Candidate genes for disease inducible Promoter:

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members of GSL family of genes in wheat have some role to play in resistance against fungal pathogen F. graminearum. In terms of plant defence in arabidopsis At-GSL5, At-GSL-6 and At-GSL-11 have shown their involvement. It is reported that the transcript levels of these three arabidopsis genes increases in the leaves upon inoculation of Blumeria graminis spores. The functional analysis of At-GSL5 by gene knock down showed that the Glucan Synthase encoded by this gene is required in the papillary callose formation and also in the salicylic acid formation pathway. The plant growth was slightly stunted in general in the knock downs.

The growth of the several species of powdery mildews and Perrenospora parasitica was ceased in At-GSL5 knock downs. This is some thing reverse to our results. The results found with At-GSL5 reject the commonly found model of the involvement of callose in disease resistance (Jacobs et al., 2003, Nishimura et al., 2003, Enns et al., 2005 and Dong et al., 2005) while our results support the model.

There are a lot of things to be found about the GSL gene family of plants. Results of functional analysis of the some members of this family in Arabidopsis indicate their involvement in cell division, male gametogenesis and plant development and disease resistance. In wheat, according to our knowledge this is the first report about functional analysis of any members of GSL gene family. Our results indicate the involvement of this family both towards disease resistance and the plant development. Future studies with other genes of the family and further details of the presently discussed genes will uncover what this family offers for wheat in general.

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expressed genes upon disease infection some studies have reported the micro array analysis using c-DNA from the control and infected plants at different time points. These experiments revealed the up-regulation of verious biotic and abiotic stress related genes in wheat (Pritsch et al., 2000 , Kruger et al., 2003, Kong et al., 2003 and Boddu et al., 2005).

In the present experiment 10 contigs were selected from barley gene chip reported by Eichmann et al., 2006 and provided by Prof. Dr. W. Schaeffer (University of Hamburg, Germany). These ten sequences were used to find out the homologous genes/sequences in wheat by BLAST search in the genomic data base. Aditionally two ESTs were selected from Kruger et al., 2002 which are highly expessed upon F. graminearum infection at early time points in semi-resistant wheat genotype Sumai-3. The homologous sequences found in wheat mainly were reported as pathogenesis and defence related genes. All the twelve genes were tested at first with rt-PCR using non infected control; mock inoculated control and F.

graminearum inoculated wheat spikes (susceptible genotype Florida) at 12 hai, 24 hai, 48 hai, 72 hai, 96 hai, 120 hai and 144 hai. The results showed the over expression of all the 10 wheat genes on F.graminearum inoculation but there was no reduction or increase in both of the genes reported from Sumai-3. May be these two genes which are up-rregulated at early time points are the part of ―Sumai-3‖ semi resistance mechanism against FHB compared to

―Florida‖.

Out of the 10 genes 4 genes were selected for further analysis with Northern Blot analysis.

The rest of the 6 genes were discarded based on relatively low expression levels or the expression on mock inoculation along with up-regulation on F. graminearum infection. The results of the Northern Blot analysis showed that all the four genes are up-regulated upon F.

graminearum infection and the maximum level of the transcript is achieved at 72 hai and 96 hai which keep on decreasing till 144hai. Genes/sequences ―pk0023‖ and ―oxalate oxidase precursor gene‖ showed no expression on the mock control and till 24 hai and 48 hai respectively of F. graminearum while ―Ta-Chitinase‖ and ―PDR like ABC transporter gene‖

showed some back ground activity on mock control. ―pk0023” and ―Ta-Chitinase‖ showed high expression as compared to the ―oxalate oxidase precursor gene‖ and ―PDR like ABC transporter gene‖. Depending upon these results it can be said that ―pk0023” and ―Ta-Chitinase‖ are the best candidates for disease inducible promoter although some background activity is present at ―Ta-Chitinase‖. When absolutely no expression is needed under normal conditions with high expression under disease then ―pk0023” is recommended. ―Oxalate oxidase precursor gene‖is although completely inducible but the expression level is too low under disease as well.

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CONCLUSION AND OUTLOOK

The goal of this study was to analyse the role of some endogenous and exogenous genes in wheat disease resistance so that a future strategy can be devised for the development of a genotype that can be resistant against more than one disease pathogens. For this purpose a couple of antifungal genes (HarChit and HarCho) from Trichoderma harzianum were co-transformed into wheat genotype ―Florida‖ under the control of constitutive as well as inducible promoter. 4 lines showed the co-expression of both the genes under constitive promoter while five lines showed co-expression under disease/stress inducible promoter.

While 2 lines and one line showed single gene integration under constitutive and inducible promoter respectively. The infection assays performed with E. graminis f.sp. tritici and F.

graminearum showed a reduction in diease development and spread of upto 75% compared to the non transgenic control. From here it can be concluded that these two anti-fungal genes do have a role in the resistance against fungal diseases in wheat and the transgenic lines developed in this project can be used in the breeding programmes (after pathological testing in the field) for the development of disease resistant cultivars.

As a second strategy three members of the Glucan Synthase Like (GSL) family of genes were tried to knock down for the function analysis with respect to fungal diseases. RNAi constructs were made for all the three genes at different sizes of the siRNA forming fragment of the constructs. Transgenic plants containing RNAi constructs with only short siRNA forming fragments could be recovered. Two out of four transgenic lines showed partial knock down of Ta-GSL3 and Ta-GSL8. Pathological analysis of all the four lines showed that only two lines with partial knock of genes showed an increase in the susceptibility to F. graminearum compared to non transgenic. From these results it can be concluded that perhaps short siRNA forming DNA fragments can only partially knock down a gene in wheat while larger siRNA forming DNA fragments in the range of 230 bp can completely knock out a gene. The complete knock down is toxic for wheat plant development. The increments in the susceptibility of the partial knock downs indicate that these genes are not only have a role in plant development but also the play a role in the resistance against fungal diseases. In future experiments it will be interesting to see if the over expression of these genes can enhance resistance against fungal diseases.

The non/less recovery of transgenic plants with RNAi cassttes under constitutive promoter gave rise to the decision of finding out some genes which can be used in the identification of disease inducible promoter in future. We found out 4 disease inducible genes whose promoter can be sequenced and used in the future experiments of such nature.

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