Sunayana Rathi

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91 9435203640

Assam Agricultural University, Jorhat, India

Martin Luther University Halle (Saale), Germany

Leibniz Institute of Plant Biochemistry Halle (Saale), Germany

Topic: Characterization of enzymes putatively involved in pathogen-induced sesquiterpene biosynthesis of Medicago truncatula. Characterization of enzymes (CYP-I, CYP-II and UGT) putatively involved in pathogen-induced sesquiterpene biosynthesis of Medicago truncatula (A17 ecotype) was done. These genes are induced in M. truncatula roots after inoculation with A. euteiches as confirmed by qRT-PCR (Heena Yadav). I also started with qRT-PCR from Medicago truncatula roots after 2 hours of infection with Aphanomyces euteiches from independent aeroponic experiment and could validate only one gene (CYP-IIinc.) which was significantly upregulated (Fig. 1). Later on from the help of Dr. David Nelson, a pioneer in Cytochrome P450, from the University of Tennessee Health Science Centre, Madison, we could get the complete sequence of CYP-II, which was later used from R108 ecotype of Medicago truncatula for construction of different combinations of constructs for Golden Gate Cloning approach. Yeast transformation: Gas chromatography–mass spectrometry (GC-MS) for metabolite analysis Golden Gate Cloning approach was followed for getting different combinations of constructs from CYP-I, CYP-IIinc. and UGT gene to be transformed in yeast (Table 1). Yeast strains were transformed with individual plasmids bearing preceding enzymes of the pathway under the control of GAL promoters. To confirm the functionality of these plasmids the transformed strains were grown in culture tubes containing 5 mL minimal medium and 20 g/L galactose as carbon source. The metabolites were analyzed with an Agilent 6890 Series gas chromatograph and an Agilent 5973 Network mass detector controlled by a GC-MSD Chemstation (G 1701EA E.02.02.1431). All the transformed strains were able to synthesize sesquiterpenes. Along with other sesquiterpenes produced, Himachalol was clearly the major product. However, the amount of himachalol was significantly reduced in case of CYP-II, but no difference was observed in other cases. Figure 2a, 2b, 2c, 2d and 2f illustrates the range of sesquiterpene products produced by different constructs. The different constructs of complete CYP-II gene (Table 1) were also transformed in yeast and metabolites analyzed in GC-MS but there was no major difference was observed as compared to the CYP-II inc. gene (Figure 2e, 2g, 2h). Subcellular localization of genes of interest Golden Gate Cloning approach was followed for getting different combinations of constructs from CYP-I, CYP-IIinc., CYP-II and UGT gene to be transformed in protoplast and Agrobacterium (Table 2). Protoplast transformation: An E. coli stock containing the plasmid was isolated in a larger concentration by PEG precipitation and transformed into protoplasts for transient expression. The GFP signal could be localized in two independent transformations, especially in the cytosol, but also in the nucleus for the gene UGT while for the two cytochrome P450 genes (CYP-I and CYP-IIinc. and CYP-II) GFP signal localized in ER (Fig. 3). Expression in N. benthamiana For CYP-I, CYP-IIinc, CYP-II and UGT, a subcellular localization was done by a fusion of the open reading frame to GFP and an expression in N. benthamiana, to get a further hint about their functionality. All the constructs could be successfully co-expressed with GFP (Fig. 4). The GFP signal shown in Fig. 4 suggests ER localization of CYP-I, CYP-IIinc. and CYP-II, while for UGT it suggests cytosol yet the interpretation of the pictures is not precise, due to a slight GFP signal in the nucleus, that cannot be explained. To confirm the results, a western blot analysis was performed with the infiltrated tissue to test if the gene of interest fused to GFP was really expressed in the infiltrated N. benthamiana leaves, and the outcome indicates that there is no free GFP. But there are strong bands which appears just below 26 kDa, which does not represents GFP bands and we are not sure of what these bands are as these bands are also present in positive control (only GFP) and not in negative control (only protoplast). In planta expression: M. truncatula transient root transformation mediated by Agrobacterium rhizogenes The selected candidate gene (CYP-IIinc.) was analyzed by knock-down or knock-out of the corresponding genes. The method for gene silencing (amiRNA) was used, to knock down the function of the gene of interest. The transformation of Agrobacterium rhizogenes (strain Arqua1) was performed by electroporation. Transformed A. rhizogenes was cultivated on Yeast-Tryptone plates with selection antibiotics (Spectinomycin) and treated with 200 μl Acetosyringone (1 mM/mL). Then Agrobacteria were plated and plates were closed with parafilm and incubated in dark at 28°C for 2 days. In parallel M. truncatula seeds (R108 ecotype) were sterilized and plated on 0.7 % plant agar. The lids of the petri dishes were filled with 1 ml sterile H2O, closed with Leucopore and kept for 4 days at 4°C and then for 2 days at 12°C in the dark. The seedlings were freed from the seed coat and 3 mm of the root tip was cut with a scalpel. The cut root of the seedling was then dipped through the bacterial lawn and was directly transferred to square plates containing Fahraeus medium with spectinomycin. Only 2/3 of the plates were filled with medium, so that plant shoots had space to develop in the upper section of the plate. 7 plants were transferred on each plate. As reference plants for the amiRNA analysis, plants were transformed with an empty vector (EV), not containing any target gene sequence, so that any effect induced by the transformation process could be excluded for later data analysis. Also, seedlings with plates containing Fahraeus medium with and without spectinomycin was also kept as control to see the effect of spectinomycin on plants. The sealed plates were cultivated in a climate chamber for 1 week (16 h light period, 90 μM light intensity, 20°C day temperature, 17°C night temperature). Afterwards plants were transferred to a climate chamber (with 24°C day and 20°C night temperature) for 3 weeks. Two weeks after seedling transfer, the plates were watered with 2 ml sterile H2O. But we observed that the plants without spectinomycin grew well but all other plants with empty vector as well as the constructs did not grew well, that is root system was not developed at all but shoots were growing. From this it can be concluded that spectinomycin is hampering the root growth in the plants and the experiment was unsuccessful. Later on the healthy plants were shifted to Fahraeus medium plates but without spectinomycin and it was observed that the roots started growing and when observed in fluorescence microscopy the roots were transformed and those plants were potted in pots for further experiments just for trial as the number of plants were very less for experimental purpose and this experiment was repeated this time by growing seeding after transformation in Fahraeus medium plates but without spectinomycin and the experiment will be continued by Heena Yadav for selection of plants containing transgenic roots and infection by A. euteiches and determination of infection rate by staining and qRT-PCR. Conclusion:  Two genes (CYP-I & UGT) under study could not be validated from independent aeroponics, only one gene (CYP-II) was significantly up-regulated.  Localization studies seems to show that CYP-II and CYP-I located in ER while UGT located in nucleus and cytoplasm.  Western blot was done to exclude a putative effect of free GFP.  Significantly reduced amount of one of the main product (himachalol) as compared to positive control was observed in construct with CYP-II.  CYP-II was considered as target for gene silencing, but the experiment was not successful due to the effects of spectinomycin which hampered the root growth.

Assistant Professot Department of Biochemistry & Agricultural Chemistry Assam Agricultural University, Jorhat-785013, Assamm India