Genetic and physical mapping of Lrk10-like receptor kinase sequences in hexaploid oat (Avena sativa L.).

Oat receptor-like kinase gene sequences, homologous to the Lrk10 gene from wheat (Triticum aestivum L.), were mapped in oat (Avena sativa L.). PCR primers designed from the wheat Lrk10 were used to produce ALrk10 from oat. Two DNA sequences, ALrk1A1 and ALrk4A5, were produced from primers designed from coding and noncoding regions of ALrk10. Their use as RFLP probes indicated that the kinase genes mapped to four loci on different hexaploid oat 'Kanota' x 'Ogle' linkage groups (4_12, 5, 6, and 13) and to a fifth locus unlinked to other markers. Three of these linkage groups contain a region homologous to the short arm of chromosome I of wheat and the fourth contains a region homologous to chromosome 3 of wheat. Analysis with several nullisomics of oat indicated that two of the map locations are on satellite chromosomes. RFLP mapping in a 'Dumont' x 'OT328' population indicated that one map location is closely linked to Pg9, a resistance gene to oat stem rust (Puccinia graminis subsp. avenae). Comparative mapping indicates this to be the region of a presumed cluster of crown rust (Puccinia coronata subsp. avenae) and stem rust resistance genes (Pg3, Pg9, Pc44, Pc46, Pc50, Pc68, Pc95, and PcX). The map position of several RGAs located on KO6 and KO3_38 with respect to Lrk10 and storage protein genes are also reported.     

Nitric oxide and reactive oxygen species do not elicit hypersensitive cell death but induce apoptosis in the adjacent cells during the defense response of oat

Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2 probe DCF demonstrated that NO and H2O2 were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2 accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2 by interacting with O2- in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2- but not H2O2 are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.     

A restriction fragment length polymorphism based linkage map of a diploid Avena recombinant inbred line population.

A population of 100 F6-derived recombinant inbred lines was developed from the cross of two diploid (2n = 14) Avena accessions, CI3815 (A. strigosa) and C11994 (A. wiestii). Restriction fragment length polymorphism (RFLP) probes previously mapped in other grass species were used to develop a framework linkage map suitable for comparative genetics. Nine linkage groups were identified among the 181 loci mapped, with an average interlocus distance of 5 cM, and a total genetic map length of 880 cM. A cluster of five tightly linked crown rust resistance genes (Pca) was localized on the map, as were five loci identified by disease resistance gene analogs from maize, sorghum, and wheat. None of the five loci identified by the gene analogs were linked to the Pca locus. The linkage map was compared with previously published diploid and hexaploid linkage maps in an attempt to identify homologous or homoeologous chromosomes between populations. Locus orders and linkage relationships were poorly conserved between the A. strigosa x A. wiestii map and other Avena maps. In spite of mapping complications due to duplications within a basic genome a well as the allopolyploid constitution of many Avena species, such map comparisons within Avena provide further evi dence of substantial chromosomal rearrangement between species within Avena.     

Autoactive alleles of the flax L6 rust resistance gene induce non-race-specific rust resistance associated with the hypersensitive response

L6 is a nucleotide binding site-leucine rich repeat (NBS-LRR) gene that confers race-specific resistance in flax (Linum usitatissimum) to strains of flax rust (Melampsora lini) that carry avirulence alleles of the AvrL567 gene but not to rust strains that carry only the virulence allele. Several mutant and recombinant forms of L6 were made that altered either the methionine-histidine-aspartate (MHD) motif conserved in the NBS domain of resistance proteins or exchanged the short domain C-terminal to the LRR region that is highly variable among L allele products. In transgenic flax some of these alleles are autoactive; they cause a gene dosage-dependent dwarf phenotype and constitutive expression of genes that are markers for the plant defense response. Their effects and penetrance ranged from extreme to mild in their degree of plant stunting, survival, and reproduction. Dwarf plants were also resistant to flax rust strains virulent to wild-type L6 plants, and this nonspecific resistance was associated with a hypersensitive response (HR) at the site of rust infection. The strongest autoactive allele, expressed in Arabidopsis from an ethanol-inducible promoter, gave rise to plant death dependent on the enhanced disease susceptibility 1 (EDS1) gene, which indicates that the mutant flax (Linaceae) L6 gene can signal cell death through a defined disease-resistance pathway in a different plant family (Brassicaceae).     

