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.

Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis).

BACKGROUND: The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. RESULTS: Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. CONCLUSIONS: The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.

Protective effects of a cysteine proteinase propeptide expressed in transgenic soybean roots.

Sedentary endoparasitic nematodes cause extensive damage to a large number of ornamental plants and food crops, with estimated economical losses over 100 billion US$ worldwide. Various efforts have put forth in order to minimize nematode damage, which typically involve the use of nematicides that have high cost and enhanced toxicity to humans and the environment. Additionally, different strategies have been applied in order to develop genetically modified plants with improved nematode resistance. Among the strategies are anti-invasion and migration, feeding-cell attenuation, and anti-nematode feeding. In the present study, we focus on anti-nematode feeding, which involves the evaluation and potential use of the cysteine proteinase (CPs) propeptide as a control alternative. The cysteine proteinase prodomain, isolated from Heterodera glycines (HGCP prodomain), is a natural inhibitory peptide used to transform soybean cotyledons using Agrobacterium rhizogenes. Genetically modified soybean roots expressing the propeptide were detected by Western blot and expression levels were measured by ELISA (around 0.3%). The transgenic roots expressing the propeptide were inoculated with a thousand H. glycines at the second juvenile stage, and a remarkable reduction in the number of females and eggs was observed. A reduction of female length and diameter was also observed after 35 days post-inoculation. Furthermore, the H. glycines mature protein was detected in females fed on soybean transformed root expressing or not expressing the propeptide. The data presented here indicate that the HGCP propeptide can reduce soybean cyst nematode infection and this strategy could be applied in the near future to generate resistant crop cultivars.

Involvement of g proteins and camp in the production of chitinolytic enzymes by Trichoderma harzianum

The effect of G protein modulators and cyclic AMP (cAMP) on N-acetylglucosaminidase (NAGase) production was investigated during 84 h of growth of a Trichoderma harzianum strain in chitin-containing medium. Caffeine (5 mM), N6¾2'-O-dibutyryladenosine 3'5'-cyclic monophosphate sodium salt (dBcAMP) (1 mM) and 3-isobutyl-1-methylxanthine (IBMX) (2 mM) decreased extracellular NAGase activity by 80(per cent), 77(per cent) and 37(per cent), respectively. AlCl3/KF (100 µM/10 mM and 200 µM/ 20 mM) decreased the activity by 85(per cent)and 95(per cent), respectively. Cholera (10 µ/mL) and pertussis (20 µ/mL) toxins also affected NAGase activity, causing a decrease of approximately 75(per cent). Upon all treatments, protein bands of approximately 73 kDa, 68 kDa and 45 kDa had their signals diminished whilst a 50 kDa band was enhanced only by treatment with cholera and pertussis toxins. N-terminal sequencing analysis identified the 73 kDa and 68 kDa proteins as being T. harzianum NAGase in two different truncated forms whereas the 45 kDa band comprised a T. harzianum endochitinase. The 50 kDa protein showed sequence similarity to Coriolus vesicolor cellobiohydrolase. The above results suggest that a signaling pathway comprising G-proteins, adenylate cyclase and cAMP may be involved in the synthesis of T. harzianum chitinases.