Haustorially Expressed Secreted Proteins from Flax Rust Are Highly Enriched for Avirulence Elicitors<sup><a href="#fn1" rid="fn1" class=" fn">W⃞</a></sup>
Abstract
Rust fungi, obligate biotrophs that cause disease and yield losses in crops such as cereals and soybean (Glycine max), obtain nutrients from the host through haustoria, which are specialized structures that develop within host cells. Resistance of flax (Linum usitatissimum) to flax rust (Melampsora lini) involves the induction of a hypersensitive cell death response at haustoria formation sites, governed by gene-for-gene recognition between host resistance and pathogen avirulence genes. We identified genes encoding haustorially expressed secreted proteins (HESPs) by screening a flax rust haustorium-specific cDNA library. Among 429 unigenes, 21 HESPs were identified, one corresponding to the AvrL567 gene. Three other HESPs cosegregated with the independent AvrM, AvrP4, and AvrP123 loci. Expression of these genes in flax induced resistance gene–mediated cell death with the appropriate specificity, confirming their avirulence activity. AvrP4 and AvrP123 are Cys-rich proteins, and AvrP123 contains a Kazal Ser protease inhibitor signature, whereas AvrM contains no Cys residues. AvrP4 and AvrM induce cell death when expressed intracellularly, suggesting their translocation into plant cells during infection. However, secreted AvrM and AvrP4 also induce necrotic responses, with secreted AvrP4 more active than intracellular AvrP4, possibly as a result of enhanced formation of endoplasmic reticulum–dependent disulfide bonds. Addition of an endoplasmic reticulum retention signal inhibited AvrM-induced necrosis, suggesting that both AvrM and AvrP4 can reenter the plant cell after secretion in the absence of the pathogen.
Acknowledgments
We thank Valerie Ryle, Patricia Atkinson, and Kim Newell for providing excellent technical assistance. The ML1 monoclonal antibody was kindly provided by Adrienne Hardham (Research School of Biological Science, Australian National University). This research was supported by grants from the Grains Research and Development Corporation. A.-M.C. was supported by an Australian Postgraduate Award funded by the Department of Education, Science, and Training and by a Commonwealth Scientific and Industrial Research Organization partial scholarship.
Notes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Peter N. Dodds (ua.orisc@sddod.retep).
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