RNA interference and crop protection against biotic stresses.

RNA interference (RNAi) is a universal phenomenon of RNA silencing or gene silencing with broader implications in important physiological and developmental processes of most eukaryotes, including plants. Small RNA (sRNA) are the critical drivers of the RNAi machinery that ensures down-regulation of the target genes in a homology-dependent manner and includes small-interfering RNAs (siRNAs) and micro RNAs (miRNAs). Plant researchers across the globe have exploited the powerful technique of RNAi to execute targeted suppression of desired genes in important crop plants, with an intent to improve crop protection against pathogens and pests for sustainable crop production. Biotic stresses cause severe losses to the agricultural productivity leading to food insecurity for future generations. RNAi has majorly contributed towards the development of designer crops that are resilient towards the various biotic stresses such as viruses, bacteria, fungi, insect pests, and nematodes. This review summarizes the recent progress made in the RNAi-mediated strategies against these biotic stresses, along with new insights on the future directions in research involving RNAi for crop protection.

Identification of suitable reference genes for expression profiling studies using qRT-PCR in an important insect pest, Maruca vitrata.

BACKGROUND: Maruca vitrata is one of the potential insect pests that cause devastating losses to legume cultivation worldwide. Gene functional studies facilitate dissecting the molecular mechanisms underlying the infection process and enable devising appropriate molecular strategies to control this insect pest. Expression profiling using quantitative real-time PCR (qRT-PCR) provides insights into the functional characterization of target genes; however, ideal reference genes should be deployed in such studies to nullify the background variation and improve the accuracy of target gene expression. An ideal reference gene should have a stable expression across developmental stages, biological conditions, tissues, or experimental conditions. METHODS AND RESULTS: Given this, the stability of eight candidate reference genes was evaluated in M. vitrata at different developmental stages, diets, and sexes by qRT-PCR method, and the data was analyzed using four independent algorithms, namely GeNorm, NormFinder, BestKeeper, and ΔCt, and one comprehensive algorithm, RefFinder. CONCLUSION: The analysis showed that RP49 and RPL13 were the best suitable reference genes for studying target gene expression at different developmental stages. Further, the study identified RP49 and RPL24, and GAPDH and RPL24 as the ideal reference genes in M. vitrata fed with different diets and sexes, respectively. The reference genes reported in the present study will ensure the accuracy of target gene expression, and thus, will serve as an important resource for gene functional studies in M. vitrata.

Genetic transformation of legumes: an update.

KEY MESSAGE: This review summarizes the recent advances in legume genetic transformation and provides an insight into the critical factors that play a major role in the process. It also sheds light on some of the potential areas which may ameliorate the transformation of legumes. Legumes are an important group of dicotyledonous plants, highly enriched in proteins and minerals. Majority of the legume plants are cultivated in the arid and semi-arid parts of the world, and hence said to be climate resilient. They have the capability of atmospheric nitrogen fixation and thus play a vital role in the ecological sphere. However, the worldwide production of legumes is somehow not up to the mark and the yields are greatly affected by various biotic and abiotic stress factors. Genetic engineering strategies have emerged as a core of plant biology and remarkably facilitate the crop improvement programmes. A significant progress has been made towards the optimization of efficient transformation system for legume plants over the years but this group is still underutilized in comparison to other crops. Among the variety of available DNA delivery systems, Agrobacterium-mediated and particle bombardment have been primarily deployed for optimization and trait improvement. However, recalcitrance and genotype-dependence are some of the major bottlenecks for successful transformation. In this context, the present review summarizes the advances taken place in the area of legume transformation and provides an insight into the present scenario. The challenges and future possibilities for yield improvement have also been discussed.

p-Benzoquinone-induced aggregation and perturbation of structure and chaperone function of α-crystallin is a causative factor of cigarette smoke-related cataractogenesis.

