Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance

Abstract

Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.

Nitric oxide affects salt-induced changes in free amino acid levels in maize

“…For the real-time PCR analysis of the genes encoding enzymes involved in the amino acid and polyamine metabolism or in the antioxidant defence and death processes, the DNase treatment of RNA and first-strand cDNA synthesis were carried out according to Altpeter et al (2005). The PCR amplification mixture (20 µl) contained 0.5 µl cDNA, 10 µl QuantiTect SYBR Green PCR Master mix (Qiagen, Hilden, Germany) and 0.8 µl forward and reverse primer (10 µM).…”

“…The primers are listed in Supplementary Table S1. The amplification of DNA and normalization against glyceraldehyde-3-phosphate dehydrogenase (Kocsy et al, 2010) were performed as described earlier (Altpeter et al, 2005).…”

Promoters of the Barley Germin-LikeGER4Gene Cluster Enable Strong Transgene Expression in Response to Pathogen Attack

“…RT-PCR was performed using the 384-well system (7900 HT Fast Real-Time PRC system from Applied Biosystems) essentially as described (Altpeter et al, 2005). The amplification reactions (10 mL) contained 1 mL cDNA, 5 mL SYBR Green PCR Master mix (Qiagen), and 100 nM forward and reverse primer specific for the tested GUS transgene or the barley internal control gene UBC (barley ubiquitin-conjugating enzyme), respectively.…”

Isolation and characterization of rubisco small subunit gene promoter from common wheat (Triticum aestivumL.)

“…As a result, the promoter of GstA1 has been used as a tool to express defense-related genes in the inner leaf tissues to confer plants with resistance against pathogens that enter through the epidermis. 8 The barley abundant protein (HVA1) gene encoding the late embryogenesis abundant 3 (LEA3), one of the proteins involved in controlling the closure of leaf stomata to prevent evaporation and thereby cellular dehydration, has been shown to be induced by ABA, and stress conditions including cold, heat, drought and salinity. 9 This result indicates the presence of ABA and stress responsive elements in the promoter of HVA1.…”