Characterisation of putative avirulence genes in flax rust, Melampsora lini

Abstract

The aim of this study was to isolate and characterise four linked avirulence genes (A-L5, A-L6, A-L7, A-Lx) from a homozygous strain of flax rlust (Melampsora lini). A 250 bp clone (pERT 5.8) was isolated by subtractive hybridisation between DNA of a rust strain, 228e99, homozygous for the 4 linked avirulence genes, and DNA from a mutant, 4ec68.1, used as tho subtractive driver (Timmis et a1.,1990). The rust strain Aec68.1 is a close relative of 228e9e but has the four linked avirulence genes deleted (Timmis et al., 1990). Genetic evidence suggested that pERT 5.8 was within, or linked to, these avirulence genes. Six clones were isolated from an EMBL4 genomic library using pERT 5.8 as a probe. One lambda clone was partially characterised in this project through a combination of sequencing, mapping, computer sequence analysis and PCR amplification of RNA transcripts. Southern hybridisation of Aec68.1 and 228e99 DNA using the subcloned EcoRI fragments of lambda clone 4 indicated that at least 2 kbp of DNA (subcloned as pMl4N) was deleted in its entirety from the genome. The extent of the deletion in Aec68.1 may be much larger because sequences flanking pMl4N apparently contain dispersed repoats present in both strains. Northern analysis of total RNA from both a compatible (growth of rust) and incompatible (no growth of rust) reaction between appropriate flax varieties and 228e9e using the insert from pMl4N as a probe was insufficiently sensitive to detect any mRNA produced by this region of the genome. Sequence analysis of the insert from pMl4N identified a large open reading frame of 840 bp to which primers were designed for PCR analysis of possible gene products. These primers amplified a specific transcript from total 11 RNA isolated from germinated 228e99 rust spores alone and from total RNA isolated from 228990 infected plants. No PCR product was detected in the incompatible reaction. Another rust strain , 27 t.26, virulent on hosts differentiating these four specificities (a-L5, a-L6, a-L7, a-Lx) contains DNA homologous to pMl4N and virulence in this strain is therefore not due to deletion of a segment of DNA. PCR analysis of total RNA from germinated 271.26 spores alone and from a compatible reaction between flax and this rust strain also indicated the presence of a transcription product of the 840 bp open reading frame. Comparisons between four cDNA clones of 228e9e and27l.26 mRNAs indicated that 228999 produces transcripts from at least two different genes and271.26 produces transcripts from four different genes. This may indicate the possibility that a small gene family produces several different mRNA products.