Restriction fragment length polymorphisms linked to genes for resistance to crown rust (Puccinia coronata) in near-isogenic lines of hexaploid oat (Avena sativa).

Crown rust, perhaps the most important fungal disease of oat, is caused by Puccinia coronata. An examination of near-isogenic lines (NILs) of hexaploid oat (Avena sativa) was conducted to identify markers linked to genes for resistance to crown rust. These lines were created such that a unique resistance gene is present in each of the two recurrent parent backgrounds. The six NILs of the current study, X434-II, X466-I, and Y345 (recurrent parent C237-89) and D486, D494, and D526 (recurrent parent Lang), thus provide a pair of lines to study each of three resistance genes. Restriction fragment length polymorphisms and resistance loci were mapped using BC1F2 populations. Three markers were found linked to a locus for resistance to crown rust race 203, the closest at 1.9 cM in line D494 and 3.8 cM in line X466-I. In lines D526 and Y345 a marker was placed 1.0 and 1.9 cM, respectively, from the locus conferring resistance to crown rust race 345, and in D486 and X434-II a marker mapped at 8.0 and 10.2 cM from the locus for resistance to rust race 264B.     

Reactive Oxygen Species are not Increased in Resistant Oat Genotypes Challenged by Crown Rust Isolates

Crown rust (Puccinia coronata Corda f.sp. avenae) can devastate oats (Avena sativa). Oxidative stress is part of the resistance mechanism in several pathosystems, but in the oat–crown rust system, it is unclear, especially regarding partial resistance. We evaluated the effects of P. coronata on oxidative stress in oat cultivars: URS 21 (partially resistant), Leggett (race‐specific resistant), URS22 and Clintland 64 (susceptibles). Seedlings and plants were inoculated with P. coronata uredospores. Cultivars were assessed for antioxidant enzyme activity and the reactive oxygen species (ROS) hydrogen peroxide and superoxide. Due to the importance of the partial resistance of URS21, this cultivar and URS 22 were also appraised for total phenolics and the relative expression of oxidative stress genes. Postinoculation, Leggett and URS 21 showed no increased peroxide levels. The susceptible cultivars increased ROS and ascorbate peroxidase activity. Clintland 64 increased also catalase activity, whereas URS 22 increased glutathione reductase and the expression of genes encoding antioxidant enzymes. URS 21 showed almost no antioxidant enzyme induction. Shortly after inoculation, URS 21 showed increased expression of genes encoding lipoxygenase and peroxidase. Cultivars URS 21 and Leggett accumulated cell wall fluorescent compounds, phenolics being detected in the former. Oxidative stress appears not to cause the hypersensitive response in this pathosystem, but late ROS accumulation did occur in the susceptible cultivars. Cultivar URS 21 may, differently from other known mechanism to date, reduce ROS accumulation by increasing the level of phenolics, resulting in later pathogen and cell death, showing non‐specific resistance to races of the pathogen also at seedling stage.     

Incidence of crown rust and virulence of puccinia coronata cda. f.sp. avenae eriks. and the effectiveness of pc genes for resistance in europe,middle east and north africa: Inzidenz von kronenrost und virulenz von puccinia coronata cda. f.sp. avenae eriks. und die wirksamkeit von pc-genen für resistenz in europa,nahost und nordafrika

Crown rust on oat incited by Puccinia coronata Cda f.sp. avenae is a wide-spread disease in Europe, Middle East (Israel) and North Africa (Morocco). High natural levels of the disease were recorded in Austria, Bulgaria, Czech Republic, Estonia, Israel, Italy, Poland, Russia and the former Yugoslavia. The severity of the disease in the European and Mediterranean Oat Disease Nursery (EMODN) between 1995 and 1999 showed that it occurred at damaging levels in a number of countries. Considerable differences in a disease resistance index (DRI) to crown rust among 67 oat lines in the period 1995?–?2000 were found. The values of the DRI ranged from18 (KR 8122) to over 290 (Pc 59, Pc 68). Detailed studies of the virulence combinations of P.coronata f.sp. avenae in Europe, Middle East and North Africa were carried out between 1995?–?2001. There were considerable differences in the average number of virulence among countries. Virulence to Pc 39 and 68 was found for the first time and is a significant finding. Nevertheless the major genes Pc 68, Pc 39, Pc 50-2, Pc Pc 59, Pc 60, Pc 61 proved to be highly effective.     

