A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat

Abstract

The generation and characterization of transgenic wheat plants is a tedious and time-consuming process that limits the number of putatively important transgenes that can be tested. We therefore established a transient assay system based on wheat leaves to study the effect of transiently expressed genes on the interaction with the wheat powdery mildew fungus Erysiphe (syn. Blumeria) graminis f. sp. tritici. Young wheat leaves were bombarded with tungsten particles coated with a mixture of plasmids carrying the β-glucuronidase (GUS) reporter gene and a test gene. Leaves were subsequently challenge inoculated with E. graminis and the fungus was allowed to develop for 40 h. After being stained for GUS enzymatic activity as well as for epiphytic fungal structures, the phenotype of transformed epidermal cells was evaluated by bright-field microscopy. The fungus was routinely found to penetrate cells transiently expressing GUS with an efficiency of approximately 35%, which should suffice to detect putative transgene effects. Transgenes encoding a low-molecular-weight cell-wall protein of wheat (WIR1), a thaumatin-like protein, and a glucanase had no effect on fungal penetration of transformed epidermal cells. On the other hand, trans-genes encoding a pathogen-induced wheat protein of unknown function (WCI5), a chitinase, a glucose oxidase, and a putative peroxidase significantly reduced fungal penetration.

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

“…pUBI-GUS-MU108H 2 pUbi-GUS (Schweizer et al, 1999) was digested by HindIII, to release a 4.2 Kb fragment containing ubiquitin-1 promoter from maize, the uidA gene and 35S terminator. This fragment was cloned in the HindIII digested pMU108H 2 transformation vector (M. Uze´, unpublished) carrying the PP-108 genomic locus (Schaefer and Zryd, 1997) and a hygromycin resistance cassette driven by 35S promoter (Details provided upon request).…”

Functional analysis of barley RAC/ROP G‐protein family members in susceptibility to the powdery mildew fungus

“…For transient transformation assays complete open-reading frames of the Rac/Rop cDNAs were subcloned into pGY1 (Schweizer et al, 1999) that contains a 540-bp fragment of the CaMV35S promoter and terminator separated by a multiple cloning site. The constructs were cloned using the restriction sites linked to primers mentioned in Table 2.…”

“…For expression of GFP:HvRAC/ROP fusion proteins, cDNAs of GFP (GFPemd-b in pGFP; Schweizer et al, 1999) were ampli®ed from plasmids by PCR using primers with attached BamHI restriction sites in all three frames under elimination of the GFP stop codon (GFP-5 H primer: 5 H -GGATCCATGGTGAGCAAGGGCGAG-3 H ; GFP-3 H primer (frame 1): 5 H -GGATCCTTGTACAGCTCGTCCAT-3 H ; GFP-3 H primer (frame 2): 5 H -GGATCCCTTGTACAGCTCGTCCAT-3 H ; GFP-3 H primer (frame 3): 5 H -GGATCCCCTTGTACAGCTCGTCCAT-3 H ). GFP-PCR products were cloned in frame into the appropriate pGY1-RAC/ROP-construct (linearised with BamHI).…”

“…A transient transformation protocol, originally developed for wheat to assess gene function in the interaction with powdery mildew, was used to deliver overexpression constructs of the RAC/ ROP proteins into epidermal cells of barley leaf segments as described by Schweizer et al (1999) and .…”

The Arabidopsis ROP-activated receptor-like cytoplasmic kinase RLCK VI_A3 is involved in control of basal resistance to powdery mildew and trichome branching

“…All constructs for the microscopic subcellular localization studies were based on the pUC18-based plant expression vector pGY1 (Schweizer et al 1999) that contains the CaMV 35S promoter and CaMV 35S terminator site. The fluorescence tag containing constructs pGY1-GFP and pGY1-RFP have been described elsewhere AtROP mutants were obtained by site-directed mutagenesis using overlap extension PCR.…”