Genetic and Phenotypic Evaluation of European Maize Landraces as a Tool for Conservation and Valorization of Agrobiodiversity.

The ECPGR European Evaluation Network (EVA) for Maize involves genebanks, research institutions, and private breeding companies from nine countries focusing on the valorization of maize genetic resources across Europe. This study describes a diverse collection of 626 local landraces and traditional varieties of maize (Zea mays L.) from nine European genebanks, including criteria for selection of the collection and its genetic and phenotypic diversity. High-throughput pool genotyping grouped the landraces into nine genetic groups with a threshold of 0.6 admixture, while 277 accessions were designated admixed and likely to have resulted from previous breeding activities. The grouping correlated well with the geographic origins of the collection, also reflecting the various pathways of introduction of maize to Europe. Phenotypic evaluations of 588 accessions for flowering time and plant architecture in multilocation trials over three years confirmed the great diversity within the collection, although phenotypic clusters only partially correlated with the genetic grouping. The EVA approach promotes conservation of genetic resources and opens an opportunity to increase genetic variability for developing improved varieties and populations for farmers, with better adaptation to specific environments and greater tolerance to various stresses. As such, the EVA maize collection provides valuable sources of diversity for facing climate change due to the varieties' local adaptation.

Development and application of Single Primer Enrichment Technology (SPET) SNP assay for population genomics analysis and candidate gene discovery in lettuce.

Single primer enrichment technology (SPET) is a novel high-throughput genotyping method based on short-read sequencing of specific genomic regions harboring polymorphisms. SPET provides an efficient and reproducible method for genotyping target loci, overcoming the limits associated with other reduced representation library sequencing methods that are based on a random sampling of genomic loci. The possibility to sequence regions surrounding a target SNP allows the discovery of thousands of closely linked, novel SNPs. In this work, we report the design and application of the first SPET panel in lettuce, consisting of 41,547 probes spanning the whole genome and designed to target both coding (~96%) and intergenic (~4%) regions. A total of 81,531 SNPs were surveyed in 160 lettuce accessions originating from a total of 10 countries in Europe, America, and Asia and representing 10 horticultural types. Model ancestry population structure clearly separated the cultivated accessions (Lactuca sativa) from accessions of its presumed wild progenitor (L. serriola), revealing a total of six genetic subgroups that reflected a differentiation based on cultivar typology. Phylogenetic relationships and principal component analysis revealed a clustering of butterhead types and a general differentiation between germplasm originating from Western and Eastern Europe. To determine the potentiality of SPET for gene discovery, we performed genome-wide association analysis for main agricultural traits in L. sativa using six models (GLM naive, MLM, MLMM, CMLM, FarmCPU, and BLINK) to compare their strength and power for association detection. Robust associations were detected for seed color on chromosome 7 at 50 Mbp. Colocalization of association signals was found for outer leaf color and leaf anthocyanin content on chromosome 9 at 152 Mbp and on chromosome 5 at 86 Mbp. The association for bolting time was detected with the GLM, BLINK, and FarmCPU models on chromosome 7 at 164 Mbp. Associations were detected in chromosomal regions previously reported to harbor candidate genes for these traits, thus confirming the effectiveness of SPET for GWAS. Our findings illustrated the strength of SPET for discovering thousands of variable sites toward the dissection of the genomic diversity of germplasm collections, thus allowing a better characterization of lettuce collections.

Publisher Correction: A downy mildew effector evades recognition by polymorphism of expression and subcellular localization.

The original version of this article contained an error in the author affiliations. Oliver J. Furzer was incorrectly associated with Department of Plant Sciences, College of Life Sciences, Wuhan University, 430072, Wuhan, China.This has now been corrected in the HTML version of the article. The PDF version of the article was correct at the time of publication.Furthermore, the original version of this article stated that correspondence and requests for materials should be addressed to Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany S.A. (email: shuta.asai@riken.jp) or to J.D.G.J. (email: jonathan.jones@tsl.ac.uk). The words "Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany" were introduced inadvertently.This has now been corrected in the PDF version of the article. The HTML version of the article was correct at the time of publication.

A downy mildew effector evades recognition by polymorphism of expression and subcellular localization.

Pathogen co-evolution with plants involves selection for evasion of host surveillance systems. The oomycete Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis, and race-specific interactions between Arabidopsis accessions and Hpa isolates fit the gene-for-gene model in which host resistance or susceptibility are determined by matching pairs of plant Resistance (R) genes and pathogen Avirulence (AVR) genes. Arabidopsis Col-0 carries R gene RPP4 that confers resistance to Hpa isolates Emoy2 and Emwa1, but its cognate recognized effector(s) were unknown. We report here the identification of the Emoy2 AVR effector gene recognized by RPP4 and show resistance-breaking isolates of Hpa on RPP4-containing Arabidopsis carry the alleles that either are not expressed, or show cytoplasmic instead of nuclear subcellular localization.

Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein.

KEY MESSAGE: Using fine mapping techniques, the genomic region co-segregating with Restorer - of - fertility ( Rf ) in pepper was delimited to a region of 821 kb in length. A PPR gene in this region, CaPPR6 , was identified as a strong candidate for Rf based on expression pattern and characteristics of encoding sequence. Cytoplasmic-genic male sterility (CGMS) has been used for the efficient production of hybrid seeds in peppers (Capsicum annuum L.). Although the mitochondrial candidate genes that might be responsible for cytoplasmic male sterility (CMS) have been identified, the nuclear Restorer-of-fertility (Rf) gene has not been isolated. To identify the genomic region co-segregating with Rf in pepper, we performed fine mapping using an Rf-segregating population consisting of 1068 F2 individuals, based on BSA-AFLP and a comparative mapping approach. Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence. Prediction of expressed genes in this region based on transcription analysis revealed four candidate genes. Among these, CaPPR6 encodes a pentatricopeptide repeat (PPR) protein with PPR motifs that are repeated 14 times. Characterization of the CaPPR6 protein sequence, based on alignment with other homologs, showed that CaPPR6 is a typical Rf-like (RFL) gene reported to have undergone diversifying selection during evolution. A marker developed from a sequence near CaPPR6 showed a higher prediction rate of the Rf phenotype than those of previously developed markers when applied to a panel of breeding lines of diverse origin. These results suggest that CaPPR6 is a strong candidate for the Rf gene in pepper.

Fine mapping and identification of candidate genes for the sy-2 locus in a temperature-sensitive chili pepper (Capsicum chinense).

KEY MESSAGE: The sy - 2 temperature-sensitive gene from Capsicum chinense was fine mapped to a 138.8-kb region at the distal portion of pepper chromosome 1. Based on expression analyses, two putative F-box genes were identified as sy - 2 candidate genes. Seychelles-2 ('sy-2') is a temperature-sensitive natural mutant of Capsicum chinense, which exhibits an abnormal leaf phenotype when grown at temperatures below 24 °C. We previously showed that the sy-2 phenotype is controlled by a single recessive gene, sy-2, located on pepper chromosome 1. In this study, a high-resolution genetic and physical map for the sy-2 locus was constructed using two individual F2 mapping populations derived from a cross between C. chinense mutant 'sy-2' and wild-type 'No. 3341'. The sy-2 gene was fine mapped to a 138.8-kb region between markers SNP 5-5 and SNP 3-8 at the distal portion of chromosome 1, based on comparative genomic analysis and genomic information from pepper. The sy-2 target region was predicted to contain 27 genes. Expression analysis of these predicted genes showed a differential expression pattern for ORF10 and ORF20 between mutant and wild-type plants; with both having significantly lower expression in 'sy-2' than in wild-type plants. In addition, the coding sequences of both ORF10 and ORF20 contained single nucleotide polymorphisms (SNPs) causing amino acid changes, which may have important functional consequences. ORF10 and ORF20 are predicted to encode F-box proteins, which are components of the SCF complex. Based on the differential expression pattern and the presence of nonsynonymous SNPs, we suggest that these two putative F-box genes are most likely responsible for the temperature-sensitive phenotypes in pepper. Further investigation of these genes may enable a better understanding of the molecular mechanisms of low temperature sensitivity in plants.

Structurally distinct Arabidopsis thaliana NLR immune receptors recognize tandem WY domains of an oomycete effector.

Nucleotide-binding leucine-rich repeat (NB-LRR, or NLR) receptors mediate pathogen recognition. The Arabidopsis thaliana NLR RPP1 recognizes the tandem WY-domain effector ATR1 from the oomycete Hyaloperonospora arabidopsidis through direct association with C-terminal LRRs. We isolated and characterized homologous NLR genes RPP1-EstA and RPP1-ZdrA from two Arabidopsis ecotypes, Estland (Est-1) and Zdarec (Zdr-1), responsible for recognizing a novel spectrum of ATR1 alleles. RPP1-EstA and -ZdrA encode nearly identical NLRs that are phylogenetically distinct from known immunity-activating RPP1 homologs and possess greatly expanded LRR domains. Site-directed mutagenesis and truncation analysis of ATR1 suggests that these homologs recognize a novel surface of the 2(nd) WY domain of ATR1, partially specified by a C-terminal region of the LRR domain. Synteny comparison with RPP1 loci involved in hybrid incompatibility suggests that these functions evolved independently. Closely related RPP1 homologs have diversified their recognition spectra through LRR expansion and sequence variation, allowing them to detect multiple surfaces of the same pathogen effector. Engineering NLR receptor specificity may require a similar combination of repeat expansion and tailored amino acid variation.

Recognition and activation domains contribute to allele-specific responses of an Arabidopsis NLR receptor to an oomycete effector protein.

In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function.

Computational prediction and molecular characterization of an oomycete effector and the cognate Arabidopsis resistance gene.

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses.

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition.

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.

A novel role for protein farnesylation in plant innate immunity.

Plants utilize tightly regulated mechanisms to defend themselves against pathogens. Initial recognition results in activation of specific Resistance (R) proteins that trigger downstream immune responses, in which the signaling networks remain largely unknown. A point mutation in SUPPRESSOR OF NPR1 CONSTITUTIVE1 (SNC1), a RESISTANCE TO PERONOSPORA PARASITICA4 R gene homolog, renders plants constitutively resistant to virulent pathogens. Genetic suppressors of snc1 may carry mutations in genes encoding novel signaling components downstream of activated R proteins. One such suppressor was identified as a novel loss-of-function allele of ENHANCED RESPONSE TO ABSCISIC ACID1 (ERA1), which encodes the beta-subunit of protein farnesyltransferase. Protein farnesylation involves attachment of C15-prenyl residues to the carboxyl termini of specific target proteins. Mutant era1 plants display enhanced susceptibility to virulent bacterial and oomycete pathogens, implying a role for farnesylation in basal defense. In addition to its role in snc1-mediated resistance, era1 affects several other R-protein-mediated resistance responses against bacteria and oomycetes. ERA1 acts partly independent of abscisic acid and additively with the resistance regulator NON-EXPRESSOR OF PR GENES1 in the signaling network. Defects in geranylgeranyl transferase I, a protein modification similar to farnesylation, do not affect resistance responses, indicating that farnesylation is most likely specifically required in plant defense signaling. Taken together, we present a novel role for farnesyltransferase in plant-pathogen interactions, suggesting the importance of protein farnesylation, which contributes to the specificity and efficacy of signal transduction events.

The ubiquitin pathway is required for innate immunity in Arabidopsis.

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 (snc1) carries a point mutation in a Resistance (R)-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1-mediated constitutive resistance is modifier of snc1 5 (mos5), which carries a 15-bp deletion in UBA1, one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1, suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1-mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1-related R-genes RPP4 and RPP5, are dispensable in snc1-mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins.

A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1.

Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades. Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR). In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2. The snc1 mutation was mapped to the RPP4 cluster. In snc1, one of the TIR-NB-LRR-type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR. Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance. The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1. Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens. To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed. Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.