The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells

Abstract

The Linum usitatissimum (flax) L gene alleles, which encode nucleotide binding site-Leu rich repeat class intracellular receptor proteins, confer resistance against the Melampsora lini (flax rust) fungus. At least 11 different L resistance specificities are known, and the corresponding avirulence genes in M. lini map to eight independent loci, some of which are complex and encode multiple specificities. We identified an M. lini cDNA marker that cosegregates in an F2 rust family with a complex locus determining avirulence on the L5, L6, and L7 resistance genes. Two related avirulence gene candidates, designated AvrL567-A and AvrL567-B, were identified in a genomic DNA contig from the avirulence allele, whereas the corresponding virulence allele contained a single copy of a related gene, AvrL567-C. Agrobacterium tumefaciens-mediated transient expression of the mature AvrL567-A or AvrL567-B (but not AvrL567-C) proteins as intracellular products in L. usitatissimum and Nicotiana tabacum (tobacco) induced a hypersensitive response-like necrosis that was dependent on coexpression of the L5, L6, or L7 resistance gene. An F1 seedling lethal or stunted growth phenotype also was observed when transgenic L. usitatissimum plants expressing AvrL567-A or AvrL567-B (but not AvrL567-C) were crossed to resistant lines containing L5, L6, or L7. The AvrL567 genes are expressed in rust haustoria and encode 127 amino acid secreted proteins. Intracellular recognition of these rust avirulence proteins implies that they are delivered into host cells across the plant membrane. Differences in the three AvrL567 protein sequences result from diversifying selection, which is consistent with a coevolutionary arms race.

Figure 1.

An M. lini Family Segregating for 16 Avirulence Specificities. The rust strains C and H were crossed together to produce the F1 rust CH5, which was then selfed to produce 74 F2 progeny (Lawrence et al., 1981). Avirulence specificities segregating in the F2 family are indicated next to the parental rust from which they were inherited. Some avirulence specificities, such as A-L5, A-L6, and A-L7, cosegregate in this family and are listed next to each other. The A-P gene from rust C segregates as an alternative allele at the same locus as A-P1, A-P2, and A-P3 from rust H. The A-N gene was present in both parents but segregates in the F2. An inhibitor gene (I) that suppresses resistance reactions triggered by some avirulence genes was inherited from rust C.

Figure 2.

Characterization of the M. lini AvrL567 Locus. (A) Schematic diagram of the AvrL567 locus. A 26.5-kb region from the avirulence allele (H) contains the AvrL567-A and AvrL567-B genes, whereas the AvrL567-C gene occurs in an 11.5-kb region at the virulence allele (C). A second gene related to yeast Sec14 (Bankaitis et al., 1989) also occurs in this region along with a third predicted gene (orf-3; no similarity to database sequences). The position of a 7.4-kb duplication in the H allele is indicated, as is a 6.3-kb inserted sequence similar to gypsy class retrotransposons, with 174-bp identical direct repeats flanking a long open reading frame encoding a putative polyprotein. A repeat region containing 12 or 13 copies of an 82-bp sequence, which occurs close to the 5′ ends of the AvrL567 genes, is shown as a closed box. An ∼1-kb region in which three recombination events were resolved is indicated at the 3′ end of the contigs. (B) Amino acid sequence alignment of the predicted AvrL567-A, AvrL567-B, and AvrL567-C proteins. Only those amino acids that differ from the consensus (top line) are shown, with identical residues indicated by a dotted line. The signal peptide is underlined, and the first amino acid of the predicted mature protein (Met-24) is shown in bold.

Figure 3.