Comparative mapping of homoeologous group 1 regions and genes for resistance to obligate biotrophs in Avena, Hordeum, and Zea mays.

The colinearity of markers linked with resistance loci on linkage group A of diploid oat, on the homoeologous groups in hexaploid oat, on barley chromosome 1H, and on homoeologous maize chromosomes was determined. Thirty-two DNA probes from homoeologous group 1 chromosomes of the Gramineae were tested. Most of the heterologous probes detected polymorphisms that mapped to linkage group A of diploid oat, two linkage groups of hexaploid oat, barley chromosome 1H, and maize chromosomes 3, 6, and 8. Many of these DNA markers appeared to have conserved linkage relationships with resistance and prolamin loci in Avena, Hordeum, and Zea mays. These resistance loci included the Pca crown rust resistance cluster in diploid oat, the R203 crown rust resistance locus in hexaploid oat, the Mla powdery mildew resistance cluster in barley, and the rp3, wsm1, wsm2, mdm1, ht2, and htn1 resistance loci in maize. Prolamin encoding loci included Avn in diploid oat and Hor1 and Hor2 in barley. A high degree of colinearity was revealed among the common RFLP markers on the small chromosome fragments among these homoeologous groups. Key words : disease resistance, colinearity, Gramineae, cereals.     

Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust

Seven-day-old seedlings of the near-isogenic wheat ( Triticum aestivum L.) lines Prelude and Prelude-Sr5, susceptible and resistant to wheat stem rust, respectively, were inoculated with uredospores of the oat crown rust fungus Puccinia coronata Cda. f. sp. avenae Fraser & Led. Fluorescence microscopy revealed that the majority of colonies developed intercellular infection structures including haustorial mother cells and haustoria after penetration of wheat mesophyll cells. All penetrated cells became necrotic, and exhibited bright yellow autofluorescence. This autofluorescence was not extractable with alkali, and fluorescent cells stained positively with phloroglucinol/HCI, suggesting that hypersensitive cell death was correlated with cellular lignification. Accordingly, the lignin biosynthetic enzymes phenylalanine ammonia-lyase (EC4.3.1.5). 4-coumarate:CoA ligase (EC6.2.1.12), cinnamyl-alcohol dehydrogenase (EC1.1.1.149), and peroxidases (EC1.11.1.7) increased in activity during the expression of resistance. The induced pattern of peroxidase iso/ymes closely resembled that observed for highly incompatible wheat/wheat stem rust interactions. Furthermore, an elieitor was extracted from oat crown rust germlings. which induces lignification when injected into the intercellular space of wheat leaves. This elieitor appears to be functionally similar to that isolated from wheat stem rust germlings. The results suggest that the non-host resistance of wheat to the xenopara-site oat crown rust closely resembles the race/cullivar-speeific resistant mechanism of highly resistant wheat varieties to wheat stem rust.     

Identification and genetic characterisation of adult plant resistance to crown rust in diploid and tetraploid accessions of Avena

The identification and genetic characterisation of adult plant resistance (APR) to crown rust, caused by Puccinia coronata f. sp. avenae (Pca), was carried out in diploid Avena strigosa and tetraploid Avena barbata accessions from diverse geographical regions. Seven accessions were found to carry APR to Pca, six of which (CIav6956, CIav7280, CIav8089, CIav9020, PI292226, PI436082) were diploid and one (PI337865) a tetraploid. All six diploid A. strigosa accessions were postulated to carry the ‘Saia’ seedling resistance to Pca (Pc15, Pc16, Pc17) in addition to the APR. Three of these six accessions (CIav6956, CIav9020, PI292226) were used to study both seedling resistance and APR, using two Pca pathotypes, one avirulent on seedlings and the second virulent on seedlings but avirulent on adult plants. The seedling resistance in each was shown to be inherited independently of the APR. In each case, APR was conferred by a single major dominant gene, based on hypersensitivity, coupled with low infection types. Allelism tests are required to determine if these three APR genes are different. This is the first report of APR to crown rust in A. strigosa and A. barbata.     

