Advances in exogenous RNA delivery techniques for RNAi-mediated pest control.

Climate change imposes a great threat to world food security and encourages insect pest proliferation and spreading. Because of these challenges, identifying novel biotechnology pest management and its applications is inevitable. RNA interference (RNAi) is a gene regulatory process used for the maintenance and regulation of host defences against invading viruses. Nevertheless, it is widely used for the analysis of gene function. In recent years, the potential use of RNA interference (RNAi) as a tool for manipulating crop traits, as well as an alternative for crop protection, has undergone outstanding developments. In this review, we describe some genes involved in insect dsRNA uptake and discuss the reasons for varying RNAi response in insect pests, emphasizing the presence of nucleases and double-stranded RNA binding protein. We explore recent breakthroughs in innovative dsRNA delivery for efficient and effective knockdown in insect pests. Conclusively, topical delivery of dsRNA combined with a nanoparticle complex holds great potential for RNAi-mediated pest control.

Resistance to Chilo infuscatellus (Lepidoptera: Pyraloidea) in transgenic lines of sugarcane expressing Bacillus thuringiensis derived Vip3A protein.

Sustainable agriculture requires management of insect pests through resistance development. The biological potential of Cry toxins and Vip protein, derived from Bacillus species, is widely recognized in this context. The identification, evaluation of new insecticidal protein genes with different mode of action and entomotoxicity against sugarcane stem borer (Chilo infuscatellus) is important to overcome evolved insect resistance. In this study, we reported the generation of transgenic sugarcane lines expressing Vip3A toxin driven by polyubiquitin promoter for resistance against sugarcane stem borer. The V0 transgenic sugarcane plants were initially characterized by GUS histochemical staining, PCR and Southern blot assays that confirmed genetic transformation of twelve independent sugarcane lines. Variable transgene expression was found among transgenic sugarcane lines when revealed through Realtime quantitative PCR (RT-qPCR) with highest in S10 line while minimum was observed in V5 line. A similar expression pattern was observed in transgenic sugarcane lines for Vip3A protein concentration which ranged from 5.35 to 8.89 µg/mL. A direct correlation was observed between the Vip3A protein and Vip3A transgene expression in the transgenic sugarcane lines. In in-vitro insect bioassay on V1, Vip3A transgenic sugarcane lines exhibited high resistance to C. infuscatellus with upto 100% mortality compared to the control sugarcane line. Our findings suggest that a single copy insertion of Vip3A gene in transgenic sugarcane lines render them resistant to borer and these lines can be potentially used for generation of insect resistant transgenic sugarcane and could also be employed in gene pyramiding with Bt toxin to prolong resistance.

Mini Review - Epigenetics: Quest for no-escape to HIV, a persistent pathogen.

Acquired Immune Deficiency Syndrome (AIDS) is a disease infection mix, which is primarily because of 'deficient' immune system. Human Immune-deficiency Virus (HIV) makes the immune system susceptible to many infections by infiltrating it. Many researchers believe that HIV is a mutated form of Simian Immune-deficiency Virus (SIV). After being clinically discovered in 1981 in America, it is said to have caused 36 million deaths. Treatment of AIDS has been a 'burning ' issue ever since its discovery. There is no cure for AIDS! Although, Recombinant Transcriptase Inhibitors (RTis) are being considered a major treatment against HIV that can not only lessen the effect of HIV but also can prolong the life of HIV positive patients. More recent advancement includes 'transplantation of transgenic stem cells' in HIV positive patients. As latency of HIV provirus in host genome is the preeminent weapon of this virus against RTis that compel it to hide from host immune system and a persistent pathogen thereof. Thus, epigenetic activation of latent provirus pool by methyl inhibitors along with non¬toxic chemical drugs seems to be a more promising treatment to avoid the burden of lifelong RTI.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32kDa recombinant chitinase in Escherichia coli host.

Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35kDa exhibited highest expression at 0.5mM concentration of IPTG. Expressed recombinant protein of 35kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80µg and 200µg.

Antifungal activity of chitinase II against Colletotrichum falcatum Went. causing red rot disease in transgenic sugarcane.

We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T 0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.