Transformation of the flax rust fungus, Melampsora lini: selection via silencing of an avirulence gene

Abstract

Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium-mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non-transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.

Figure 1. Analysis of transgenic flax rust isolates

(a) Schematic representation (not to scale) of the siAvrL567 T-DNA construct used to transform flax rust strain CH5–89. The AvrL567 promoter region (yellow) drives expression of the inverted repeat AvrL567-A transcript (orange). Two intron sequences are shown in grey, while a 120-bp non-repeated region is hatched orange. The T-DNA also contains a spectinomycin resistance gene (SpecR) under the control of the 35S promoter and OCS terminator sequences (green). The left (LB) and right (RB) borders of the T-DNA and internal XbaI (X) restriction sites are indicated, as is the location of the AvrL567 probe. The location on the T-DNA of oligonucleotide primers 10-1.15, 10-1.16, 10-1.19 and 35Srev are indicated by small arrows. (b) Genomic DNA of non-transgenic flax rust strain CH5–89 and six transgenic (T1–T6) isolates, each from a separate pot, was amplified by PCR with primers directed to the siAvrL567 T-DNA construct (v). The primer pair 10–1.15/10–1.16 (15/16) amplifies a 600-bp fragment from the T-DNA and a 900-bp fragment from genomic DNA, while the 10-1.15/10-1.19 (15/19) and 35Srev/10-1.16 (35Srev/16) primer pairs amplify 900-bp fragments from the T-DNA only. (c) Genomic DNA of non-transgenic flax rust strain CH5–89 and six transgenic (T1–T6) isolates, each from a separate pot, was digested with the restriction enzyme XbaI, separated by agarose gel electrophoresis, transferred to nylon membranes and hybridised with a probe derived from the coding region of AvrL567-A. This probe detects two fragments in CH5–89 derived from the endogenous AvrL567 locus. In the transgenic lines the probe hybridises additionally to a 2.6-kb fragment originating from within the T-DNA and to a single T-DNA–fungal DNA junction fragment of variable size, indicating that each of the transgenic isolates contains a single T-DNA insert.

Figure 2. RNA gel blot analysis of transgenic rust lines

RNA extracted from flax leaves heavily infected with non-transgenic flax rust strain CH5–89 and three transgenic (T1, T3, T4) isolates was separated by gel electrophoresis, transferred to nylon membranes and hybridised with probes derived from AvrL567-A or from the flax rust tubulin gene.