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

Genome and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control.

Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth

A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

Background:We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. Results:Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. Conclusions:We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay and Robbie Waug

A wheat kinase and immune receptor form host-specificity barriers against the blast fungus

Since emerging in Brazil in 1985, wheat blast has spread throughout South America and recently appeared in Bangladesh and Zambia. Here we show that two wheat resistance genes, Rwt3 and Rwt4, acting as host-specificity barriers against non-Triticum blast pathotypes encode a nucleotide-binding leucine-rich repeat immune receptor and a tandem kinase, respectively. Molecular isolation of these genes will enable study of the molecular interaction between pathogen effector and host resistance genes

LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains

Tracing the ancestry of modern bread wheats

For more than 10,000 years, the selection of plant and animal traits that are better tailored for human use has shaped the development of civilizations. During this period, bread wheat (Triticum aestivum) emerged as one of the world’s most important crops. We use exome sequencing of a worldwide panel of almost 500 genotypes selected from across the geographical range of the wheat species complex to explore how 10,000 years of hybridization, selection, adaptation and plant breeding has shaped the genetic makeup of modern bread wheats. We observe considerable genetic variation at the genic, chromosomal and subgenomic levels, and use this information to decipher the likely origins of modern day wheats, the consequences of range expansion and the allelic variants selected since its domestication. Our data support a reconciled model of wheat evolution and provide novel avenues for future breeding improvement.</p

The rice NLR pair Pikp-1/Pikp-2 initiates cell death through receptor cooperation rather than negative regulation

Plant NLR immune receptors are multidomain proteins that can function as specialized sensor/helper pairs. Paired NLR immune receptors are generally thought to function via negative regulation, where one NLR represses the activity of the second and detection of pathogen effectors relieves this repression to initiate immunity. However, whether this mechanism is common to all NLR pairs is not known. Here, we show that the rice NLR pair Pikp-1/Pikp-2, which confers resistance to strains of the blast pathogen Magnaporthe oryzae (syn. Pyricularia oryzae) expressing the AVR-PikD effector, functions via receptor cooperation, with effector-triggered activation requiring both NLRs to trigger the immune response. To investigate the mechanism of Pikp-1/Pikp-2 activation, we expressed truncated variants of these proteins, and made mutations in previously identified NLR sequence motifs. We found that any domain truncation, in either Pikp-1 or Pikp-2, prevented cell death in the presence of AVR-PikD, revealing that all domains are required for activity. Further, expression of individual Pikp-1 or Pikp-2 domains did not result in cell death. Mutations in the conserved P-loop and MHD sequence motifs in both Pikp-1 and Pikp-2 prevented cell death activation, demonstrating that these motifs are required for the function of the two partner NLRs. Finally, we showed that Pikp-1 and Pikp-2 associate to form homo- and hetero-complexes in planta in the absence of AVR-PikD; on co-expression the effector binds to Pikp-1 generating a tri-partite complex. Taken together, we provide evidence that Pikp-1 and Pikp-2 form a fine-tuned system that is activated by AVR-PikD via receptor cooperation rather than negative regulation