Flax Rust Resistance Research Articles

EVALUATION OF THE LATVIAN FLAX GENETIC RESOURCES AND PERSPECTIVE OF THEIR UTILISATION

Flax (Linum usitatissimum L.) is one of the oldest fibre and oil crop in Europe, including Latvia. In the middle of the last century growing areas of this crop was reduced dramatically. Nowadays flax attracted a great interest again as a fibre and oil processor, as well as a crop for diversification of agriculture and, in the same time, as a crop for producing high quality fibre for industry, high quantity oil for biofuel and high quality oil for feeding, food, and pharmacy (source of α-linolenic acid, linoleic acid and vitamins). We evaluated all available flax accessions of the Latvian origin and number of their hybrid lines. Agronomically important qualitative and quantitative traits, such as total plant height, technical plant height, number of seed vessels, logging resistance, vegetation period, yield of straws and seeds, 1000 seeds weight, bast fibre, number of seeds in a seed vessel, oil content and rust resistance gene L alleles L2 and L9 were recorded. Results of the field trials of two last years were summarized. Local accessions are useful as a source for breeding to adaptation of flax varieties to the local Latvian conditions. With use of molecular methods flax rust resistance allele L2 and L9 were found in eleven accessions.

CRT1, an Arabidopsis ATPase that Interacts with Diverse Resistance Proteins and Modulates Disease Resistance to Turnip Crinkle Virus

Plant immunity frequently involves the recognition of pathogen-encoded avirulence (avr) factors by their corresponding plant resistance (R) proteins. This triggers the hypersensitive response (HR) where necrotic lesions formed at the site(s) of infection help restrict pathogen spread. HRT is an Arabidopsis R protein required for resistance to turnip crinkle virus (TCV). In a genetic screen for mutants compromised in the recognition of TCV's avr factor, we identified crt1 (compromised recognition of TCV), a mutant that prematurely terminates an ATPase protein. Following TCV infection, crt1 developed a spreading HR and failed to control viral replication and spread. crt1 also suppressed HR-like cell death induced by ssi4, a constitutively active R protein, and by Pseudomonas syringae carrying avrRpt2. Furthermore, CRT1 interacts with HRT, SSI4, and two other R proteins, RPS2 and Rx. These data identify CRT1 as an important mediator of defense signaling triggered by distinct classes of R proteins.

Pyramiding of alleles with different rust resistance specificities in Linum usitatissimum L.

Transgenic flax plants expressing flax rust resistance specificities conferred by L2 and L10 alleles of L locus were produced through Agrobacterium tumefaciens-mediated transformation of hypocotyl segments or anther culture derived-calli. Transgenic plants were characterized by PCR amplification and Southern hybridization. Homozygous transgenic lines containing a single locus of effective transgene were isolated by consecutive progeny analyses of transgenic plants. In addition, homozygous lines were directly obtained from transformation of haploid cells followed by spontaneous or artificial chromosome doubling when anther culture derived-calli were used as the explants. All transgenic plants containing L2 transgene expressed L2 flax rust resistance specificity and had unambiguous infection type (IT) “0” (immune) reactions to flax rust race 22, which is virulent to endogenous L6 and K1 genes present in the untransformed Linola™1084. Transgenic plants containing L10 transgene exhibited various levels of resistance to flax rust race 191. Five plants had IT “fleck” (immune) reactions, similar to the reactions with the L10 rust differential line, Bolley Golden Selection. The enhanced resistance to rust race 191 in these transgenic plants was attributed to the expression of L10 rust resistance specificity. Further evaluation of the transgenic plants containing L2 or L10 transgene to flax rust race 258 and race 247 respectively showed that the endogenous rust resistance specificities were not modified. The implication of this study in producing transgenic flax plants with multiple resistance specificities and for crop improvement using molecular breeding strategy is discussed.

Flax Rust Resistance Gene Specificity is Based on Direct Resistance-Avirulence Protein Interactions

Genetic studies of the flax-flax rust interaction led to the formulation of the gene-for-gene hypothesis and identified resistance genes (R) in the host plant and pathogenicity genes, including avirulence (Avr) and inhibitor of avirulence genes (I), in the rust pathogen. R genes have now been cloned from four of the five loci in flax and all encode proteins of the Toll, Interleukin-1 receptor, R gene-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) class. Avr genes have been cloned from four loci in flax rust and encode small secreted proteins with no between locus similarity and no close homologs in current data bases. It is postulated that Avr proteins enter the host cell, have virulence effector functions, and in resistant host genotypes, are recognized by direct and specific interaction with host R proteins, leading to activation of rust resistance defense responses. Direct interaction between R and Avr proteins is the basis of gene-for-gene specificity in the flax-flax rust system and both R and Avr genes have the signatures of diversifying selection, suggesting the existence of a coevolutionary arms race between the host plant and its obligate rust pathogen.

