Mohan Kamthan

A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis

Plant often responds to fungal pathogens by expressing a group of proteins known as pathogenesis-related proteins (PRs). The expression of PR is mediated through pathogen-induced signal-transduction pathways that are fine-tuned by phytohormones such as methyl jasmonate (MeJA). Here, we report functional characterization of an Ocimum basilicum PR5 family member (ObTLP1) that was identified from a MeJA-responsive expression sequence tag collection. ObTLP1 encodes a 226 amino acid polypeptide that showed sequence and structural similarities with a sweet-tasting protein thaumatin of Thaumatococcus danielli and also with a stress-responsive protein osmotin of Nicotiana tabacum. The expression of ObTLP1 in O. basilicum was found to be organ-preferential under unstressed condition, and responsive to biotic and abiotic stresses, and multiple phytohormone elicitations. Bacterially-expressed recombinant ObTLP1 inhibited mycelial growth of the phytopathogenic fungi, Scleretonia sclerotiorum and Botrytis cinerea; thereby, suggesting its antifungal activity. Ectopic expression of ObTLP1 in Arabidopsis led to enhanced tolerance to S. sclerotiorum and B. cinerea infections, and also to dehydration and salt stress. Moreover, induced expression of the defense marker genes suggested up-regulation of the defense-response pathways in ObTLP1-expressing Arabidopsis upon fungal challenge. Thus, ObTLP1 might be useful for providing tolerance to the fungal pathogens and abiotic stresses in crops.

Expression of a fungal sterol desaturase improves tomato drought tolerance, pathogen resistance and nutritional quality

Crop genetic engineering mostly aims at improving environmental stress (biotic and abiotic) tolerance as well as nutritional quality. Empowering a single crop with multiple traits is highly demanding and requires manipulation of more than one gene. However, we report improved drought tolerance and fungal resistance along with the increased iron and polyunsaturated fatty acid content in tomato by expressing a single gene encoding C-5 sterol desaturase (FvC5SD) from an edible fungus Flammulina velutipes. FvC5SD is an iron binding protein involved in ergosterol biosynthesis. Morphological and biochemical analyses indicated ≈23% more epicuticular wax deposition in leaves of transgenic plants that provides an effective waterproof barrier resulting in improved protection from drought and infection by phytopathogenic fungus Sclerotinia sclerotiorum. Furthermore, the transgenic fruits have improved nutritional value attributed to enhanced level of beneficial PUFA and 2-3 fold increase in total iron content. This strategy can be extended to other economically important crops.

Quantitative proteomics and metabolomics approaches to demonstrate N-acetyl-D-glucosamine inducible amino acid deprivation response as morphological switch in Candida albicans.

Candida albicans is a life threatening polymorphic pathogen for immunocompromised patients, causing superficial as well as invasive systemic diseases. The mucosal membranes of the host, which are the primary sites of its infection, are rich in amino sugars like N-acetylglucosamine (GlcNAc). GlcNAc is also one of the potent inducers of morphological transition, an important pathogenic trait of C. albicans. We thus performed proteomic analysis on total soluble proteins to identify the molecules involved in this response. Proteomic analysis using 2-DE demonstrated reproducible upregulation of 36 spots from a total of 585 matched spots. Mass spectroscopy (MS/MS) analyses of upregulated proteins revealed that carbohydrate and amino acid metabolism were the most prominent functional classes. Metabolite profiling using GC-MS allowed a quantitative comparison of 58 metabolites in GlcNAc or glucose grown cells. We observed a significant decrease in the intracellular amino acid pool of GlcNAc grown cells. Moreover, GlcNAc induces both bZIP transcription factor (GCN4) and eIF2α kinase (GCN2) which are responsible for the activation of general amino acid control response in C. albicans. Inactivation of these genes blocks GlcNAc induced morphogenesis. Altogether these results suggest that amino acid starvation is the morphogenetic signal in presence of GlcNAc in C. albicans.

Mapping of functional domains and characterization of the transcription factor Cph1 that mediate morphogenesis in Candida albicans.