Four QTLs determine crown rust (Puccinia coronata f. sp. lolii) resistance in a perennial ryegrass (Lolium perenne) population

Crown rust resistance is an important selection criterion in ryegrass breeding. The disease, caused by the biotrophic fungus Puccinia coronata, causes yield losses and reduced quality. In this study, we used linkage mapping and QTL analysis to unravel the genomic organization of crown rust resistance in a Lolium perenne population. The progeny of a pair cross between a susceptible and a resistant plant were analysed for crown rust resistance. A linkage map, consisting of 227 loci (AFLP, SSR, RFLP and STS) and spanning 744 cM, was generated using the two-way pseudo-testcross approach from 252 individuals. QTL analysis revealed four genomic regions involved in crown rust resistance. Two QTLs were located on LG1 (LpPc4 and LpPc2) and two on LG2 (LpPc3 and LpPc1). They explain 12.5, 24.9, 5.5 and 2.6% of phenotypic variance, respectively. An STS marker, showing homology to R genes, maps in the proximity of LpPc2. Further research is, however, necessary to check the presence of functional R genes in this region. Synteny at the QTL level between homologous groups of chromosomes within the Gramineae was observed. LG1 and LG2 show homology with group A and B chromosomes of oat on which crown rust-resistance genes have been identified, and with the group 1 chromosomes of the Triticeae, on which leaf rust-resistance genes have been mapped. These results are of major importance for understanding the molecular background of crown rust resistance in ryegrasses. The identified markers linked to crown rust resistance have the potential for use in marker-assisted breeding.     

An anchored AFLP- and retrotransposon-based map of diploid Avena.

A saturated genetic map of diploid oat was constructed based on a recombinant inbred (RI) population developed from a cross between Avena strigosa (Cereal Introduction, C.I. 3815) and A. wiestii (C.I. 1994). This 513-locus map includes 372 AFLP (amplified fragment length polymorphism) and 78 S-SAP (sequence-specific-amplification polymorphism) markers, 6 crown-rust resistance loci, 8 resistance-gene analogs (RGAs), one morphological marker, one RAPD (random amplified polymorphic DNA) marker, and is anchored by 45 grass-genome RFLP (restriction fragment length polymorphism) markers. This new A. strigosa x A. wiestii RI map is colinear with a diploid Avena map from an A. atlantica x A. hirtula F2 population. However, some linkage blocks were rearranged as compared to the RFLP map derived from the progenitor A. strigosa x A. wiestii F2 population. Mapping of Bare-1-like sequences via sequence-specific AFLP indicated that related retrotransposons had considerable heterogeneity and widespread distribution in the diploid Avena genome. Novel amplified fragments detected in the RI population suggested that some of these retrotransposon-like sequences are active in diploid Avena. Three markers closely linked to the Pca crown-rust resistance cluster were identified via AFLP-based bulk-segregant analysis. The derived STS (sequence-tagged-site) marker, Agx4, cosegregates with Pc85, the gene that provides resistance specificity to crown-rust isolate 202 at the end of the cluster. This framework map will be useful in gene cloning, genetic mapping of qualitative genes, and positioning QTL (quantitative trait loci) of agricultural importance.     

Systemic acquired resistance is induced by R gene-mediated responses independent of cell death

On infection by pathogens, plants initiate defence responses that are able to curtail infection locally. These responses are mediated either by receptor-like proteins that recognize pathogen-associated molecular patterns or by the protein products of disease resistance ( R ) genes. At the same time, primary defence responses often result in the generation of signals that induce what is known as systemic acquired resistance (SAR), such that defence responses are enhanced on secondary pathogen challenge in distal tissues. R protein-mediated SAR induction is normally accompanied by a type of programmed cell death known as the hypersensitive response (HR) and, in some instances, cell death alone has been implicated in the induction of SAR. This has raised the question of whether R protein-mediated signalling per se induces SAR or whether SAR is an indirect result of the induction of HR. Using the Rx gene of potato, which confers resistance to Potato Virus X in the absence of cell death, we have shown that the HR is dispensable for R protein-mediated induction of SAR and that Rx-induced SAR is mediated by the same salicylate-dependent pathway induced by other R proteins.     