The M flax rust resistance pre-mRNA is alternatively spliced and contains a complex upstream untranslated region

Alternative splicing is an important step in controlling gene expression and has been shown to occur for a number of plant disease resistance (R) genes. The specific biological role of alternatively spliced transcripts from most R genes is unknown, yet in two cases it is clear that functional disease resistance cannot be activated without them. We report 12 splice isoforms of the M flax rust resistance gene, a TIR-NBS-LRR class of R gene. Collectively, these isoforms are predicted to encode at least nine different polypeptide products, only one of which is a full length peptide believed to confer functional M gene-specific disease resistance. An additional intron to that previously described was found in the 5' untranslated region. Splicing of this leader intron removes an upstream ORF (muORF) sequence. In some transcripts the leader intron is retained and in this case we predict negligible translation initiation of the full length M gene-encoding ORF. The majority of the alternatively spliced isoforms of M would encode truncated TIR and TIR-NBS containing proteins. Although the role of alternative splicing and the existence and function of the products they encode is still unclear, the complexities of the splicing profile, and the 5' UTR of the M gene, are likely to serve in mechanisms to regulate R protein levels.

Purification of the M flax-rust resistance protein expressed in Pichia pastoris

The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.

Further analysis of gene‐for‐gene disease resistance specificity in flax

SUMMARY The flax rust resistance gene L, a nucleotide binding site, leucine-rich repeat (NBS-LRR) class of plant resistance gene, has 12 characterized alleles with different gene-for-gene resistance specificities. Here the specificities of presumptive L1, L5, L8 and L11 genomic clones are confirmed by transgenic expression. L6 and L11 differ by 33 amino acids, 32 in the LRR region and one in the C-terminal non-LRR region, and recognize unrelated avirulence proteins, AvrL567 and AvrL11, respectively. To analyse the specificity differences, 13 L6L11 recombinant genes were constructed in vitro and tested in transgenic flax for resistance to F2 progeny of rust strain CH5, in which the unlinked avirulence genes AvrL567 and AvrL11 segregate. The data show that the single C-terminal non-LRR region polymorphism is not involved in L6-L11 specificity differences, that polymorphisms necessary for specificity are spread throughout the LRR region and that some polymorphisms essential for L11 are not essential for L6. Seven 'null' recombinants expressed no resistance when tested with CH5-derived rusts. These were tested for new resistance specificities by inoculation with a strain of rust, Bs-1, which is distantly related to CH5 and which potentially carries a different range of avirulence specificities. The 'null' recombinant L6L11RV, which differs from L6 and L11 by its susceptibility to CH5, was resistant to strain Bs-1. The specificity difference is due to a reduction in the number of AvrL567 variants recognized by L6L11RV compared with L6 and not due to recognition of an unrelated Avr gene product in strain Bs-1.

Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation of defense responses.

Tobacco was transformed with three different alleles (L2, L6, and L10) of the flax rust resistance gene L, a member of the toll interleukin-1 receptor, nucleotide-binding site, leucine-rich repeat (TIR-NBS-LRR) class of plant disease resistance genes. L6 transgenics had a stunted phenotype, expressed several defense response genes constitutively, and had increased resistance to the fungus Cercospora nicotianae and the oomycete Phytophthora parasitica pv. nicotianae. L2 and L10 transgenics, with one exception for L10, did not express these phenotypes, indicating that the activation of tobacco defense responses is L6 allele-specific. The phenotype of the exceptional L10 transgenic plant was associated with the presence of a truncated L10 gene resulting from an aberrant T-DNA integration. The truncated gene consisted of the promoter, the complete TIR region, and 39 codons of the NBS domain fused inframe to a tobacco retrotransposon-like sequence. A similar truncated L10 gene, constructed in vitro, was transiently expressed in tobacco leaves and gave rise to a strong localized necrotic reaction. Together, these results suggest that defense signaling properties of resistance genes can be expressed in an allele-specific and pathogen-independent manner when transferred between plant genera.

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.

