Coat Protein Gene based Characterization of Cucumber Mosaic Virus Isolates Infecting Banana in India

Banana plants showing typical yellow stripes on leaves as symptoms, in addition to leaf distortion and stunting of plant were collected from Karnataka (KAR), Maharashtra (MH) and Uttar Pradesh (UP) in India. The causal agent was identified as Cucumber mosaic virus (CMV) on the basis of transmission electron microscopy and reverse transcription polymerase chain reaction (RT-PCR). Complete coat protein (CP) gene of all isolates were amplified using gene specific primers for coat protein (CP), followed by cloning into desired cloning vector for sequencing. Sequenced region were found containing complete single open reading frame of 657 nucleotides, potentially coding 219 amino acids. Sequence analysis of CP gene showed 93%-98% (at nucleotide) and 94%-99% (at amino acid) sequence identity between all three Indian isolates. On comparing CP gene sequences of CMV KAR, CMV MH and CMV UP with CMV P isolate (Physalis minima); we got 94%, 99% and 96% identity respectively. High degree identity at nucleotide level between these isolates of banana and Physalis minima (a weed) suggest that Physalis minima could be an alternate host of CMV banana. Phylogenetic analysis of nucleotide along with amino acid sequence of coat protein gene revealed that all our isolates belong to IB subgroup.  In short, it appears that there occurs a high incidence of CMV infecting banana belonging to IB subgroup in most parts of Indian subcontinent.Key words: Banana, CMV, CP gene, RT-PC

Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux

Objectives: To investigate the role of transmembrane segment 11 (TMS11) of Candida albicans drug resistance protein (Cdr1p) in drug extrusion. Methods: We replaced each of the 21 putative residues of TMS11 with alanine by site-directed mutagenesis. The Saccharomyces cerevisiae AD1-8u− strain was used to overexpress the green fluorescent protein tagged wild-type and mutant variants of TMS11 of Cdr1p. The cells expressing mutant variants were functionally characterized. Results: Out of 21 residues of TMS11, substitution of seven residues, i.e. A1346G, A1347G, T1351A, T1355A, L1358A, F1360A and G1362A, affected differentially the substrate specificity of Cdr1p, while 14 mutants had no significant effect on Cdr1p function. TMS11 projection in an α-helical configuration revealed with few exceptions (A1346 and F1360), a distinct segregation of mutation-sensitive residues (A1347, T1351, T1355, L1358 and G1362) towards the more hydrophilic face. Interestingly, mutation-insensitive residues seem to cluster towards the hydrophobic side of the helix. Competition of rhodamine 6G efflux, in the presence of excess of various substrates in the cells expressing native Cdr1p, revealed for the first time the overlapping binding site between azoles (such as ketoconazole, miconazole and itraconazole) and rhodamine 6G. The ability of these azoles to compete with rhodamine 6G was completely lost in mutants F1360A and G1362A, while it was selectively lost in other variants of Cdr1p. We further confirmed that fungicidal synergism of calcineurin inhibitor FK520 with azoles is mediated by Cdr1p; wherein in addition to conserved T1351, substitution of T1355, L1358 and G1362 of TMS11 also resulted in abrogation of synergism. Conclusions: Our study for the first time provides an insight into the possible role of TMS11 of Cdr1p in drug efflux

Synthesis and mechanistic studies of diketo acids and their bioisosteres as potential antibacterial agents

A series of diketo esters and their pertinent bioisosteres were designed and synthesized as potent antibacterial agents by targeting methionine amino peptidases (MetAPs). In the biochemical assay against purified MetAPs from Streptococcus pneumoniae (SpMetAP1a), Mycobacterium tuberculosis (MtMetAP1c), Enterococcus faecalis (EfMetAP1a) and human (HsMetAP1b), compounds 3a, 4a and 5a showed more than 85% inhibition of all the tested MetAPs at 100 μM concentration. Compounds 4a and 5a also exhibited antibacterial potential with MIC values 62.5 μg/mL (S. pneumoniae), 31.25 μg/mL (E. faecalis), 62.5 μg/mL (Escherichia coli) and 62.5 μg/mL (S. pneumoniae), 62.5 μg/mL (E. coli), respectively. Moreover, 5a also significantly inhibited the growth of multidrug resistant E. coli strains at 512 μg/mL conc., while showing no cytotoxic effect towards healthy CHO cells and thus being selected. Growth kinetics study showed significant inhibition of bacterial growth when treated with different conc. of 5a. TEM analysis also displayed vital damage to bacterial cells by 5a at MIC conc. Moreover, significant inhibition of biofilm formation was observed in bacterial cells treated with MIC conc. of 5a as visualized by SEM micrographs. Interestingly, 5a did not cause an alteration in the hemocyte density in Galleria mellonella larvae which is considered in vivo model for antimicrobial studies and was non-toxic up to a conc. of 2.5 mg/mL

