Small RNAs in plants: recent development and application for crop improvement

The phenomenon of RNA interference (RNAi) which involves sequence specific gene regulation by small non-coding RNAs i.e small interfering RNA (siRNA) and micro RNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits & vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. Micro RNAs are key regulators of important plant processes like growth, development and response to various stresses. In spite of similarity in size (20-24nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. Micro RNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA based transgenics are much safer for consumption than those over expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of small RNAs and its application for crop improvement

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

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

<div><p>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 <i>Flammulina velutipes</i> 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 <i>F</i>. <i>velutipes</i>, 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 <i>S</i>. <i>pombe</i> 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.</p></div

Expression of HA tagged FvC5SD in <i>S</i>. <i>pombe</i>.

<p>FvC5SD was cloned in yeast expression vector pSLF173 (thiamine repressible nmt1 promoter) with triple HA tag at N terminus and transformed in S. <i>pombe</i> strain BJ7468. (a) Analysis of colony morphology of <i>S</i>. <i>pombe</i> expressing FvC5SD/ empty vector or untransformed BJ7468 cells through thiamine repressible nmt1 promoter. (b) i. Typical staining of filipin in tips and septum of <i>S</i>. <i>pombe</i> cells marked by arrows. ii. Filipin staining of S. <i>pombe</i> expressing FvC5SD/ empty vector or untransformed cells. (c) Indirect immunofluorescence staining of <i>S</i>. <i>pombe</i> expressing HA-FvC5SD/ empty vector was performed with primary antibodies against HA tag followed by incubation with FITC conjugated secondary antibodies (anti-mouse). Confocal microscopy images to show immunolocalization of FvC5SD at 60X magnification. EMM medium with or without thiamine (15μM) was used for repression and re-repression of gene respectively. Uracil (40μg/ml) was added to the EMM medium for growth of untransformed <i>S</i>. <i>pombe</i> cells (uracil auxotroph). T, thiamine; Un, untransformed.</p

Expression analysis of FvC5SD at different temperature and pH.

<p>(a) Northern blot analysis to determine the expression of FvC5SD under low pH condition.15-day-old <i>F</i>. <i>velutipes</i> mycelia was treated with oxalic acid (to bring down the pH of the medium to 3.0) for desired time points. (b) Northern blot analysis to determine the effect of temperature on FvC5SD expression. <i>F</i>. <i>velutipes</i> mycelia were grown at 23°C for 15-days followed by exposure to different temperatures (40°C and 4°C) for 6 hours. Upper panel represent autoradiogram of Northern blot analysis done with 20 μg of total RNA and after transfer, blot was probed with P³² labelled FvC5SD cDNA. Equal loading was confirmed by ethidium bromide staining of rRNA. Lower panel depicts mRNA levels on autoradiograms quantified by densitometry scanning using flour-S-(Bio-Rad) software. Error bars represents standard error between 3.0 separate biological replicates. Statistical analysis was performed using GraphPad PRISM 5 software. One way analysis of variance (ANNOVA) through Newman Kyle method was used to determine statistical difference between means of three independent groups where <b>*** = P<0.0001 and ** = P<0.001. (c)</b> Western blot analysis of total protein isolated from <i>F</i>. <i>velutipes</i> mycelia exposed to different conditions was performed using peptide- based polyclonal antibody (raised in rabbit) against FvC5SD. Un, uninduced.</p

Expression of C-5 sterol desaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance - Fig 6

<p><b>GC-MS based comparative lipid profile of <i>S</i>. <i>pombe</i> expressing (a) Empty pSLF173 vector and (b) FvC5SD</b>. Peak specific to ergosterol was detected at retention time (Rt) of 88.8; oleic acid peak was detected at Rt of 54.1. Two peaks specific to linoleic acid were detected (Rt 54.4 & 55.9) only in <i>S</i>. <i>pombe</i> expressing FvC5SD. Graph comparing the levels of different type of sterols (c) episterol, ergosta 5,7, 22, 24 tetraene and ergosterol in <i>S</i>. <i>pombe</i> expressing FvC5SD and empty vector. Graph comparing level of (d) oleic acid and linoleic acid and (e) oleic acid and stearic acid in <i>S</i>. <i>pombe</i> expressing FvC5SD with empty vector control. Area of internal standard (5α-cholest7en-3β-ol) peak was used to determine the concentration of identified lipids in the given sample. Graph represents average value of three biological replicates.</p

Gene organization of FvC5SD.

<p>(a) Southern blot analysis to determine the copy number of FvC5SD in <i>Flammulina velutipes</i> genome. Genomic DNA (10μg) was digested with the selected restriction enzymes Msc I, Sal I and Xba I. Blot was hybridized with P³² labelled FvC5SD cDNA (891bp) used as probe. Positions of different fragments of 1Kb ladder are shown on left. (b) FvC5SD gene lies upstream to oxalate decarboxylase (OXDC) in the genome. Both the genes have independent promoters. <b>(</b>c) OXDC promoter has binding sites of ECM22/ UPC2 transcription factor (-42), which are characteristic of many sterol biosynthesis genes. <i>In silico</i> analysis for detection of cis-regulatory element was done by TFsitescan (Ghosh 2000).</p

Spot dilution assay to determine tolerance of <i>S</i>. <i>pombe</i> expressing FvC5SD to pH, temperature and cell wall inhibitors.

<p>(a) Growth of cells at different incubation temperature (16°C, 40°C and 30°C). (b) Growth of cells on low pH medium created by adding oxalic acid/ HCl to bring pH of EMM medium to 3.0. (c) Growth of cells in presence of cell wall inhibitors Congo red (50 μg/ml) and Calcoflour White (40 μg/ml). Cells were grown overnight in EMM+ thiamine and washed twice with sterile milliQ water. Serial dilutions were made and indicated dilutions were spotted on EMM plate in presence or absence of thiamine (15 μg/ml) and incubated for 4 days. Uracil (40μg/ml) was added to the EMM medium for growth of untransformed <i>S</i>. <i>pombe</i> cells. All experiment were performed in three biological replicates. CR, Congo red; Cal, Calcoflour.</p

C-5 sterol desaturase characterized from various organisms.

<p>C-5 sterol desaturase characterized from various organisms.</p

<i>S</i>. <i>pombe</i> expressing FvC5SD shows ethanol tolerance.

<p>(a) Growth of cells on EMM agar presence of different concentrations of ethanol (5% and 10%.). Cells were grown overnight in EMM + thiamine and washed twice with sterile water. Serial dilutions were made and indicated dilutions were spotted on EMM plate in presence or absence of thiamine (15μM) and incubated for 4 days. (b) Growth of cells in liquid EMM media consisting of 5% ethanol in the absence of thiamine. Optical density (O.D) of the cell was monitored by measuring absorbance at 600nm. Uracil (40μg/ml) was added to the EMM medium for growth of untransformed <i>S</i>. <i>pombe</i> cells. The experiment was performed in three biological replicates.</p