Pathogenesis-related (PR-1) proteins in cereals challenged with fungal pathogens

Systemic acquired resistance (SAR) is a broad physiological immunity in uninfected parts of the plant that results from local infection by a variety of pathogens. In the SAR response, a group of genes is expressed that encodes pathogenesis related (PR) proteins of which the PR-1 protein is the most abundant and is induced up to 1-2% of the total leaf protein. The biochemical function of the barley PR-1 protein is still unclear. The main objective of these studies was to characterize the nature of barley PR-1 protein and evaluate its importance in SAR and plant defence. The following aspects of the PRb-1 protein were investigated: 

1) A specific antibody for barley PRb-1 was raised in rabbit and this antibody was used to identify the presence and induction of the PRb-1 protein and to screen the transgenic wheat & barley transformed with the prb-1 gene. 

2) The local and systemic expression of the prb-1 gene and PRb-1 protein were examined in barley cultivar Psaknon plants following treatment of the primary leaves with either 2,6- dichloro-isonicotinic acid (INA) or Erysiphe graminis f.sp. hordei (Egh). A correlation between expression of PRb-1 protein and PRb-1 mRNA was established. Both the INA treated plants and Egh inoculated plants exhibited a similar strong systemic induction of the mRNA that was followed by a similar pattern of PRb-1 protein accumulation. Although the proteins accumulated more slowly and less transiently than the mRNA, they showed similar expression patterns. The induction of the PRb-1 protein after INA treatment was tissue specific with high levels of PRb-1 protein induced locally in primary leaves and systemically in secondary leaves while a low level of PRb-1 protein was induced in crown tissue after INA treatment but no induction of PRb-1 protein was detected in the roots. INA treated barley Psaknon plants induced strong SAR to subsequent Egh infection, the onset of which was correlated with induced expression of the PRb-1 protein in primary and secondary leaves. These results provided indirect evidence for antifungal activity of the PRb-1 protein against Egh. 

3) To study the effect of PRb-1 protein on fungal growth the coding region of barley prb-1 gene corresponding to putative mature PRb-1 protein was cloned and expressed in Escherichia coli. The in vitro expressed barley PRb-1 protein significantly reduced the germination of Egh spores. A direct fungicidal activity of PRb-1 protein against Egh was demonstrated by in-vivo tests in which the reduction of infected leaf surface was scored. This assay also showed that the inhibition on fungal growth by PRb-1 protein was dose dependent. 

4) Attempts were made to enhance the resistance against fungal pathogens in transgenic wheat and barley by introducing barley prb-1 gene and thereby strengthening the natural defence system of these crops. Transformation was performed by the bombardment of immature embryos with a plasmid containing both prb-1 and the marker bar gene under the constitutive ubiquitin promoter. Four PPT resistant ST₀ wheat plants were selected from 6 separate experiments. In the T₀ generation two plants were demonstrated to contain both prb-1 and bar genes. In addition, one plant was positive for the bar gene only and while another plant was negative for both the genes. Transmission of the bar and prb-1 gene to the T₁ and T₂ generation occurred but the segregation patterns of the genes varied among the progeny plants. Transgenic wheat plants expressed the prb-1 transgene in the T₁ generation but the T₂ generation of these plants failed to express the prb-1 transgene.