Resistance to Bremia lactucae in natural populations of Lactuca saligna from some Middle Eastern countries and France

The results of the first detailed screening of a resistance to Bremia lactucae in naturally growing populations of Lactuca saligna are presented here. In total, 146 accessions from 25 populations of L. saligna originating in Israel (N = 136), France (N = 8), Jordan (N = 1) and Turkey (N = 1) were tested at seedling stage for their resistance to 10 highly virulent isolates (races) of B. lactucae from Lactuca sativa (DEG2, Bl:5, Bl:15, Bl:16, Bl:17, Bl:18, Bl:21, Bl:22, Bl:24 and Bl:25). Our study strongly supports the suggestion that L. saligna is indeed generally highly resistant to B. lactucae. However, our results provide evidence that at least at a seedling stage L. saligna may not be a non‐host plant for B. lactucae, as was hypothesised for approximately the last 30 years. Some accessions expressed a differential (i.e. race‐specific) response, which accords with other recently published data for this Lactuca species. Furthermore, some geographical differences in race‐specific resistance were observed, too. Tests performed at an adult‐plant stage, however, did not prove race‐specificity of the respective accessions. To summarise, what is behind the race‐specific character of the responses observed at a seedling stage is still uncertain, as is its comparability with the race‐specific resistance of some other Lactuca species such as L. sativa or L. serriola. The presence of plant stage‐dependent resistance, governed by a combined effect of different quantitative trait loci in young and adult plants of L. saligna, is discussed.     

The genetic relationship between races of Bremiae lactucae and cultivars of Lactuca sativa

The reactions of lettuce cultivars to physiologic races of Bremia lactucae are interpreted in terms of a gene-for-gene relationship between pathogen and host. The hypothesis takes into account the parentage of cultivars and the origins of their resistance, the characteristics of the resistance reactions and data available from detailed genetical analysis of various race/cultivar combinations. Cultivars are classified with respect to ten postulated resistance genes and B. lactucae races are defined by the virulence genes present. The practical significance of these studies is discussed in relation to both future lettuce breeding programmes and to the choice of cultivars available to counteract any given local race situation.     

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor

Plant‐pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface‐localized receptor Ve1 confers race‐specific resistance against race 1 strains of the soil‐borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.     

Further work on the genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae)

Data are presented on the segregation of resistance to four races of Bremia lactucae in the F2 progenies of crosses involving 15 resistant and various susceptible lettuce cultivars. Most of these data and those recently published by other workers fit the systematic model for the genetics of race specific resistance to B. lactucae proposed by Crute & Johnson(1976). Seven different dominant resistance genes of major effect were found. There was also evidence of a pair of dominant genes with complementary effect, one of which may sometimes be effective on its own. Two of the genes may be linked and another may have two different alleles for resistance at the same locus. The resistance conferred by these genes is specified in relation to two British, two Dutch and four French races of the fungus. Resistance genotypes are proposed for 16 cultivars.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

The genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae)

Data are presented on the segregation of resistance to five British races and two Dutch races of Bremia lactucae in the F₂ progenies of crosses involving seven resistant and several susceptible lettuce cultivars. These data and also those previously published by other workers are considered in relation to the systematic model proposed by Crute & Johnson (1976) to explain the genetics of race specific resistance to B. lactucae in lettuce. It is shown that, with minor modifications, the model accommodates almost all of the previously published data and correctly predicts the new data, except for one set which cannot at present be interpreted. It is concluded that genetic evidence exists for the presence, among various cultivars of lettuce, of at least four and possibly five different dominant resistance genes of major effect designated Dm₂, Dm₃, Dm₄, Dm₆ and Dm₈; and of a pair of dominant genes with complementary effect designated Dm7/1 and Dm7/2. The resistance conferred by these genes is specified in relation to five British races, five Dutch, three Israeli and one United States race of the fungus. Resistance genotypes are proposed for cultivars Avoncrisp, Avondefiance, Calmar, Great Lakes 659, Kares, Meikoningen, Mildura, Proeftuins Blackpool, Solito, Valverde, Ventura and the USDA line PI 164937.     

Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis,causal agent of the black leaf streak disease of banana

Previously, we have determined the nonhost‐mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf‐4 and Cf‐Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2‐2 and MfEcp2‐3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf‐4 and Cf‐Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co‐evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South‐East Asia relative to strains from other banana‐producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian‐Pacific region. Furthermore, transient co‐expression of the MfAvr4 effector alleles and the tomato Cf‐4 resistance gene, as well as of MfEcp2, MfEcp2‐2 and MfEcp2‐3 and the putative Cf‐Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field.     

Sunflower resistance to multiple downy mildew pathotypes revealed by recognition of conserved effectors of the oomycete Plasmopara halstedii

Over the last 40 years, new sunflower downy mildew isolates (Plasmopara halstedii) have overcome major gene resistances in sunflower, requiring the identification of additional and possibly more durable broad‐spectrum resistances. Here, 354 RXLR effectors defined in silico from our new genomic data were classified in a network of 40 connected components sharing conserved protein domains. Among 205 RXLR effector genes encoding conserved proteins in 17 P. halstedii pathotypes of varying virulence, we selected 30 effectors that were expressed during plant infection as potentially essential genes to target broad‐spectrum resistance in sunflower. The transient expression of the 30 core effectors in sunflower and in Nicotiana benthamiana leaves revealed a wide diversity of targeted subcellular compartments, including organelles not so far shown to be targeted by oomycete effectors such as chloroplasts and processing bodies. More than half of the 30 core effectors were able to suppress pattern‐triggered immunity in N. benthamiana, and five of these induced hypersensitive responses (HR) in sunflower broad‐spectrum resistant lines. HR triggered by PhRXLRC01 co‐segregated with Pl22 resistance in F3 populations and both traits localized in 1.7 Mb on chromosome 13 of the sunflower genome. Pl22 resistance was physically mapped on the sunflower genome recently sequenced, unlike all the other downy mildew resistances published so far. PhRXLRC01 and Pl22 are proposed as an avirulence/resistance gene couple not previously described in sunflower. Core effector recognition is a successful strategy to accelerate broad‐spectrum resistance gene identification in complex crop genomes such as sunflower.     

Identification of specificity-defining amino acids of the wheat immune receptor Pm2 and powdery mildew effector AvrPm2

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) act as intracellular sensors for pathogen-derived effector proteins and trigger an immune response, frequently resulting in the hypersensitive cell death response (HR) of the infected host cell. The wheat (Triticum aestivum) NLR Pm2 confers resistance against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) if the isolate contains the specific RNase-like effector AvrPm2. We identified and isolated seven new Pm2 alleles (Pm2e–i) in the wheat D-genome ancestor Aegilops tauschii and two new natural AvrPm2 haplotypes from Bgt. Upon transient co-expression in Nicotiana benthamiana, we observed a variant-specific HR of the Pm2 variants Pm2a and Pm2i towards AvrPm2 or its homolog from the AvrPm2 effector family, BgtE-5843, respectively. Through the introduction of naturally occurring non-synonymous single nucleotide polymorphisms and structure-guided mutations, we identified single amino acids in both the wheat NLR Pm2 and the fungal effector proteins AvrPm2 and BgtE-5843 responsible for the variant-specific HR of the Pm2 variants. Exchanging these amino acids led to a modified HR of the Pm2–AvrPm2 interaction and allowed the identification of the effector head epitope, a 20-amino-acid long unit of AvrPm2 involved in the HR. Swapping of the AvrPm2 head epitope to the non-HR-triggering AvrPm2 family member BgtE-5846 led to gain of a HR by Pm2a. Our study presents a molecular approach to identify crucial effector surface structures involved in the HR and demonstrates that natural and induced diversity in an immune receptor and its corresponding effectors can provide the basis for understanding and modifying NLR–effector specificity.     

Role of the penetration‐resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race‐specific resistance in Arabidopsis thaliana

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host‐encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall‐based defense against the non‐adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run‐away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall‐based defense against non‐host pathogens.     

