Gregory J. Lawrence

Identification of Regions in Alleles of the Flax Rust Resistance Gene L That Determine Differences in Gene-for-Gene Specificity

Thirteen alleles (L, L1 to L11, and LH) from the flax L locus, which encode Toll/interleukin-1 receptor homology–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) rust resistance proteins, were sequenced and compared to provide insight into their evolution and into the determinants of gene-for-gene resistance specificity. The predicted L6 and L11 proteins differ solely in the LRR region, whereas L6 and L7 differ solely in the TIR region. Thus, specificity differences between alleles can be determined by both the LRR and TIR regions. Functional analysis in transgenic plants of recombinant alleles constructed in vitro provided further information: L10–L2 and L6–L2 recombinants, encoding the LRR of L2, conferred L2 resistance specificity, and an L2–L10 recombinant, encoding the LRR of L10, conferred a novel specificity. The sequence comparisons also indicate that the evolution of L alleles has probably involved reassortment of variation, resulting from accumulated point mutations, by intragenic recombination. In addition, large deletion events have occurred in the LRR-encoding regions of L1 and L8, and duplication events have occurred in the LRR-encoding region of L2.

Haustorially Expressed Secreted Proteins from Flax Rust Are Highly Enriched for Avirulence Elicitors

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.

The Melampsora lini AvrL567 Avirulence Genes Are Expressed in Haustoria and Their Products Are Recognized inside Plant Cells

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.

Six Amino Acid Changes Confined to the Leucine-Rich Repeat β-Strand/β-Turn Motif Determine the Difference between the P and P2 Rust Resistance Specificities in Flax

At least six rust resistance specificities (P and P1 to P5) map to the complex P locus in flax. The P2 resistance gene was identified by transposon tagging and transgenic expression. P2 is a member of a small multigene family and encodes a protein with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains and an N-terminal Toll/interleukin-1 receptor (TIR) homology domain, as well as a C-terminal non-LRR (CNL) domain of ∼150 amino acids. A related CNL domain was detected in almost half of the predicted Arabidopsis TIR-NBS-LRR sequences, including the RPS4 and RPP1 resistance proteins, and in the tobacco N protein, but not in the flax L and M proteins. Presence or absence of this domain defines two subclasses of TIR-NBS-LRR resistance genes. Truncations of the P2 CNL domain cause loss of function, and evidence for diversifying selection was detected in this domain, suggesting a possible role in specificity determination. A spontaneous rust-susceptible mutant of P2 contained a G→E amino acid substitution in the GLPL motif, which is conserved in the NBS domains of plant resistance proteins and the animal cell death control proteins APAF-1 and CED4, providing direct evidence for the importance of this motif in resistance gene function. A P2 homologous gene isolated from a flax line expressing the P resistance specificity encodes a protein with only 10 amino acid differences from the P2 protein. Chimeric gene constructs indicate that just six of these amino acid changes, all located within the predicted β-strand/β-turn motif of four LRR units, are sufficient to alter P2 to the P specificity.

Regions outside of the Leucine-Rich Repeats of Flax Rust Resistance Proteins Play a Role in Specificity Determination

Multiple alleles controlling different gene-for-gene flax rust resistance specificities occur at the L locus of flax. At least three distinct regions can be recognized in the predicted protein products: the Toll/interleukin-1 receptor homology (TIR) region, a nucleotide binding site (NBS) region, and a leucine-rich repeat (LRR) region. Replacement of the TIR-encoding region of the L6 allele with the corresponding regions of L2 or LH by recombination changed the specificity of the allele from L6 to L7. Replacement of the TIR and most of the NBS-encoding region of L10 with the equivalent region of L2 or L9 generated recombinant alleles having a novel specificity. However, replacement of the L10 TIR-encoding region with the TIR-encoding region of L2 gave rise to an allele with no detectable specificity. These data indicate that non-LRR regions can determine specificity differences between allelic gene products and that functional specificity involves interactions between coadapted polymorphic regions in the protein products of the alleles. Evidence for the action of diversifying selection on the TIR region is observed.

Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

This work examines translocation of the flax rust effector protein AvrM, showing that it accumulates in the haustorial wall and extracellular matrix and is delivered into the host cytoplasm. Protein fusions expressed in the plant showed that AvrM can be secreted by the plant and subsequently internalized in the absence of pathogen. Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Diversity and evolution of effector loci in natural populations of the plant pathogen Melampsora lini

Genetic variation for pathogen infectivity is an important driver of disease incidence and prevalence in both natural and managed systems. Here, we use the interaction between the rust pathogen, Melampsora lini, and two host plants, Linum marginale and Linum usitatissimum, to examine how host-pathogen interactions influence the maintenance of polymorphism in genes underlying pathogen virulence. Extensive sequence variation at two effector loci (AvrP123, AvrP4) was found in M. lini isolates collected from across the native range of L. marginale in Australia, as well as in isolates collected from a second host, the cultivated species L. usitatissimum. A highly significant excess of nonsynonymous compared with synonymous polymorphism was found at both loci, suggesting that diversifying selection is important for the maintenance of the observed sequence diversity. Agrobacterium-mediated transient transformation assays were used to demonstrate that variants of both the AvrP123 and AvrP4 genes are differentially recognized by resistance genes in L. marginale. We further characterized patterns of nucleotide variation at AvrP123 and AvrP4 in 10 local populations of M. lini infecting the wild host L. marginale. Populations were significantly differentiated with respect to allelic representation at the Avr loci, suggesting the possibility of local selection maintaining distinct genetic structures between pathogen populations, whereas limited diversity may be explained via selective sweeps and demographic bottlenecks. Together, these results imply that interacting selective and nonselective factors, acting across a broad range of scales, are important for the generation and maintenance of adaptively significant variation in populations of M. lini.

The genome sequence and effector complement of the flax rust pathogen Melampsora lini

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

A Plant Gene Up-Regulated at Rust Infection Sites

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 L6resistance 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-induciblefis1 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 Δ1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1homolog 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.