Transient Expression of AvrL567 Genes in L. usitatissimum and N. tabacum Causes R Gene–Dependent Necrosis. (A) Leaves of rust-susceptible L. usitatissimum var Hoshangabad were infiltrated with A. tumefaciens cultures containing an empty binary vector, an L6 T-DNA vector, a T-DNA vector encoding AvrL567-A¹²⁷, or a mixture of two cultures containing the L6 and AvrL567-A¹²⁷ T-DNAs. (B) Leaves of near-isogenic L. usitatissimum lines containing the L9, L5, L6, or L7 resistance gene were infiltrated with A. tumefaciens cultures containing T-DNA expression vectors encoding AvrL567-A¹²⁷, AvrL567-B¹²⁷, or AvrL567-C¹²⁷. Images were prepared 12 d after infiltration. (C) W38 N. tabacum leaves were infiltrated with A. tumefaciens cultures containing either an L6 T-DNA vector, a T-DNA vector encoding AvrL567-A¹²⁷, or a mixture of two cultures containing the L6 and AvrL567-A¹²⁷ T-DNAs. Each A. tumefaciens culture was infiltrated into separate regions of the leaf bounded by the major veins, and the leaf was photographed 11 d after infiltration.

Figure 4.

Phenotypes Resulting from Crossing AvrL567 Transgenes into L. usitatissimum Lines with Different L Genes. (A) A single T0 transgenic plant containing one copy of an AvrL567-A¹⁵⁰ transgene was crossed to L. usitatissimum lines homozygous for the L5, L6, L7, or Lx resistance gene as well as to the rust susceptible line Hoshangabad (Hosh). Twelve seeds from each cross as well as self-fertilized seeds were planted in soil. Photographs were taken 2 weeks after planting. (B) Seeds resulting from a cross between a T0 transgenic plant containing one copy of the AvrL567-A¹²⁷ transgene and a L. usitatissimum line homozygous for L5. Approximately half of the seeds have a shrunken appearance (left) and are unable to germinate, whereas the phenotypically wild-type seeds (right) give rise to normal plants. (C) Segregation of wild-type and dwarf phenotypes among 3-week-old seedlings resulting from a cross between a T0 transgenic plant containing one copy of the AvrL567-B¹²⁷ transgene and a L. usitatissimum line homozygous for L6.

Figure 5.

AvrL567 Transcripts Are Induced during Infection and Are Expressed in Haustoria. (A) RNA samples (5 μg) from uninfected L. usitatissimum leaves (F), rust CH5–infected L. usitatissimum leaves 5 to 8 d after infection (5d to 8d), and in vitro–germinated CH5 rust spores (Sp) were separated on a 1.5% agarose gel, transferred to nylon membranes, and hybridized with probes for the AvrL567, Sec14, orf-3, and tubulin genes of M. lini. Note that the infected leaf samples contain largely L. usitatissimum RNA with an increasing proportion of rust RNA as the fungal biomass increases during infection. The AvrL567 probe also was hybridized to RNA from leaves infected with rust strain CH5-F2-89 (homozygous for the avirulence allele) or CH5-F2-112 (homozygous for the virulence allele). RNA extracted from 50 mg of haustoria purified from L. usitatissimum leaves 6 d after infection with rust CH5 (Haus) was run alongside 2.5 μg of RNA from rust CH5 infected leaves (6d) and hybridized to the AvrL567 probe. RNA filters were stained with methylene blue to detect rRNA loading (top panel). Given the low amount of haustorial RNA, the AvrL567 signal in haustoria reflects a considerable enrichment compared with the infected leaf RNA. (B) RNA from in vitro–germinated rust spores (Sp) and infected L. usitatissimum leaves after 1 to 5 d was reversed transcribed and amplified by PCR using primers specific for the AvrL567 (Avr), Sec14, or tubulin (Tub) gene. RT-PCR products were separated on a 2% low melting point agarose gel and visualized by ethidium bromide staining and UV irradiation. (C) Haustoria isolated from L. usitatissimum leaves 6 d after infection were fixed on glass slides and labeled with monoclonal antibody mL1 (Murdoch et al., 1998), followed by a goat anti-mouse secondary antibody coupled to the fluorophore Alexa-488 and visualized by fluorescence under illumination by 488-nm light.