Race non-specific resistance to Bremia lactucae

Three ways of measuring race non-specific resistance to Bremia lactucae in lettuce were studied to determine the most important components of this form of resistance. Tests were conducted on leaf discs from 4–6 wk-old plants that were quantitatively inoculated and incubated under controlled conditions. Observations were made of disease intensity, latent period and production of conidia. Inoculum concentration markedly influenced disease intensity and latent period. Cv. Iceberg exhibited race non-specific resistance and this was characterised by a longer latent period, a lower disease intensity and reduced production of conidia compared to the susceptible control cv. Cobham Green. On older, intact plants, the differences in latent period and disease intensity between cultivars were even greater. Production of conidia was a particularly good criterion for discriminating levels of resistance.     

Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.     

Ve1‐mediated resistance against Verticillium does not involve a hypersensitive response in Arabidopsis

The recognition of pathogen effectors by plant immune receptors leads to the activation of immune responses that often include a hypersensitive response (HR): rapid and localized host cell death surrounding the site of attempted pathogen ingress. We have demonstrated previously that the recognition of the Verticillium dahliae effector protein Ave1 by the tomato immune receptor Ve1 triggers an HR in tomato and tobacco. Furthermore, we have demonstrated that tomato Ve1 provides Verticillium resistance in Arabidopsis upon Ave1 recognition. In this study, we investigated whether the co‐expression of Ve1 and Ave1 in Arabidopsis results in an HR, which could facilitate a forward genetics screen. Surprisingly, we found that the co‐expression of Ve1 and Ave1 does not induce an HR in Arabidopsis. These results suggest that an HR may occur as a consequence of Ve1/Ave1‐induced immune signalling in tomato and tobacco, but is not absolutely required for Verticillium resistance.     

Evidence for a race-specific resistance factor in some lettuce (Lactuca sativa L.) cultivars previously considered to be universally susceptible to Bremia lactucae regel

Summary Previously undetected race-specific resistance to Bremia lactucae (downy mildew) was located in many lettuce cultivars hitherto considered to be universally susceptible to this disease. This resistance factor(s) may also be widely distributed in other cultivars known to carry combinations of already recognised factors R1 to R11. Specific virulence to match this resistance is almost invariably present in pathogen collections. This situation may be either a relic of the evolutionary history of the B. lactucae — L. sativa asssociation or may reflect a rare mutation in B. lactucae for avirulence on all but a few specialised L. sativa genotypes.     

Comparative genomics reveals the in planta-secreted Verticillium dahliae Av2 effector protein recognized in tomato plants that carry the V2 resistance locus

Plant pathogens secrete effector molecules during host invasion to promote colonization. However, some of these effectors become recognized by host receptors to mount a defence response and establish immunity. Recently, a novel resistance was identified in wild tomato, mediated by the single dominant V2 locus, to control strains of the soil-borne vascular wilt fungus Verticillium dahliae that belong to race 2. With comparative genomics of race 2 strains and resistance-breaking race 3 strains, we identified the avirulence effector that activates V2 resistance, termed Av2. We identified 277 kb of race 2-specific sequence comprising only two genes encoding predicted secreted proteins that are expressed during tomato colonization. Subsequent functional analysis based on genetic complementation into race 3 isolates and targeted deletion from the race 1 isolate JR2 and race 2 isolate TO22 confirmed that one of the two candidates encodes the avirulence effector Av2 that is recognized in V2 tomato plants. Two Av2 allelic variants were identified that encode Av2 variants that differ by a single acid. Thus far, a role in virulence could not be demonstrated for either of the two variants.     

