A Plant Gene Up-Regulated at Rust Infection Sites
Abstract
Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a β-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%–82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a Δ-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.
For successful parasitism of plants by fungal pathogens, particularly in the case of obligate, biotrophic phytopathogens such as rust fungi, it is apparent that the pathogen has evolved strategies for manipulating host cellular metabolism, morphology, and development (Clancy and Coffey, 1980; Sutton and Shaw, 1982, 1986; Scholes, 1992; Roy, 1993; Chou et al., 2000; Hall and Williams, 2000). Successful infection of the host plant by the pathogen requires the induction of a subset of pathogen genes that are essential for pathogenicity (Pieterse et al., 1993; Talbot et al., 1993; Rogers et al., 1994; Hwang et al., 1995; Hahn and Mengden, 1997; Hahn et al., 1997). Less clear is the role and expression of specific host genes that may be required for successful pathogen infection of the plant. Identification of host genes induced during a successful pathogen infection may provide insight into the compatible host/pathogen interaction at a molecular level.
The fis1 gene of flax (Linum usitatissimum) was identified as a host gene up-regulated during a successful or compatible flax rust (Melampsora lini) infection (Roberts and Pryor, 1995). RNA-blot analysis of fis1 demonstrated that this gene was induced during a compatible flax rust infection, but its induction was not detectable during an incompatible infection when rust growth was halted by the action of the L6 resistance gene. Homology searches suggested that the fis1 gene encoded an aldehyde dehydrogenase enzyme of unknown substrate specificity (Roberts and Pryor, 1995). Deuschle et al. (2001) recently have isolated a gene from Arabidopsis, AtP5CDH, which encodes a protein with 82% identity to the predicted FIS1 protein. The AtP5CDH gene was isolated by screening an Arabidopsis cDNA library in a yeast (Saccharomyces cerevisiae) mutant deficient for the enzyme Δ-pyrroline-5-carboxylate dehydrogenase. The AtP5CDH enzyme catalyzes the second step in the degradation of Pro to Glu, specifically the conversion of pyrroline-5-carboxylate to Glu (Deuschle et al., 2001). Pyrroline-5-carboxylate is potentially a toxic intermediate and thus the Δ-pyrroline-5-carboxylate dehydrogenase enzyme may provide protection from Pro toxicity (Deuschle et al., 2001).
In this report, we describe the characterization of the promoter and expression pattern of the fis1 gene in response to rust infection and the identification of related genes in a diverse range of plant species. Homologs of the fis1 gene in both maize (Zea mays) and barley (Hordeum vulgare) are also shown to be up-regulated in response to rust infection, demonstrating the generality of this response. The highly localized up-regulation of this promoter in response to a compatible rust infection makes it a candidate for controlling the expression of synthetic rust disease resistance genes. In addition, the similarity of the protein products of these genes to Δ-pyrroline-5-carboxylate dehydrogenase suggests that Pro catabolism plays an as yet undefined role in the interaction between rust fungi and the host plant.
Each EST was translated and the similarity and identity to the predicted FIS1 protein determined. The region of the FIS1 protein that was homologous to these translated ESTs is also indicated. This table is not exhaustive and homologues were identified in a no. of other plant species. Wheat and barley ESTs were identified in the International Tritici EST Consortium database.
The no. of codons in each exon is given. Exon 8 of the mis1 gene appears to be a composite of exons 8 and 9 of the AtP5CDH gene.
ACKNOWLEDGMENTS
We wish to thank Mr. Luch Hac and Ms. Kim Newell for technical assistance, Dr. Sylvie Cloutier for provision of a wheat EST clone, and Ms. Sandie McIntosh for assistance in manuscript preparation.
Footnotes
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010940.