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.

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.

Stabilized Double-Stranded RNA Strategy Improves Cotton Resistance to CBW (Anthonomus grandis).

Cotton is the most important crop for fiber production worldwide. However, the cotton boll weevil (CBW) is an insect pest that causes significant economic losses in infested areas. Current control methods are costly, inefficient, and environmentally hazardous. Herein, we generated transgenic cotton lines expressing double-stranded RNA (dsRNA) molecules to trigger RNA interference-mediated gene silencing in CBW. Thus, we targeted three essential genes coding for chitin synthase 2, vitellogenin, and ecdysis-triggering hormone receptor. The stability of expressed dsRNAs was improved by designing a structured RNA based on a viroid genome architecture. We transformed cotton embryos by inserting a promoter-driven expression cassette that overexpressed the dsRNA into flower buds. The transgenic cotton plants were characterized, and positive PCR transformed events were detected with an average heritability of 80%. Expression of dsRNAs was confirmed in floral buds by RT-qPCR, and the T1 cotton plant generation was challenged with fertilized CBW females. After 30 days, data showed high mortality (around 70%) in oviposited yolks. In adult insects fed on transgenic lines, chitin synthase II and vitellogenin showed reduced expression in larvae and adults, respectively. Developmental delays and abnormalities were also observed in these individuals. Our data remark on the potential of transgenic cotton based on a viroid-structured dsRNA to control CBW.

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.

Discovery and functional characterization of novel cotton promoters with potential application to pest control.

KEY MESSAGE: pGhERF105 and pGhNc-HARBI1 promoters are highly responsive to CBW infestation and exhibit strong activity in vegetative and reproductive tissues, increasing their potential application in GM crop plants for pest control. The main challenge to cotton (Gossypium hirsutum) crop productivity is the constant attack of several pests, including the cotton boll weevil (CBW, Anthonomus grandis), which uses cotton floral buds for feeding and egg-laying. The endophytic nature of the early developmental stages of CBW makes conventional pesticide-based control poorly efficient. Most biotechnological assets used for pest control are based on Bacillus thurigiensis insecticidal Cry toxins or the silencing of insect-pest essential genes using RNA-interference technology. However, suitable plant promoter sequences are required to efficiently drive insecticidal molecules to the target plant tissue. This study selected the Ethylene Responsive Factor 105 (GhERF105) and Harbinger transposase-derived nuclease (GhNc-HARBI1) genes based on available transcriptome-wide data from cotton plants infested by CBW larvae. The GhERF105 and GhNc-HARBI1 genes showed induction kinetics from 2 to 96 h under CBW's infestation in cotton floral buds, uncovering the potential application of their promoters. Therefore, the promoter regions (1,500 base pairs) were assessed and characterized using Arabidopsis thaliana transgenic plants. The pGhERF105 and pGhNc-HARBI1 promoters showed strong activity in plant vegetative (leaves and roots) and reproductive (flowers and fruits) tissues, encompassing higher GUS transcriptional activity than the viral-constitutive Cauliflower Mosaic Virus 35S promoter (pCaMV35S). Notably, pGhERF105 and pGhNc-HARBI1 promoters demonstrated more efficiency in driving reporter genes in flowers than other previously characterized cotton flower-specific promoters. Overall, the present study provides a new set of cotton promoters suitable for biotechnological application in cotton plants for pest resistance.

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.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.