Differential induction of two different cystatin genes during pathogenesis of Karnal bunt (Tilletia indica) in wheat under the influence of jasmonic acid.

In the present study, expression patterns of two different wheat cystatins (WCs) were studied under the influence of jasmonate signaling in triggering resistance against Karnal bunt (KB). Cystatins are cysteine proteinase inhibitors (CPI) constituting a multigene family which regulate the activity of endo- and/or exogenous cysteine proteinases (CP). Two wheat varieties HD-29 (resistant, R) and WH-542 (susceptible, S) were pre-conditioned with jasmonate and then artificially inoculated with sporidial suspension of Tilletia indica to study its influence in inducing defense by regulating cystatin genes. On the transcriptional level, WC4 and WC5 gave different temporal expression patterns. Expression of WC4 was higher in boot emergence stage which is most susceptible to KB and then slowly declined in both varieties. Expression of WC5 showed an entirely reverse pattern of expression, which kept on rising as the grains matured. Cystatin activity determination by inhibitor assay gave higher activity in resistant variety and under JA treatment. Estimation of specific activity of total cystatin at different days after inoculation (DAI) showed that JA positively induced cystatin expression in both varieties but R variety always registered a greater cystatin expression than the susceptible one (P<0.05). In plants inoculated with pathogen, initially there was a rise in cystatin activity which gradually decreased 7 DAI when compared with the un-inoculated plants. Based on these findings it is clearly demonstrated that jasmonate acts as a potential activator of induced resistance by up-regulating cystatin expression and provides the conditioning effect prior to infection through the maintenance of critical balance of CP/CPI interaction. However, different cystatin genes show different temporal expression patterns and may play different roles at various developmental stages of the grain.

Jasmonate signal induced expression of cystatin genes for providing resistance against Karnal bunt in wheat.

Two wheat varieties HD-29 (resistant, R) and WH-542 (susceptible, S) were pretreated with jasmonic acid (JA) or jasmonate and then artificially inoculated with sporidial suspension of Tilletia indica to study its influence in reducing Karnal bunt (KB) infection by regulating cystatin gene expression. JA was found to improve the plant defense against KB as its exogenous application resulted in decrease in coefficient of infection (CI) in both susceptible and resistant varieties following pathogen inoculation. Transcript profiling of wheat cystatin genes at different days after inoculation (DAI) showed that JA pretreatment positively induced cystatin gene expression in both varieties with greater induction of expression in resistant variety than the susceptible one (P< 0.05). Different temporal expression of three wheat cystatin genes, WC2, WC3 and WCMD was observed with their increased expression at 1DAI in the boot emergence stage which is most susceptible to KB and then slowly declined gradually at 3, 7 and 15 DAI in both the varieties. Except WC2, higher expression of other two cystatins viz. WC3 and WCMD at 1DAI showed higher response (P< 0.05) to KB pathogenesis at the disease-prone boot emergence stage as also evident by decrease of CI in both varieties. The results of determination of specific activity of cystatin by inhibitor assay were found to be consistent with those of transcript profiling. These findings suggest that jasmonic acid (JA) may act as a potential activator of induced resistance against Karnal bunt of wheat by upregulating cystatin gene expression.

In silico analysis of sequential, structural and functional diversity of wheat cystatins and its implication in plant defense.

Phytocystatins constitute a multigene family that regulates the activity of endogenous and/or exogenous cysteine proteinases. Cereal crops like wheat are continuously threatened by a multitude of pathogens, therefore cystatins offer to play a pivotal role in deciding the plant response. In order to study the need of having diverse specificities and activities of various cystatins, we conducted comparative analysis of six wheat cystatins (WCs) with twelve rice, seven barley, one sorghum and ten corn cystatin sequences employing different bioinformatics tools. The obtained results identified highly conserved signature sequences in all the cystatins considered. Several other motifs were also identified, based on which the sequences could be categorized into groups in congruence with the phylogenetic clustering. Homology modeling of WCs revealed 3D structural topology so well shared by other cystatins. Protein-protein interaction of WCs with papain supported the notion that functional diversity is a con-sequence of existing differences in amino acid residues in highly conserved as well as relatively less conserved motifs. Thus there is a significant conservation at the sequential and structural levels; however, concomitant variations maintain the functional diversity in this protein family, which constantly modulates itself to reciprocate the diversity while counteracting the cysteine proteinases.