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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.

Black rot is a severe disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), which can lead to substantial losses in cruciferous vegetable production worldwide. Although the use of resistant cultivars is the main strategy to control this disease, there are limited sources of resistance. In this study, we used the LC-MS/MS technique to analyze young cabbage leaves and chloroplast-enriched samples at 24 h after infection by Xcc, using both susceptible (Veloce) and resistant (Astrus) cultivars. A comparison between susceptible Xcc-inoculated plants and the control condition, as well as between resistant Xcc-inoculated plants with the control was performed and more than 300 differentially abundant proteins were identified in each comparison. The chloroplast enriched samples contributed with the identification of 600 additional protein species in the resistant interaction and 900 in the susceptible one, which were not detected in total leaf sample. We further determined the expression levels for 30 genes encoding the identified differential proteins by qRT-PCR. CHI-B4 like gene, encoding an endochitinase showing a high increased abundance in resistant Xcc-inoculated leaves, was selected for functional validation by overexpression in Arabidopsis thaliana. Compared to the wild type (Col-0), transgenic plants were highly resistant to Xcc indicating that CHI-B4 like gene could be an interesting candidate to be used in genetic breeding programs aiming at black rot resistance.

The cotton boll weevil (CBW, Anthonomus grandis) is the most destructive cotton insect pest affecting cotton crops. To overcome this problem, CBW-resistant genetically modified cotton plants overexpressing Bacillus thuringiensis entomotoxins were successfully obtained. Previous results showed that the overexpression of Cry10Aa protoxin led to high mortality of the CBW larvae in greenhouse conditions. In this study, we advanced three more generations (T2 to T4), with several cotton events constitutively overexpressing the Cry10Aa protoxin, and the transgene stability and agronomic performance were investigated. In addition, stable transgenic cotton overexpressing the Cry10Aa active (Cry10Aa protoxin lacking the -helix N-terminal) driven by cotton flower bud-specific promoters were generated and characterized. Cotton events constitutively or tissue-specifically overexpressing the Cry10Aa protein (protoxin or active) represented mortality percentages of the CBW larva of up to 85 % in plants under greenhouse conditions. Events overexpressing the Cry10Aa active under control of the flower bud-specific promoter showed higher protein accumulation in stamens and carpels compared to the events with constitutive expression. Our findings suggested that the high stability of the Cry10Aa transgene and the elevated expression level and protein accumulation in flower bud tissues (primarily in stamen and carpels) contribute to improved resistance to CBW larvae. Finally, some notable events were selected with potential for future field trials in different cotton- producing regions of Brazil. Therefore, cotton events overexpressing high levels of the Cry10Aa protein in flower bud tissue may have a strong potential for commercial use in the integrated management of CBW. Made available in DSpace on 2020-10-20T09:13:24Z (GMT). No. of bitstreams: 1 Stability-and-tissue-2020.pdf: 2369502 bytes, checksum: aa3a6d66a284ec2f521627b47581e7f2 (MD5) Previous issue date: 2020

[EN] 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., We thank Durvalina Felix and Alexandre Garcez by assist in samples preparation. We are grateful Fabia Guimaraes-Dias by valuable suggestions on the Manuscript. This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Fundacao de Amparo a Pesquisa do Rio de Janeiro (FAPERJ), Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) and European Community (Evolutionary Conservation of Regulatory Network Controlling Flower Development, EVOCODE).

Background Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae (Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae. Results The analysis generated a total of 327,489,418 sequence reads that were aligned to the G. hirsutum reference transcriptome. The total number of expressed genes was over 21,697 per sample with an average length of 1,063 bp. The DEGseq analysis identified 443 differentially expressed genes (DEG) in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by “in situ” qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD). Conclusion A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, Ca2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-854) contains supplementary material, which is available to authorized users.

Background Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. Results We transformed C. arabica with the α-amylase inhibitor-1 gene (α-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the α-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against α-AI1 inhibitor showed a maximum α-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the α-AI1 protein against H. hampei α-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. Conclusions This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

Background Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. Results A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Conclusions Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

Background Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Results Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. Conclusions uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

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.