Study of genes modifying morphology, pathogen interactions and MEP-derived metabolites during barley root colonization by Piriformospora indica via stable root transformation system
In agricultural cropping systems, roots are frequently subjected to a series of abiotic stress as well as biotic stress caused by microbial pathogens and pests which lead seriously yield reduction for crop food. The significance of the root for plant health is in disagreement with the availability of root systems for functional studies. This is more alarming as our knowledge and technology on protective cultivation methods, resistant germplasms, or chemical control strategies to ward off root disease and root stress in crops is limited. Therefore, Stable Root Transformation System (STARTS) was developed in barley on the basis of the conventional stable transformation method. STARTS accelerates functional studies in roots by the continued culture of calli on root induction medium thereby producing large amounts of roots. STARTS allows functional analysis of proteins in roots in about six weeks. The method was proven to be effective to overexpress (GFP, GFP-BI-1) and silence (HvEXPANSIN B1, GFP) genes. Moreover, STARTS was identified to be suitable for the analysis of protein sub-cellular localization by transforming the scutella with a modified version of GFP (mGFP5-ER). Finally, we studied the impact of BAX INHIBITOR-1 (BI-1) overexpression on root colonization by hemi-biotrophic and biotrophic microbes. Results confirmed that STARTS is applicable to study the effect of barley proteins in root microbe interactions. Most importantly, the method is suitable to pre-screen the effect of candidate genes on root stress resistance and root development.STARTS provided a good foundation for the second part of my work referred to the function kaurene synthase-like (KSL) genes during barley root colonization byPiriformospora indica. Sequence alignment of barley KSL genes indicated that all these genes contained the aspartate-rich domain DDFFD which is supposed to function as a divalent metal ion-diphosphate complex binding site in terpene cyclases (Pinheiro et al. 2005). Further experiment showed that barley KS-like genes (HvAK370792, HvKSL1, HvKS4) were differently up-regulated at 1, 3 and 7 dai and KS-like silencing in roots resulted in reduced colonization by P. indica. Silencing of the KSL genes HvAK370792, HvKSL1 lead to less dark green leaves and slower plant development. Further, I observed reduced spikelet fertility in progenies of RNAi plants heterozygous for HvAK370792 and HvKSL1. The data suggests that HvAK370792 and HvKSL1 are involved in gibberellin (GA) biosynthesis. I was successful in cloning HvCPSL1, HvKSL1, Hvsyn-CPS and HvAK370792 as well as the heterlogous expression of Hvsyn-CPS and HvAK370792 in E. coli. This work therefore build the basis to decipher a presumed function of these proteins in GA and/or phytoalexin metabolism.
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