Identification and functional characterisation of N-acetylglucosamine kinase from Helicoverpa armigera divulge its potential role in growth and development via UDP-GlcNAc salvage pathway.

N-acetylglucosamine kinase (NAGK), a major enzyme of sugar-kinase/Hsp70/actin superfamily, catalyses the conversion of N-acetylglucosamine to N-acetylglucosamine-6-phosphate, the first step leading to the salvage synthesis of uridine diphosphate N-acetylglucosamine. Here, we present the first report on identification, cloning, recombinant expression and functional characterisation of NAGK from Helicoverpa armigera (HaNAGK). The purified soluble HaNAGK exhibited a molecular mass of ∼39 kDa with monomeric conformation. It catalysed the sequential transformation of GlcNAc into UDP-GlcNAc, indicating its role as the initiator of UDP-GlcNAc salvage pathway. HaNAGK exhibited ubiquitous expressions across all the developmental stages and major tissues of H. armigera. The gene was significantly upregulated (80 %; p < 0.01) by the moulting hormone 20-hydroxyecdysone and significantly downregulated (89 %; p < 0.001) by the chitin synthesis inhibitor novaluron, indicating its involvement in ecdysis and chitin metabolism. Furthermore, RNAi of HaNAGK caused poor weight gain, deformed insect bodies, aberrant metamorphosis and pronounced wing abnormalities in >55 % of surviving adults, while recording 7.79 ± 1.52 % and 24.25 ± 7.21 % mortality during larval and pupal stages, respectively. Altogether, the present findings suggest that HaNAGK plays a crucial role in the growth and development of H. armigera and thus, could be considered as a compelling gene of interest while formulating novel pest management strategies.

Development of loop mediated isothermal amplification (LAMP): A new tool for rapid diagnosis of cotton leaf curl viral disease.

Cotton leaf curl disease (CLCuD) ranks top among all endemic diseases transmitted by whitefly (Bemisia tabaci) affecting cotton (Gossypium hirsutum) causing severe economic losses to the cotton growers in the Indian subcontinent. For its effective management, robust tools for detection are a prerequisite and it is important to diagnose the virus titre in early stage of infection in plants as well as in the disease transmitting vector. Considering the limitations in current PCR-based techniques we have standardised rapid and sensitive Loop Mediated Isothermal Amplification (LAMP) protocol for the diagnosis of cotton leaf curl virus (CLCuV) in cotton leaves and in its transmitting vector whitefly. Perhaps, this is the first report of use of LAMP tool for rapid diagnosis of CLCuV in cotton and its transmitting vector the whitefly. Further, the colorimetric detection for diagnostic simplicity of amplified LAMP product by using different dyes lead to enhanced applicability of this technique in the field of disease diagnostics. The merit of present study is that the diagnostic failure of PCR and LAMP due to low virus titre in the infected leaf has been circumvented through the combination of rolling circle amplification (RCA) with LAMP. Thus RCA-LAMP can be an option for ultra-sensitive detection of samples with low virus titre. The potential applications of this advanced diagnostic tool in laboratory research on diagnosis of CLCuV, an important viral pathogen of cotton have been discussed.

Genome-wide identification and expression analysis of the plant specific LIM genes in Gossypium arboreum under phytohormone, salt and pathogen stress.