MicroRNAs as biomarkers in tomato leaf curl virus (ToLCV) disease

MicroRNAs are ∼21- 25 nt long RNA species that are critical regulators of transcriptome across theeukaryotes. Growing number of evidences clearly supports their involvement in plant leaf development. ToLCV infection severely affects the morphology of mature tomato leaves. To investigate the mechanism underlying the virus- host interaction, we focussed our studies on expression of microRNAs and the irrespective targets under normal and ToLCV infection. We have cloned Myb33, ARF4 homolog, Argonaute1, Apetala2, SBP transcription factor and RBOH from tomato and checked their expression by RT-PCR. Our work suggests that miR159 is upregulated while miR164 and miR171 are downregulated under viral infection. Our studies shed light on the impact of ToLCV infection on host transcriptome

In silico analysis reveals that several tomato microRNA/microRNA∗ sequences exhibit propensity to bind to tomato leaf curl virus (ToLCV) associated genomes and most of their encoded open reading frames (ORFs)

Tomato leaf curl virus (ToLCV) is a member of family geminiviridae that constitute rapidly emerging group of phytopathogens posing threat to a large number of vegetable crops worldwide. Three different genomes are found to be associated with ToLCV viz., DNA-A, DNA-B and beta satellite DNA. MicroRNAs (miRs) are known to govern several fundamental processes in eukaryotes, including basal defense mechanisms. In animals, it has been demonstrated that certain host miRs prevent viral establishment by directly interfering with pathogen replication or by binding to viral transcripts. However, in spite of the existence of huge families of phytopathgenic viruses, no such mechanism has been observed in plants. In the present study, we performed in silico analysis to investigate whether tomato encoded miR/miR∗ sequences possess any potential to bind to viral genome and/or encoded ORFs. We observed that different sequences can bind to ToLCNDV DNA-A, ToLCNDV DNA-B and ToLCNDV associated DNA beta genomes and most of the encoded ORFs. Interestingly, our analysis revealed that several miR species could similarly target genome and ORFs of ToLCNDV suggesting novel role of miR∗ in host defense response. This observation holds much importance as miR molecules are presently thought to follow degradation pathway and are not assigned with any function, till now. Moreover, we could predict targets for these miR∗ sequences that are generally involved in plant metabolism. Overall, these results shed light on new paradigm of intricate host-pathogen interactions via miRNA pathway

Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India

Abstract Mercury, which is ubiquitous and recalcitrant to biodegradation processes, threatens human health by escaping to the environment via various natural and anthropogenic activities. Non-biodegradability of mercury pollutants has necessitated the development and implementation of economic alternatives with promising potential to remove metals from the environment. Enhancement of microbial based remediation strategies through genetic engineering approaches provides one such alternative with a promising future. In this study, bacterial isolates inhabiting polluted sites were screened for tolerance to varying concentrations of mercuric chloride. Following identification, several Pseudomonas and Klebsiella species were found to exhibit the highest tolerance to both organic and inorganic mercury. Screened bacterial isolates were examined for their genetic make-up in terms of the presence of genes (merP and merT) involved in the transport of mercury across the membrane either alone or in combination to deal with the toxic mercury. Gene sequence analysis revealed that the merP gene showed 86–99% homology, while the merT gene showed >98% homology with previously reported sequences. By exploring the genes involved in imparting metal resistance to bacteria, this study will serve to highlight the credentials that are particularly advantageous for their practical application to remediation of mercury from the environment

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus indica cv. K2, by constitutive and inducible expression of tobacco osmotin

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought