The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.     

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.     

The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes

Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single-gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.     

A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection

Late blight caused by the oomycete Phytophthora infestans is the most important fungal disease in potato cultivation worldwide. Resistance to late blight is controlled by a few major genes (R genes) which can be easily overcome by new races of P. infestans and/or by an unknown number of genes expressing a quantitative type of resistance which may be more durable. Quantitative resistance of foliage to late blight was evaluated in five F₁ hybrid families originating from crosses among seven different diploid potato clones. Tuber resistance was evaluated in four of the families. Two of the families were scored for both foliage maturity and vigour. The five families were genotyped with DNA-based markers and tested for linkage with the traits analysed. QTL (quantitative trait locus) analysis identified at least twelve segments on ten chromosomes of potato having genes that affect reproducibly foliage resistance. Two of those segments also have major R genes for resistance to late blight. The segments are tagged by 21 markers that can be analyzed based on PCR (polymerase chain reaction) with specific oligonucleotide primers. One QTL was detected for tuber resistance and one for foliage vigour. Two QTLs were mapped for foliage maturity. Major QTL effects on foliage and tuber resistance to late blight and on foliage maturity and vigour were all linked with marker GP179 on linkage group V of potato. Plants having alleles at this QTL, which increased foliage resistance, exhibited decreased tuber resistance, later maturity and more vigour.     

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.     

Trafficking arms: oomycete effectors enter host plant cells

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.     

Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.     

Potato late blight in Morocco: characterization of Phytophthora infestans populations (virulence and mating type)

Late blight caused by Phytophthora infestans, is the most important disease of potato in Morocco. Use of partially resistant cultivars should be an essential component of a sustainable management strategy of potato late blight, provided the durability of this form of resistance. It is therefore important to determine the nature of P. infestans Moroccan populations. Mating types were determined for 91 strains of P. infestans collected in the northern (Larache-northern plain), north western (Kénitra) and north eastern (Méknès, Middle Atlas) potato cropping areas of Morocco in 1999-2000, 2000-2001 and 2003-2004. They showed a clear regional structure of these populations, with the presence of both mating types (A1 and A2). Of all isolates collected since 1999, A2 mating type constituted 56% (54 isolates), following by A1 mating type (40.7%, 31 isolates) and A1-A2 (self-fertile) mating type (3.30%, 3 isolates). Populations from Méknès and Kénitra consisted mainly of A2 mating type, whereas populations from Larache predominantly included A1 mating type. Physiological race study revealed the presence of 19 races of P. infestans in the first collection of 25 isolates tested between 1999 and 2001. All known virulence genes were detected in western and northern Moroccan isolates, except virulence for resistance genes R2, R5, and R6 which were absent. All isolates were able to overcome two or more R genes except one isolate (5-1) corresponding to race 1.     

Genetic architecture of qualitative and quantitative Melampsora larici-populina leaf rust resistance in hybrid poplar: genetic mapping and QTL detection

In order to elucidate the genetic control of resistance to Melampsora larici-populina leaf rust in hybrid poplars, a Populus deltoides x P. trichocarpa F(1) progeny was analysed for qualitative and quantitative rust resistances. This progeny was evaluated for three components of quantitative resistance (latent period, uredinia number and uredinia size) to seven M. larici-populina strains in controlled conditions, and for one component of field susceptibility (rust colonization on the most infected leaf). One qualitative resistance locus inherited from P. deltoides, R(1), was localized on the genetic map. It segregates 1 : 1 in the F(1) progeny and is effective against four of the studied strains. QTL analysis was performed separately on R(1) and r(1) genotype subsets. An additional detection was conducted on the entire F(1) progeny for the three strains able to overcome R(1) and for MAX2. A total of nine QTLs were detected. Two had large, broad-spectrum effects. One (R(US)) is inherited from the P. trichocarpa parent; the other is inherited from P. deltoides and colocalized with R(1). Seven QTLs had only limited and specific effects. Significant interaction effects were detected mainly between the two major QTLs. Implications of these results for durable resistance breeding strategies, and possible benefits from the Populus genome sequence, are discussed.     

Changes in Photosynthetic Electron Transport in Potato Cultivars with Different Field Resistance after Infection with Phytophthora infestans

The estimation of field resistance of potato cultivars to Phytophthora infestans are usually carried out in expensive and time consuming field experiments over several years. Therefore, a procedure is required for the fast and objective determination of qualitative and quantitative field resistance of new cultivars. This study correlated level of field resistance to P. infestans in leaf discs or leaflets of various potato cultivars to characteristic changes of Chlorophyll-a fluorescence (CF) parameters F _m (maximal fluorescence) and F _v (maximal variable fluorescence). Two different inocula, both containing virulence genes 1-11 were tested. The results were achieved when leaf discs from greenhouse or field plants were each inoculated with a P. infestans spore suspension and incubated for 24 h. In field measurements, comparable results were obtained 48 h after inoculation. The estimation of field resistance by measuring specific CF parameters could be an economical and rapid procedure to reduce or substitute visual lesion assessment for determining cultivar field resistance.     

利用抑制差减杂交技术分离马铃薯晚疫病抗性相关基因

以晚疫病病原菌混合小种接种处理48h的马铃薯水平抗性材料(R-gene-free)叶片为目的材料,以未处理材料作为对照,用抑制差减杂交技术构建了一个富集晚疫病抗性相关基因的差减文库。应用反向Northern技术对840个克隆进行斑点杂交筛选,筛选出150个病原诱导后信号明显增强的克隆。26个片段测序结果表明：部分片段基因功能与抗病性明显相关。7个差异表达片段与GenBank EST数据库中已有晚疫病原诱导马铃薯叶片得到的EST有很高同源性(达95％～100％);部分片段核苷酸或氨基酸序列分别与番茄、烟草、拟南芥等的EST序列或氨基酸序列有较高同源性;另有4个基因片段在GenBank EST数据库中未找到明显的同源序列,可能为新发现的基因片段。     

Inducible Positive Mutant Screening System to Unveil the Signaling Pathway of Late Blight Resistance

Late blight is the most devastating potato disease and it also causes serious yield loss in tomato.Several disease resistance genes (R genes) to late blight have been cloned from potato in the past decade.However,the resistance mechanisms remain elusive.Tomato and potato belong to the botanical family Solanaceace and share remarkably conserved genome structure.Since tomato is a model system in genetic and plant pathology research,we used tomato to develop a powerful mutant screening system that will greatly facilitate the analysis of the signaling pathway of resistance to Phytophthora infestans.First we proved that the R3a transgenic tomatoes developed specific hypersensitive cell death response (HR) to P.infestans strains carrying the corresponding avirulence gene Avr3a,indicating that the signaling pathway from the R3a-Avr3a recognition to HR is conserved between potato and tomato.Second,we generated transgenic tomatoes carrying both R3a and Avr3a genes,with the latter under the control of a glucocorticiod-inducible promoter.Dexamethasone induced expression of Avr3a and resulted in localized HR.This versatile system can be used to construct a mutant library to screen surviving mutants whose resistance signal transduction was interrupted,providing the basis to identify key genes involved in the resistance to late blight in Solanaceae.     

Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ～44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.     

CMPG1-dependent cell death follows perception of diverse pathogen elicitors at the host plasma membrane and is suppressed by Phytophthora infestans RXLR effector AVR3a

? Little is known about how effectors from filamentous eukaryotic plant pathogens manipulate host defences. Recently, Phytophthora infestans RXLR effector AVR3a has been shown to target and stabilize host E3 ligase CMPG1, which is required for programmed cell death (PCD) triggered by INF1. We investigated the involvement of CMPG1 in PCD elicited by perception of diverse pathogen proteins, and assessed whether AVR3a could suppress each. ? The role of CMPG1 in PCD events was investigated using virus-induced gene silencing, and the ability of AVR3a to suppress each was determined by transient expression of natural forms (AVR3a(KI) and AVR3a(EM)) and a mutated form, AVR3a(KI/Y147del) , which is unable to interact with or stabilize CMPG1. ? PCD triggered at the host plasma membrane by Cf-9/Avr9, Cf-4/Avr4, Pto/AvrPto or the oomycete pathogen-associated molecular pattern (PAMP), cellulose-binding elicitor lectin (CBEL), required CMPG1 and was suppressed by AVR3a, but not by the AVR3a(KI/Y147del) mutant. Conversely, PCD triggered by nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins R3a, R2 and Rx was independent of CMPG1 and unaffected by AVR3a. ? CMPG1-dependent PCD follows perception of diverse pathogen elicitors externally or in association with the inner surface of the host plasma membrane. We argue that AVR3a targets CMPG1 to block initial signal transduction/regulatory processes following pathogen perception at the plasma membrane.     

Detection of the Virulent Form of AVR3a from Phytophthora infestans following Artificial Evolution of Potato Resistance Gene R3a

Engineering resistance genes to gain effector recognition is emerging as an important step in attaining broad, durable resistance. We engineered potato resistance gene R3a to gain recognition of the virulent AVR3a^EM effector form of Phytophthora infestans. Random mutagenesis, gene shuffling and site-directed mutagenesis of R3a were conducted to produce R3a* variants with gain of recognition towards AVR3a^EM. Programmed cell death following gain of recognition was enhanced in iterative rounds of artificial evolution and neared levels observed for recognition of AVR3a^KI by R3a. We demonstrated that R3a*-mediated recognition responses, like for R3a, are dependent on SGT1 and HSP90. In addition, this gain of response is associated with re-localisation of R3a* variants from the cytoplasm to late endosomes when co-expressed with either AVR3a^KI or AVR3a^EM a mechanism that was previously only seen for R3a upon co-infiltration with AVR3a^KI. Similarly, AVR3a^EM specifically re-localised to the same vesicles upon recognition by R3a* variants, but not with R3a. R3a and R3a* provide resistance to P. infestans isolates expressing AVR3a^KI but not those homozygous for AVR3a^EM.     

Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance

Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.