Specific domains of plant defensins differentially disrupt colony initiation,cell fusion and calcium homeostasis in Neurospora crassa

MsDef1 and MtDef4 from Medicago spp. are small cysteine‐rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ‐core motif (GXCX_3–9C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ‐core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ‐core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane/cell wall. Using N. crassa expressing the Ca²⁺ reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca²⁺ homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca²⁺ were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ‐core motifs play important roles in their different modes of antifungal action.     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

Cytokine Network in Scrub Typhus: High Levels of Interleukin-8 Are Associated with Disease Severity and Mortality

Background

Scrub typhus, caused by Orientia tsutsugamushi, is endemic in the Asia-Pacific region. Mortality is high if untreated, and even with treatment as high as 10–20%, further knowledge of the immune response during scrub typhus is needed. The current study was aimed at comparing plasma levels of a variety of inflammatory mediators in scrub typhus patients and controls in South India in order to map the broader cytokine profile and their relation to disease severity and clinical outcome.

Methodology/Principal Findings

We examined plasma levels of several cytokines in scrub typhus patients (n = 129) compared to healthy controls (n = 31) and infectious disease controls (n = 31), both in the acute phase and after recovery, by multiplex technology and enzyme immunoassays. Scrub typhus patients were characterized by marked changes in the cytokine network during the acute phase, differing not only from healthy controls but also from infectious disease controls. While most of the inflammatory markers were raised in scrub typhus, platelet-derived mediators such as RANTES were markedly decreased, probably reflecting enhanced platelet activation. Some of the inflammatory markers, including various chemokines (e.g., interleukin-8, monocyte chemoattractant peptide-1 and macrophage inflammatory protein-1β) and downstream markers of inflammation (e.g., C-reactive protein and pentraxin-3), were also associated with disease severity and mortality during follow-up, with a particular strong association with interleukin-8.

Conclusions/Significance

Our findings suggest that scrub typhus is characterized by a certain cytokine profile that includes dysregulated levels of a wide range of mediators, and that this enhanced inflammation could contribute to disease severity and clinical outcome.     

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.     

Hydrophobicity and surface electrostatic charge of conidia of the mycoparasitic <Emphasis Type="Italic">Trichoderma</Emphasis> species

The present investigation was done to understand the fungal-fungal interactions mechanisms based on level of nonspecific adhesion of a potential fungal mycoparasite (Trichoderma) to their fungal host (Macrophomina phaseolina). The relative cell surface hydrophobicity (CSH) and cell surface electrostatic charge (CSEC) of 29 isolates of Trichoderma species, analyzed by bacterial adhesion to hydrocarbon (BATH), hydrophobic interaction chromatography (HIC), microelectrophoresis and contact angle, revealed a large degree of variability. CSH and CSEC of conidia depended on culture age, pH and temperature. Maximum CSH and CSEC were recorded in 25–28 °C range, and both declined significantly with increasing temperature. Isolate Trichoderma hazianum (Th)-23/98 expressed surface hydrophobicity at 25–28 °C and hydrophilicity at 40 °C. Surface hydrophobicity of the isolate was susceptible to various proteases (trypsin, pepsin, proteinase k and a-chymotrypsin) and inhibitors (SDS, mercaptoethanol and Triton X-100) and a significant reduction in CSH was recorded in hydrophobic conidia. Hydrophilic conidia remained more or less unaffected by such treatments. SDS-PAGE analysis of the hydrophobic and hydrophilic conidia exhibited several protein bands in the 25 to 61 kDa range. However, each protein population contained one protein that was not observed in the other population. For hydrophobic conidia, the unique protein had an apparent molecular mass of 49 kDa, while the unique protein associated with hydrophilic conidia had a molecular mass of 61 kDa. Our findings suggest that CSH and CSEC of mycoparasitic Trichoderma may contribute to non-specific adhesion on to the sclerotial surfaces of Macrophomina phaseolina that may be influenced by growth and environmental conditions.     

Bioemulsifier production by an oleaginous yeastRhodotorula glutinis IIP-30

Summary A locally isolated oleaginous strain ofRhodotorula glutinis strain IIP-30 produced a growth associated extracellular emulsifying agent while utilizing glucose during fed batch fermentation under nitrogen limitation at 30°C and pH 4. 0. Similar optimum conditions were also noted for intracellular lipid accumulation.     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

Breeding lines of the Indian mega-rice variety,MTU 1010, possessing protein kinase <Emphasis Type="Italic">OsPSTOL</Emphasis> (<Emphasis Type="Italic">Pup1</Emphasis>), show better root system architecture and higher yield in soils with low phosphorus

MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F₁ and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC₂F₁, the best backcross plant was selfed to generate BC₂F₂s. Among them, the plants homozygous for Pup1 (n?=?22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC₂F₆ generation. A total of five elite BC₂F₆ lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (>?390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.