Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

Global gene expression profile and functional analysis reveal the conservation of reproduction-associated gene networks in Gossypium hirsutum.

KEY MESSAGE: Lastly, the bZIP gene family encompasses genes that have been reported to play a role in flower development, such as bZIP14 (FD). Notably, bZIP14 is essential for Flowering Locus T (FT) initiation of floral development in Arabidopsis (Abe et al. 2005). Cotton (Gossypium hirsutum L.) is the world's most extensively cultivated fiber crop. However, its reproductive development is poorly characterized at the molecular level. Thus, this study presents a detailed transcriptomic analysis of G. hirsutum at three different reproductive stages. We provide evidence that more than 64,000 genes are active in G. hirsutum during flower development, among which 94.33% have been assigned to functional terms and specific pathways. Gene set enrichment analysis (GSEA) revealed that the biological process categories of floral organ development, pollen exine formation, and stamen development were enriched among the genes expressed during the floral development of G. hirsutum. Furthermore, we identified putative Arabidopsis homologs involved in the G. hirsutum gene regulatory network (GRN) of pollen and flower development, including transcription factors such as WUSCHEL (WUS), INNER NO OUTER (INO), AGAMOUS-LIKE 66 (AGL66), SPOROCYTELESS/NOZZLE (SPL/NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), ABORTED MICROSPORES (AMS), and ASH1-RELATED 3 (ASHR3), which are known crucial genes for plant reproductive success. The cotton MADS-box protein-protein interaction pattern resembles the previously described patterns for AGAMOUS (AG), SEEDSTICK (STK), SHATTERPROOF (SHP), and SEPALLATA3 (SEP3) homolog proteins from Arabidopsis. In addition to serving as a resource for comparative flower development studies, this work highlights the changes in gene expression profiles and molecular networks underlying stages that are valuable for cotton breeding improvement.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Functional characterization of the pUceS8.3 promoter and its potential use for ectopic gene overexpression.

MAIN CONCLUSION: The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (ß-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher ß-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression of this reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

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.

Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.

MAIN CONCLUSION: Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum. Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early-EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate-IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late-LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY, AtAPETALA 3, AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1, which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

Physiological and molecular responses of Setaria viridis to osmotic stress.

Drought-tolerant species, such as Setaria viridis, a C4 model plant, make physiological and biochemical adjustments water limitation and recover from the stress upon its release. We investigated S. viridis (A10.1 accession) responses to continuing osmotic stress. The osmotic stress was imposed using polyethylene glycol (PEG) 8000 (7.5%) for 10 days. Morphological traits and stomatal conductance were measured daily for the 10 days. On days 6 and 10, the following traits were measured separately for root and shoot: relative water content (RWC), osmotic potential (OP), electrolytic leakage (EL), and proline content. qPCR analysis was used to evaluate the expression of five selected genes in roots (SvLEA, SvDREB1C, SvPIP2-1, SvHSP20, and SvP5CS2), and chlorophyll a fluorescence was measured on three key days. The morphological data demonstrated a drastic reduction in shoot biomass as an effect of water deficit caused by the osmotic stress. Shoot biomass reduction could be associated with putative ABA-dependent signaling involved in SvDREB1C expression. Stomatal conductance and photosynthesis were severely affected up until day 6, however, stomatal conductance and some photosynthetic parameters such as FV/FM, ABS/RC, and DI0/RC showed total or slight recovery on day 10. Root EL decreased in treated plants suggesting an investment in membrane protection by osmoregulator expression such as dehydrin (SvLEA) and proline (SvP5CS2) genes. Our data suggest that S. viridis exhibited a partial recovery from an imposed and constant osmotic stress within 10 days.

Genome-wide analysis of the MADS-box gene family in polyploid cotton (Gossypium hirsutum) and in its diploid parental species (Gossypium arboreum and Gossypium raimondii).

The MADS-box gene family encodes transcription factors that share a highly conserved domain known to bind to DNA. Members of this family control various processes of development in plants, from root formation to fruit ripening. In this work, a survey of diploid (Gossypium raimondii and Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton genomes found a total of 147, 133 and 207 MADS-box genes, respectively, distributed in the MIKC, Mα, Mß, Mγ, and Mδ subclades. A comparative phylogenetic analysis among cotton species, Arabidopsis, poplar and grapevine MADS-box homologous genes allowed us to evaluate the evolution of each MADS-box lineage in cotton plants and identify sequences within well-established subfamilies. Chromosomal localization and phylogenetic analysis revealed that G. raimondii and G. arboreum showed a conserved evolution of the MIKC subclade and a distinct pattern of duplication events in the Mα, Mγ and Mδ subclades. Additionally, G. hirsutum showed a combination of its parental subgenomes followed by a distinct evolutionary history including gene gain and loss in each subclade. qPCR analysis revealed the expression patterns of putative homologs in the AP1, AP3, AGL6, SEP4, AGL15, AG, AGL17, TM8, SVP, SOC and TT16 subfamilies of G. hirsutum. The identification of putative cotton orthologs is discussed in the light of evolution and gene expression data from other plants. This analysis of the MADS-box genes in Gossypium species opens an avenue to understanding the origin and evolution of each gene subfamily within diploid and polyploid species and paves the way for functional studies in cotton species.

Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones.

KEY MESSAGE: Expression analysis of the AG -subfamily members from G. hirsutum during flower and fruit development. Reproductive development in cotton, including the fruit and fiber formation, is a complex process; it involves the coordinated action of gene expression regulators, and it is highly influenced by plant hormones. Several studies have reported the identification and expression of the transcription factor family MADS-box members in cotton ovules and fibers; however, their roles are still elusive during the reproductive development in cotton. In this study, we evaluated the expression profiles of five MADS-box genes (GhMADS3, GhMADS4, GhMADS5, GhMADS6 and GhMADS7) belonging to the AGAMOUS-subfamily in Gossypium hirsutum. Phylogenetic and protein sequence analyses were performed using diploid (G. arboreum, G. raimondii) and tetraploid (G. barbadense, G. hirsutum) cotton genomes, as well as the AG-subfamily members from Arabidopsis thaliana, Petunia hybrida and Antirrhinum majus. qPCR analysis showed that the AG-subfamily genes had high expression during flower and fruit development in G. hirsutum. In situ hybridization analysis also substantiates the involvement of AG-subfamily members on reproductive tissues of G. hirsutum, including ovule and ovary. The effect of plant hormones on AG-subfamily genes expression was verified in cotton fruits treated with gibberellin, auxin and brassinosteroid. All the genes were significantly regulated in response to auxin, whereas only GhMADS3, GhMADS4 and GhMADS7 genes were also regulated by brassinosteroid treatment. In addition, we have investigated the GhMADS3 and GhMADS4 overexpression effects in Arabidopsis plants. Interestingly, the transgenic plants from both cotton AG-like genes in Arabidopsis significantly altered the fruit size compared to the control plants. This alteration suggests that cotton AG-like genes might act regulating fruit formation. Our results demonstrate that members of the AG-subfamily in G. hirsutum present a conserved expression profile during flower development, but also demonstrate their expression during fruit development and in response to phytohormones.