset of the Hypersensitive Response (HR). In order to identify genes and regulators triggering this defense response, a comprehensive root transcriptome analysis during the first stages of this incompatible interaction was conducted using Illumina Hi-Seq. Overall, eight cDNA libraries were produced generating 28.2 GB, which were de novo assembled into 44,132 contigs and 37,882 loci. Differentially expressed genes (DEGs) were identified and clustered according to their expression profile, with the majority being downregulated at 6 DAI, which coincides with the onset of the HR. Amongst these DEGs, 27 were selected for further qRT-PCR validation allowing the identification of nematode-responsive candidate genes that are putatively related to the resistance response. Those candidates are engaged in the salycilic (NBS-LRR, lipocalins, resveratrol synthase) and jasmonic (patatin, allene oxidase cyclase) acids pathways, and also related to hormonal balance (auxin responsive protein, GH3) and cellular plasticity and signaling (tetraspanin, integrin, expansin), with some of them showing contrasting expression behavior between Arachis RKN-resistant and susceptible genotypes. As these candidate genes activate different defensive signaling systems, the genetic (HR) and the induced resistance (IR), their pyramidding in one genotype via molecular breeding or transgenic strategy might contribute to a more durable resistance, thus improving the long-term control of RKN in peanut.

A survey of genes involved in Arachis stenosperma resistance to Meloidogyne arenaria race 1.

Root-knot nematodes constitute a constraint for important crops, including peanut (Arachis hypogaea L.). Resistance to Meloidogyne arenaria has been identified in the peanut wild relative Arachis stenosperma Krapov. & W. C. Greg., in which the induction of feeding sites by the nematode was inhibited by an early hypersensitive response (HR). Here, the transcription expression profiles of 19 genes selected from Arachis species were analysed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), during the early phases of an A. stenosperma-M. arenaria interaction. Sixteen genes were significantly differentially expressed in infected and non-infected roots, in at least one of the time points analysed: 3, 6, and 9 days after inoculation. These genes are involved in the HR and production of secondary metabolites related to pathogen defence. Seven genes encoding a resistance protein MG13, a helix-loop helix protein, an ubiquitin protein ligase, a patatin-like protein, a catalase, a DUF538 protein, and a resveratrol synthase, were differentially expressed in all time points analysed. Transcripts of two genes had their spatial and temporal distributions analysed by in situ hybridisation that validated qRT-PCR data. The identification of candidate resistance genes involved in wild peanut resistance to Meloidogyne can provide additional resources for peanut breeding and transgenic approaches.

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut.

BACKGROUND: Wild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated Arachis. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut. RESULTS: A set of ten reference genes were analyzed in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, ACT1 (actin depolymerizing factor-like protein), UBI1 (polyubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L10) and UBI2 (ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied. CONCLUSIONS: This first in-depth study of reference genes validation in wild Arachis species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.