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

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

Abstracts, The Canadian Phytopathological Society, annual meeting, Montreacute;al, Quebec, 1999

Abstracts, The Canadian Phytopathological Society, annual meeting, Montreacute;al, Quebec, 1999

The identification of a cleaved amplified polymorphic sequence (CAPS) marker for the flax rust resistance gene M3

A cleaved amplified polymorphic sequence (CAPS) marker has been developed for the flax rust resistance gene M3. This molecular marker was identified in a rust differential for M3 by screening primer sets, whose sequences were based on the nucleotide sequence of the M gene. One primer set was identified that co-amplified, along with other related sequences, a 2.9-kb DNA segment of M3 or an M-like gene tightly linked to the M3 locus. Restriction fragment analysis revealed several unique DNA fragments that were linked to the presence of the M3 gene in a F2 segregating population. This molecular marker along with recently developed markers for the L2, L6, L9, and L11 alleles will be useful for marker-assisted selection in breeding programs for obtaining multigenic resistance against flax rust in Canadian flax cultivars.

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.

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

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

A flax transposon identified in two spontaneous mutant alleles of the L6 rust resistance gene.

Two spontaneous mutant alleles of the L6 flax rust resistance gene, 16-X3A and 16-X117, contain the same transposable element designated dLute (defective Linum usitatissimum transposable element). The element is 314 bp long, 70% AT-rich and, because it contains no extended open reading frame, is probably non-autonomous. It has 14 bp imperfect terminal inverted repeats related to those in the Ac family of plant transposons and, like Ac, causes 8 bp target site duplications upon insertion. Multiple copies of dLute-related sequences exist in the flax genome. Rust resistant revertants were recovered amongst the progeny of both mutants and reversion was associated with excision of dLute. Excision either restored the wild-type L6 sequence or was imprecise, leaving sequence alterations ('footprints') resulting in one to three amino acid alterations in the L6 protein. No phenotypic differences were discerned between plants containing the standard and revertant L6 alleles.

Abstracts, The Canadian Phytopathological Society, annual meeting, Winnipeg, Manitoba, July 1997

Abstracts, The Canadian Phytopathological Society, annual meeting, Winnipeg, Manitoba, July 1997

Inactivation of the Flax Rust Resistance Gene M Associated with Loss of a Repeated Unit within the Leucine-Rich Repeat Coding Region

Inactivation of the Flax Rust Resistance Gene M Associated with Loss of a Repeated Unit within the Leucine-Rich Repeat Coding Region

Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region.

The M rust resistance gene from flax was cloned after two separate approaches, an analysis of spontaneous M mutants with an L6 gene-derived DNA probe and tagging with the maize transposon Activator, independently identified the same gene. The gene encodes a protein of the nucleotide binding site leucine-rich repeat class and is related (86% nucleotide identity) to the unlinked L6 rust resistance gene. In contrast to the L locus, which contains a single gene with multiple alleles, approximately 15 related genes occur at the complex M locus, with only one encoding the M resistance specificity. The M protein contains two direct repeats of 147 and 149 amino acids in the C-terminal part of the leucine-rich region. Three mutant alleles of M encoding a product containing a single repeat unit of 154 amino acids were isolated. The mutant DNA sequences probably occurred by unequal intragenic exchange in the coding region of the repeats. The recombinant alleles lost M resistance and gained no detectable new resistance specificity.

The L6 gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and the tobacco viral resistance gene N.

The L6 rust resistance gene from flax was cloned after tagging with the maize transposable element Activator. The gene is predicted to encode two products of 1294 and 705 amino acids that result from alternatively spliced transcripts. The longer product is similar to the products of two other plant disease resistance genes, the tobacco mosaic virus resistance gene N of tobacco and the bacterial resistance gene RPS2 of Arabidopsis. The similarity involves the presence of a nucleotide (ATP/GTP) binding site and several other amino acid motifs of unknown function in the N-terminal half of the polypeptides and a leucine-rich region in the C-terminal half. The truncated product of L6, which lacks most of the leucine-rich C-terminal region, is similar to the truncated product that is predicted from an alternative transcript of the N gene. The L6, N, and RPS2 genes, which control resistance to three widely different pathogen types, are the foundation of a class of plant disease resistance genes that can be referred to as nucleotide binding site/leucine-rich repeat resistance genes.

The L6 Gene for Flax Rust Resistance Is Related to the Arabidopsis Bacterial Resistance Gene RPS2 and the Tobacco Viral Resistance Gene N

The L6 Gene for Flax Rust Resistance Is Related to the Arabidopsis Bacterial Resistance Gene RPS2 and the Tobacco Viral Resistance Gene N