Cph1, a transcription factor of the Mitogen Activated Protein (MAP) kinase pathway, regulates morphogenesis in human fungal pathogen Candida albicans. Here, by following a systemic deletion approach, we have identified functional domains and motifs of Cph1 that are involved in transcription factor activity and cellular morphogenesis. We found that the N-terminal homeodomain is essential for the DNA binding activity; however, C-terminal domain and polyglutamine motif (PQ) are indispensable for the transcriptional activation function. Complementation analysis of the cph1Δ null mutant using various deletion derivatives revealed functional significance of the N- and C-terminal domains and PQ motif in filamentation process, chlamydospore formation and sensitivity to the cell wall interfering compounds. Genome-wide identification of the Cph1 binding site and quantitative RT-PCR transcript analysis in cph1Δ null mutant revealed that a number of genes which are associated with the filamentous growth, maintaining cell wall organization and mitochondrial function, and the genes of the pH response pathway are the transcriptional targets of Cph1. The data also suggest that Cph1 may function as a positive or negative regulator depending on the morphological state and physiological conditions. Moreover, differential expression of the upstream MAP kinase pathway genes in wild type and cph1Δ null mutant indicated the existence of a feedback regulation.

Characterization of a Putative Spindle Assembly Checkpoint Kinase Mps1, Suggests Its Involvement in Cell Division, Morphogenesis and Oxidative Stress Tolerance in Candida albicans

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus.

Upregulation of galactose metabolic pathway by N-acetylglucosamine induced endogenous synthesis of galactose in Candida albicans

N-Acetylglucosamine (GlcNAc) is an important signaling molecule that plays multiple roles in Candida albicans. Induction of galactose metabolic pathway by GlcNAc is an intriguing aspect of C. albicans biology. In order to investigate the role of galactose metabolic genes (GAL genes) in presence of GlcNAc, we created knockouts of galactokinase (GAL1) and UDP galactose epimerase (GAL10) genes. These mutants failed to grow on galactose and also showed lower growth rate in presence of GlcNAc. Interestingly, expression of GAL genes in presence of GlcNAc was higher in gal1Δ strain relative to that of wild type strain. Moreover, no GlcNAc induced upregulation of GAL genes was observed in the gal10Δ strain suggesting that UDP galactose epimerase is essential for GlcNAc induced activation of GAL genes. GlcNAc induced expression of GAL genes was also investigated in GlcNAc metabolic pathway triple mutant N216 (hxk1Δ nag1Δ dac1Δ). Interestingly, in this mutant the GAL genes are neither induced nor repressed and remain derepressed as found on a neutral carbon source such as glycerol, suggesting that catabolism of GlcNAc play an important role in the expression of GAL genes. GC/MS analysis of derivatized metabolites revealed a significant accumulation of galactose in the gal1Δ strain while no galactose was detected in gal10Δ and N216 strain. Solution-state NMR spectroscopy using N-acetyl-¹³C₁-glucosamine confirmed the flow of ¹³C label from GlcNAc to galactose. Thus, internal galactose synthesized via UDP galactose pathway from GlcNAc metabolites acts as the inducer of GAL genes in presence of GlcNAc.

A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter.

Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of 45Ca2+, heat stability and Ca2+ dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca2+ binding transcriptional regulators.

Expression of C-5 sterol desaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance.

Bioethanol is an environment friendly and renewable source of energy produced by the fermentation of agricultural raw material by a variety of microorganisms including yeast. Obtaining yeast strains that are tolerant to stresses like high levels of ethanol and high temperature is highly desirable as it reduces cost and increases yield during bioethanol production. Here, we report that heterologous expression of C-5 Sterol desaturase (FvC5SD)—an ergosterol biosynthesis enzyme from an edible mushroom Flammulina velutipes in fission yeast, not only imparts increased thermotolerance but also tolerance towards high ethanol concentration and low pH. This tolerance could be attributed to an increase of ≈1.5 fold in the level of ergosterol and oleic acid (C-18 unsaturated fatty acid) as analysed by gas chromatography- mass spectrometry. FvC5SD is a membrane localized iron binding enzyme that introduces double bond at C-5 position into the Δ7-sterol substrates to yield Δ5, 7- sterols as products. In F. velutipes, FvC5SD transcript was observed to be upregulated by ≈5 fold under low pH condition and by ≈ 9 folds and ≈5 fold at 40°C and 4°C respectively when compared to normal growth temperature of 23°C. Besides, susceptibility to cell wall inhibiting drugs like Congo red and Calcoflour white was also found to increase in FvC5SD expressing S. pombe strain. Alteration in membrane sterol and fatty acid composition could also lead to increase in susceptibility to cell wall inhibiting drugs. Thus, this study has immense industrial application and can be employed to ensure competitiveness of fermentation process.