Cigarette smoking is a significant risk factor for cataract. However, the mechanism by which cigarette smoke (CS) causes cataract remains poorly understood. We had earlier shown that in CS-exposed guinea pig, p-benzoquinone (p-BQ) derived from CS in the lungs is carried by the circulatory system to distant organs and induces various smoke-related pathogeneses. Here, we observed that CS exposure caused accumulation of the p-BQ-protein adduct in the eye lens of guinea pigs. We also observed accumulation of the p-BQ-protein adduct in resected lens from human smokers with cataract. No such accumulation was observed in the lens of never smokers. p-BQ is a strong arylating agent that forms Michael adducts with serum albumin and haemoglobin resulting in alterations of structure and function. A major protein in the mammalian eye lens is αA-crystallin, which is a potent molecular chaperone. αA-crystallin plays a key role in maintaining the integrity and transparency of the lens. SDS-PAGE indicated that p-BQ induced aggregation of αA-crystallin. Various biophysical techniques including UV-vis spectroscopy, fluorescence spectroscopy, FT-IR, bis-ANS titration suggested a perturbation of structure and chaperone function of αA-crystallin upon p-BQ modification. Our results indicate that p-BQ is a causative agent involved in the modification of αA-crystallin and pathogenesis of CS-induced cataract. Our findings would educate public about the impacts of smoking on eye health and help to discourage them from smoking. The study might also help scientists to develop new drugs for the intervention of CS-induced cataract at an early stage.

Subcutaneous Dirofilaria repens infestation in non-descript canines.

Dirofilaria repens is a filarial nematode which cause subcutaneous dirofilariosis. Dogs, foxes and cats are the definitive hosts and principal reservoirs of the parasite. We report cases of D. repens infestation in non-descript canines from Goa, India. The nematodes were enclosed within fibrous capsule or freely present in the tunica vaginalis of the testes, in sub-cutaneous tissue of foreleg and body cavity. The parasite showed well-developed thick multilayered cuticular ridges in the outermost layer, followed by transverse smooth muscles striations.

Spectroscopic studies of the unfolding of a multimeric protein α-crystallin.

α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol(-1) on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol(-1) on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.

The N-terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRS(apm)) is a monomer in solution.

Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRSapm) was the first reported aminoacyl-tRNA synthetase of viral origin. The previous crystal structure of TyrRSapm showed a non-canonical orientation of the dimer conformation and the CP1 domain, responsible for dimer formation, displays a major modification of a motif structurally conserved in other TyrRS structures. An earlier study reported that Bacillus stearothermophilus N-terminal TyrRS exists as a dimer under native conditions. N-terminal TyrRSapm (ΔTyrRSapm, 1-234 aa) was constructed to remove the C-terminal anticodon-binding domain. Here we show by Ferguson plot analysis and analytical ultracentrifugation that ΔTyrRSapm exists as a monomer and contains a disulfide-bridge. The ΔTyrRSapm loses the ability to bind tRNA(Tyr), however it remains active in pyrophosphate exchange with similar ligand dissociation constants as the full-length enzyme.

Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: fluorescence correlation spectroscopy and femtosecond solvation dynamics.

Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (D(t)) of the αA-crystallin oligomer (mass ~800 kDa) increases from ~35 µm(2) s(-1) to ~68 µm(2) s(-1). This corresponds to a decrease in hydrodynamic radius (r(h)) from ~6.9 nm to ~3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ~80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ~20 kDa). The steady state emission maximum and average solvation time (<τ(s)>) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.

Cigarette smoke induces p-benzoquinone-albumin adduct in blood serum: Implications on structure and ligand binding properties.

Earlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amounts of p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation and oxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-protein adducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum of human smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays a very important role in binding and transport of a variety of ligands, including fatty acids and drugs. We show in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as ɛ-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubated with p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60, the number of p-BQ incorporated is 40. The formation of HSA-p-BQ adduct has been demonstrated by absorption spectroscopy, MALDI-MS and MALDI-TOF-TOF-MS analyses. Upon complexation with p-BQ, the secondary structure and conformation of HSA are altered, as evidenced by steady state and time-resolved fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differential scanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of its ligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one of the reasons why transport and distribution of lipids and drugs are impaired in smokers.

An FCS study of unfolding and refolding of CPM-labeled human serum albumin: role of ionic liquid.

The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in D(t) and decrease in R(h). This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ~3.6 ± 0.7, ~29 ± 4.5, and 133 ± 23 µs. The faster components (~3.6 ± 0.7 and ~29 ± 4.5 µs) are ascribed to chain dynamics of the protein, while the slowest component (133 µs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (~5.1 ± 1, ~43.5 ± 2.8, and ~311 ± 2.3 µs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 µs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ~5 ± 1, ~41 ± 10, and ~230 ± 45 µs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL.