functional validation of stress responsive putative sigma factor of fi

Slide 1:

Background Finger millet ( Eleusine coracana (L)Gaertn. ) is one of the major food crop in southern parts of India. We have isolated a putative sigma factor ( Fdr-75 ) from subtracted stress cDNA library from the drought adapted species Finger millet. The BLAST search revealed that it has homology with sigma factors of Maize ( Zm SIG2), Rice (OsSIG2) and Arabidopsis ( At SIGB). In higher plants plastid encoded RNA polymerase (PEP) in chloroplast has nuclear encoded subunit called sigma factor. PEP is associated with sigma factor to bring in activation under abiotic stress conditions for transcription of photosynthetic genes in turn protecting damage occurring to photosystem. FUNCTIONAL VALIDATION OF STRESS RESPONSIVE PUTATIVE SIGMA FACTOR OF FINGER MILLET THROUGH VIRUS INDUCED GENE SILENCING APPROACH PRAKASH, V., NATARAJA KARABA K. N AND UDAYAKUMAR, M Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore, Karnataka, India, -560065, E-mail: prakashvs23@yahoo.com Hypothesis Stress responsive genes from stress adapted species will be better source for stress genes isolation. The relevance can be studied by functional genomics in heterologous system, where in the V irus I nduced G ene S ilencing (VIGS) has been standardized. Materials and Methods A specific type tobacco plant Nicotiana bethamiana (N. bent), was used as a model plant (Fig1) for studying the relevance of putative sigma factor of finger millet. The leaves of N. bent, are easy to infiltrate and it is best host system for TRV vector. Using this the functions of unknown genes can be studied through VIGS approach (Fig 2 and 3). Fig 1. N. bent, is a good model plant because its leaves are easy to infiltrate Target mRNA from plant Candidate gene Viral replication ds- RNA Dicer in plant – Chops up dsRNA dsRNA Chopped by Dicer Dicer in plant – Chops up dsRNA The plant chops up its own ds-RNA- RNA degradation Fig 3. VIGS functions via P ost T ranscriptional G ene S ilencing (PTGS), ( Lu et al., 2003) The recombinant VIGS clone developed in tobacco rattle virus (TRV) vector with sigma factor. A agro- bacterium strain GV2260 containing plasmidspTRV1andpTRV2 was grown over night in 5 ml LB media with kanamycin (50mg/L) and rifampicin (25 mg/L) at 28 o C. Incubated 5 ml O/N grown culture in to 50 ml fresh media with the above antibiotics. Grown O/N at 28 o C. Spin and resuspend the bacterial pellet in infiltration buffer to final OD of 1.0. Incubate the culture for 3 –to 4h at room temperature. Mix pTRV1 and pTRV2 in 1:1 ratio infiltrate into lower leaves of N. bent, using 1 ml needleless syringe. Controls were used to ensure that the infiltration and silencing were working. A positive control was infiltrated with (PDS) phytoene desaturase. PDS is necessary for production of chlorophyll. When PDS is silenced no chlorophyll is produced therefore making the leaves white (Fig 4). A negative control was infiltrated with a empty vector then no genes were silenced (Fig 5). Fig 4. A positive control that was infiltrated with VIGS clone PDS. When silenced, prevented chlorophyll production Fig 5. A negative control that was infiltrated with an empty vector. Other than the site of infiltration, the plant appears to be normal Results and discussion pTRV1::pTRV2 C pTRV2::SF pTRV2::SF pTRV1::pTRV2 C Silenced Silenced and maintained at 50% FC for two days Pale green leaf,leaf curling, yellowing along the margin and midrib are the phenotypic changes due to silencing of Fdr -75 (putative sigma factor) in Nicotiana banthamiana (NB) under control and moisture stress conditions. 50% FC was maintained by withholding water by gravimetric approach Upper panel: Control, Lower Panel: Stress 1 2 3 4 5 6 Northern showing the expression of putative sigma factor in N. Bent, at 15 DPI. infiltrated with pTRV2::SF. Where 1=Control, 2 to 5 = silenced plants and 6 = Mock putative sigma factor expression analysis by RT-PCR in N. bent, At 15 DPI, Infiltrated with pTRV2::SF. Where 1-= control, 2= mock and 3 to 6 = silenced with pTRV2::SF. M 1 2 3 4 5 6 Chlorophyll fluorescence Measurement of chlorophyll fluorescence by SPAD portable fluorescence meter. 1 to 5 = different infiltrated N. bent. Plants, M= mock and C= control Acknowledgements We thank Dr. Dinesh kumar, Yale University, USA for providing TRV vector to do VIGS I acknowledge AP-Netherlands Biotechnology programme for granting financial assistance to carry out the research in the Department of Crop Physiology, UAS, GKVK, Bangalore. Literature Cited Burch-Smith, T.M., Anderson, J.C., Martin, G.B., Dinesh-Kumar, S.P. 2004. Applications and advantages of virus-induced gene silencing for gene function studies in plants. The Plant Journal 39: 734-746. Lu, R., Martin-Hernandez, A.M., Peart, J.R., Malcuit, I., Baulcombe, D.C. 2003. Virus-induced gene silencing in plants. Methods 30: 296-303. Conclusions: N. bent, is the best host system for TRV vector, using which the functional relevance of heterologous genes can be studied. A stress responsive putative sigma factor transcripts were reduced in silenced plants. In silenced plants showed, appearance of Pale green leaves with reduced chlorophyll compared to mock and control. Candidate gene putative Sigma factor Clone gene in VIGS vector Virus pTRV2 in plamid vector Agrobacterium transformation Fig 2. Flow chart depicting, how VIGS is used to silence a gene in young plant (Burch-smith et al., 2004). Agro infiltrate seedling Observe phenotype and compare with control