Asiatic cotton (Gossypium arboreum) cultivated as 'desi cotton' in India, is renowned for its climate resilience and robustness against biotic and abiotic stresses. The genome of G. arboreum is therefore, considered as a valued reserve of information for discovering novel genes or gene functions for trait improvements in the present context of cotton cultivation world-wide. In the present study, we carried out genome-wide analysis of LIM gene family in desi cotton and identified twenty LIM domain proteins (GaLIMs) which include sixteen animals CRP-like GaLIMs and four plant specific GaLIMs with presence (GaDA1) or absence (GaDAR) of UIM (Ubiquitin Interacting Motifs). Among the sixteen CRP-like GaLIMs, eleven had two conventional LIM domains while, five had single LIM domain which was not reported in LIM gene family of the plant species studied, except in Brassica rapa. Phylogenetic analysis of these twenty GaLIM proteins in comparison with LIMs of Arabidopsis, chickpea and poplar categorized them into distinct αLIM1, ßLIM1, γLIM2, δLIM2 groups in CRP-like LIMs, and GaDA1 and GaDAR in plant specific LIMs group. Domain analysis had revealed consensus [(C-X2-C-X17-H-X2-C)-X2-(C-X2-C-X17-C-X2-H)] and [(C-X2-C-X17-H-X2-C)-X2-(C-X4-C-X15-C-X2-H)] being conserved as first and/or second LIM domains of animal CRP-like GaLIMs, respectively. Interestingly, single LIM domain containing GaLIM15 was found to contain unique consensus with longer inter-zinc-motif spacer but shorter second zinc finger motif. All twenty GaLIMs showed variable spatio-temporal expression patterns and accordingly further categorized into distinct groups of αLIM1, ßLIM1, γLIM2 δLIM2 and plant specific LIM (DA1/DAR). For the first time, response of GaDA1/DAR under the influence of biotic and abiotic stresses were studied in cotton, involving treatments with phytohormones (Jasmonic acid and Abscisic acid), salt (NaCl) and wilt causing pathogen (Fusarium oxysporum). Expressions patterns of GaDA1/DAR showed variable response and identified GaDA2 as a probable candidate gene for stress tolerance in G. arboreum.

Morpho-physiological and molecular characterization of drought tolerance traits in Gossypium hirsutum genotypes under drought stress.

Drought stress is one of the major abiotic stresses affecting lint yield and fibre quality in cotton. With increase in population, degrading natural resources and frequent drought occurrences, development of high yielding, drought tolerant cotton cultivars is critical for sustainable cotton production across countries. Six Gossypium hirsutum genotypes identified for drought tolerance, wider adaptability and better fibre quality traits were characterized for various morpho-physiological and biochemical characters and their molecular basis was investigated under drought stress. Under drought conditions, genotypes revealed statistically significant differences for all the morpho-physiological and biochemical traits. The interaction (genotype × treatment) effects were highly significant for root length, excised leaf water loss and cell membrane thermostability indicating differential interaction of genotypes under control and stress conditions. Correlation studies revealed that under drought stress, relative water content had significant positive correlation with root length and root-to-shoot ratio while it had significant negative correlation with excised leaf water loss, epicuticular wax, proline, potassium and total soluble sugar content. Analysis of expression of fourteen drought stress related genes under water stress indicated that both ABA dependent and ABA independent mechanisms of drought tolerance might be operating differentially in the studied genotypes. IC325280 and LRA5166 exhibited ABA mediated expression of stress responsive genes and traits. Molecular basis of drought tolerance in IC357406, Suraj, IC259637 and CNH 28I genotypes could be attributed to ABA independent pathway. Based on physiological phenotyping, the genotypes IC325280 and IC357406 were identified to possess better root traits and LRA5166 was found to have enhanced cellular level tolerance. Variety Suraj exhibited good osmotic adjustment and better root traits to withstand water stress. The identified drought component trait(s) in specific genotypes would pave way for their pyramiding through marker assisted cotton breeding.

Severe Adult-Onset Still's Disease Mimicking Systemic Infection.

Adult-onset Still's disease (AOSD) is an uncommon entity that can mimic infection. Patients present with fever for more than 1-week, joint pain, and rash. We report a case of 22-year-old male who presented with fever, sore throat, and abdominal pain. During hospitalization, he had multiple episodes of tachycardia and tachypnea requiring mechanical ventilation. The patient had elevated white blood cell count, procalcitonin, and troponin. He was extensively investigated and diagnosed as AOSD. He responded to steroids and was discharged on day 20.