Rin4 Causes Hybrid Necrosis and Race-Specific Resistance in an Interspecific Lettuce Hybrid

Some inter- and intraspecific crosses may result in reduced viability or sterility in the offspring, often due to genetic incompatibilities resulting from interactions between two or more loci. Hybrid necrosis is a postzygotic genetic incompatibility that is phenotypically manifested as necrotic lesions on the plant. We observed hybrid necrosis in interspecific lettuce (Lactuca sativa and Lactuca saligna) hybrids that correlated with resistance to downy mildew. Segregation analysis revealed a specific allelic combination at two interacting loci to be responsible. The allelic interaction had two consequences: (1) a quantitative temperature-dependent autoimmunity reaction leading to necrotic lesions, lethality, and quantitative resistance to an otherwise virulent race of Bremia lactucae; and (2) a qualitative temperature-independent race-specific resistance to an avirulent race of B. lactucae. We demonstrated by transient expression and silencing experiments that one of the two interacting genes was Rin4. In Arabidopsis thaliana, RIN4 is known to interact with multiple R gene products, and their interactions result in hypersensitive resistance to Pseudomonas syringae. Site-directed mutation studies on the necrosis-eliciting allele of Rin4 in lettuce showed that three residues were critical for hybrid necrosis.     

The hypersensitive reaction, membrane damage and accumulation of autofluorescent phenolics in lettuce cells challenged by Bremia lactucae

The expression of resistance to Bremia lactucae determined by the resistance genes Dm5/8 and Dm7 in lettuce was examined; incompatibility involved the hypersensitive reaction (HR) which occurred only within penetrated cells at early and late stages of fungal development, respectively. Autofluorescence observed under UV and blue light excitation in cells undergoing the HR was associated with the accumulation of ester-linked syringaldehyde and caffeic acid on plant cell walls. Two phases of phenolic deposition were identified. The first was highly localized around penetration points and occurred during incompatible and compatible interactions. The second and major phase was only activated after the occurrence of irreversible membrane damage in the penetrated cell and was reduced by inhibitors of mRNA synthesis. Fungal structures, primary and secondary vesicles, intercellular hyphae and haustoria also became autofluorescent during incompatible interactions. Changes in the fluorescence due to preformed phenolics located in the plant cell vacuole were found just before plasma membrane damage became irreversible during the HR. In addition to localized deposition of phenolics, increases in the concentrations of the major free phenolic esters identified as dicaffeoyl tartaric and chlorogenic acids also occurred during incompatible interactions. The results suggest that membrane damage in penetrated cells occurs at different rates in resistance controlled by Dm5/8 and Dm7 and indicate an important role for irreversible membrane damage in lettuce as a key signalling event leading to widespread activation of defence responses in surrounding cells.     

Characterization of New Highly Virulent German Isolates of Bremia lactucae and Efficiency of Resistance in Wild Lactuca spp. Germplasm

Four German isolates (FS1, SR2, SAW1 and DEG2) of Bremia lactucae originating from lettuce cultivars with R‐factors R18 and Dm6 + R36 were used for detailed characterization of virulence factors (v‐factors) and for the study of the resistance efficiency in wild Lactuca spp. germplasm. The highest complexity of v‐phenotype was recognized in isolate DEG2, which overcomes resistance in cv. Mariska (R18) and line CS‐RL (L. serriola × L. sativa, R18 + ?), until now known as resistant to all known races of B. lactucae in Europe. However, some sparse sporulation also occurred on cv. Titan (Dm6 + R36). The isolates SR2 and SAW1 overcome the resistance based on the gene R36, but are avirulent to R18. The v‐phenotype of SR2 is highly complex with the most important v‐factors being present except for v14 and v18. The isolate FS1 is the first in Germany originating from a cultivar with R18 (cv. Samourai). The search for efficient sources of resistance in 64 accessions of 11 wild Lactuca spp. and primitive forms of L. sativa showed broad variation in accession–isolate interactions. Expression of race‐specific resistance in wild Lactuca spp. (L. serriola, L. viminea, L. virosa) was recorded frequently. Lactuca indica and L. saligna could be considered as the most efficient sources of resistance against isolates FS1, SR2 and SAW1. The isolate DEG2 showed the highest level of virulence. On seedlings of L. saligna, which is considered as a most important source of resistance against B. lactucae, there was frequently recorded limited sporulation, however this response cannot be considered as a susceptible. Except for some L. saligna accessions (CGN 05310 and CGN 05315), the resistance to all studied isolates was only observed in one accession of L. serriola (PI 253467).