Chromosome location and allele-specific PCR markers for marker-assisted selection of the oat crown rust resistance gene Pc91

Race-specific seedling resistance genes are the primary means of controlling crown rust of oat caused by Puccinia coronata Corda f. sp. avenae Eriks in Canada. Pc91 is a seedling crown rust resistance gene that is highly effective against the current crown rust population in North America. A number of race-specific resistance genes have been mapped and markers that are closely linked to them have been identified. However, the use of these markers in oat breeding has been limited by the economics of marker-assisted selection (MAS). A crucial step in the successful application of MAS in breeding programs is the development of inexpensive and easy-to-use molecular markers. The primary objective of this study was to develop co-dominant KBioscience competitive allele-specific PCR (KASP) markers linked to Pc91 for deployment in high-throughput MAS in oat breeding programs. The allele-specific marker showed consistent diagnostic polymorphism between the selected 16 North American oat breeding lines. The developed co-dominant marker was also validated on three F₂ populations (AC Morgan × Stainless; SW Betania × Stainless; AC Morgan × CDC Morrison) and one recombinant inbred line population (CDC Sol-Fi × HiFi) segregating for Pc91 using KASP genotyping technology. We recommend the simple, low-cost marker as a powerful tool for pyramiding Pc91 with other effective crown rust resistance loci into a single line. The mapping results indicate that crown rust resistance gene Pc91 resides on the translocated oat chromosome 7C-17A.     

Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.     

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.     

Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria

The hypersensitive response (HR) involves rapid death of cells at the site of pathogen infection and is thought to limit pathogen growth through the plant. Ethylene regulates senescence and developmental programmed cell death, but its role in hypersensitive cell death is less clear. Expression of two ethylene receptor genes, NR and LeETR4, is induced in tomato (Lycopersicon esculentum cv. Mill) leaves during an HR to Xanthomonas campestris pv. vesicatoria, with the greatest increase observed in LeETR4. LeETR4 antisense plants previously were shown to exhibit increased sensitivity to ethylene. These plants also exhibit greatly reduced induction of LeETR4 expression during infection and an accelerated HR at inoculum concentrations ranging from 10(5) to 10(7) CFU/ml. Increases in ethylene synthesis and pathogenesis-related gene expression are greater and more rapid in infected LeETR4 antisense plants, indicating an enhanced defense response. Populations of avirulent X. campestris pv. vesicatoria decrease more quickly and to a lower level in the transgenic plants, indicating a greater resistance to this pathogen. Because the ethylene action inhibitor 1-methylcyclopropene alleviates the enhanced HR phenotype in LeETR4 antisense plants, these changes in pathogen response are a result of increased ethylene sensitivity.     

Identification of the stem rust resistance gene Pg9 and its association with crown rust resistance and endosperm proteins in 'Dumont' oat.

The Canadian oat cultivar 'Dumont' is known to have genes Pc38 and Pc39 for crown rust resistance and genes Pg2 and Pg13 for stem rust resistance. When crossed to a susceptible oat line OT328, 'Dumont' was shown to have an additional dominant gene for crown rust resistance, designated PcX. Tests of segregating progeny indicated that the stem rust resistance gene Pg9 is present and is tightly linked in coupling to PcX. The presence of Pg9 in 'Dumont' was confirmed in crosses involving the cultivar 'Ukraine', which has Pg9 and a crown rust resistance gene tightly linked to it. The association of rust resistance with endosperm proteins in 'Dumont' was investigated. The linkage of gene Pg13 with a 56.6-kDa polypeptide locus (map distance of 10.47 +/- 2.70 cM) was demonstrated using sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE). A 27.9-kDa polypeptide was shown to be associated with the linked PcX/Pg9 loci by SDS-PAGE but appeared to be more reliably separated as an avenin band, designated B4, using acid-PAGE. Another avenin band, designated B2, also was shown to be associated with the PcX/Pg9 loci using acid-PAGE. The loci conditioning the B2 and B4 bands appeared to be tightly linked or allelic and are separated from the linked PcX/Pg9 loci by a map distance of 1.03 +/- 0.36 cM. The association of Pg13 with a 56.6-kDa polypeptide and the tight linkage between PcX/Pg9 and the B2 (in coupling) and B4 (in repulsion) avenin loci offer a useful tool to breeders to detect the presence of these genes in oat breeding.     

Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.