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Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au.

Pathogens and hosts are in an ongoing arms race and genes involved in host-pathogen interactions are likely to undergo diversifying selection. Fusarium plant pathogens have evolved diverse infection strategies, but how they interact with their hosts in the biotrophic infection stage remains puzzling. To address this, we analyzed the genomes of three Fusarium plant pathogens for genes that are under diversifying selection. We found a two-speed genome structure both on the chromosome and gene group level. Diversifying selection acts strongly on the dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici and on distinct core chromosome regions in Fusarium graminearum, all of which have associations with virulence. Members of two gene groups evolve rapidly, namely those that encode proteins with an N-terminal [SG]-P-C-[KR]-P sequence motif and proteins that are conserved predominantly in pathogens. Specifically, 29 F. graminearum genes are rapidly evolving, in plant a induced and encode secreted proteins, strongly pointing toward effector function. In summary, diversifying selection in Fusarium is strongly reflected as genomic footprints and can be used to predict a small gene set likely to be involved in host-pathogen interactions for experimental verification.

Although yield and total biomass produced by annual legumes remain major objectives for breeders, other issues such as environment-friendly, resource use efficiency including symbiotic performance, resilient production in the context of climate change, adaptation to sustainable cropping systems (reducing leaching, greenhouse gas emissions and pesticide residues), adaptation to diverse uses (seeds for feed, food, non-food, forage or green manure) and finally new ecological services such as pollinator protection, imply the need for definition of new ideotypes and development of innovative genotypes to enhance their commercialization. Taken as a whole, this means more complex and integrated objectives for breeders. Several illustrations will be given of breeding such complex traits for different annual legume species. Genetic diversity for root development and for the ability to establish efficient symbioses with rhizobia and mycorrhiza can contribute to better resource management (N, P, water). Shoot architectures and phenologies can contribute to yield and biotic constraint protection (parasitic weeds, diseases or insects) reducing pesticide use. Variable maturity periods and tolerance to biotic and abiotic stresses are key features for the introduction of annual legumes to low input cropping systems and for enlarging cultivated area. Adaptation to intercropping requires adapted genotypes.Improved health and nutritional value for humans are key objectives for developing new markets. Modifying product composition often requires the development of specific cultivars and sometimes the need to break negative genetic correlations with yield. A holistic approach in legume breeding is important for defining objectives with farmers, processors and consumers. The cultivar structures are likely to be more complex, combining genotypes, plant species and associated symbionts. New tools to build and evaluate them are important if legumes are to deliver their exciting potential in

Phytohormones are central components of complex signalling networks in plants. The interplay between these metabolites, which include abscisic acid (ABA), auxin (IAA), ethylene, jasmonic acid (JA) and salicylic acid (SA), regulate plant growth and development and modulate responses to biotic and abiotic stress. Few methods of phytohormone profiling can adequately quantify a large range of plant hormones simultaneously and without the requirement for laborious or highly specialised extraction protocols. Here we describe the development and validation of a phytohormone profiling protocol, based on methyl-chloroformate derivatisation of the plant metabolites and analysis by gas chromatography/mass spectrometry (GC–MS). We describe the analysis of 11 metabolites, either plant phytohormones or intermediates of phytohormone metabolism; ABA, azelaic acid, IAA, JA and SA, and the phytohormone precursors 1-aminocyclopropane 1-carboxylic acid, benzoic acid, cinnamic acid, 13-epi-12-oxophytodienoic acid (13-epi-OPDA), linoleic acid and linolenic acid, and validate the isolation from foliar tissue of the model legume Medicago truncatula. The preparation is insensitive to the presence of water, facilitating measurement of the volatile metabolites. Quantitation was linear over four orders of magnitude, and the limits of detection between two and 10 ng/mL for all measured metabolites using a single quadrupole GC–MS.

Yield stability of legume crops is constrained by a number of pest and diseases. Major diseases are rusts, powdery and downy mildews, ascochyta blights, botrytis gray molds, anthracnoses, damping-off, root rots, collar rots, vascular wilts and white molds. Parasitic weeds, viruses, bacteria, nematodes and damages caused by chewing and sap-sucking insects add to this long list of constraints for legume production. Their incidence and relative importance together with current understanding of their interactions with the host plants are presented. State of the art of current achievements and limitations for breeding for biotic stress resistance are listed and critically discussed. The recent development of large scale phenotyping, genome sequencing and analysis of gene, protein and metabolite expressions can be of great help to further decipher plant-pathogen interactions and identify key resistance components that may be introgressed into crop plants through breeding.

The use of sweet lupins as a new food is resulting in an increasing number of cases of allergy reactions, particularly in atopic patients with other pre-existing legume allergies. We performed an extensive in silico analysis of seed ß-conglutins, a new family of major allergen proteins in lupin, and a comparison to other relevant food allergens such as Ara h 1. We analyzed surface residues involved in conformational epitopes, lineal B- and T-cell epitopes variability, and changes in 2-D structural elements and 3D motives, with the aim to investigate IgE-mediated cross-reactivity among lupin, peanut, and other different legumes. Our results revealed that considerable structural differences exist, particularly affecting 2-D elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability. Variability of residues involved in IgE-binding epitopes might be a major contributor to the observed differences in cross-reactivity among legumes.

A screening for anthracnose resistance of a set of plant genetic resources of narrow-leafed lupin (Lupinus angustifolius L.) identified the breeding line Bo7212 as being highly resistant to anthracnose (Colletotrichum lupini). Segregation analysis indicated that the resistance of Bo7212 is inherited by a single dominant locus. The corresponding resistance gene was given the designation LanrBo. Previously published molecular anchor markers allowed us to locate LanrBo on linkage group NLL-11 of narrow-leafed lupin. Using information from RNAseq data obtained with inoculated resistant vs. susceptible lupin entries as well as EST-sequence information from the model genome Lotus japonicus, additional SNP and EST markers linked to LanrBo were derived. A bracket of two LanrBo-flanking markers allows for precise marker-assisted selection of the novel resistance gene in narrow-leafed lupin breeding programs.

With the expansion of next-generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost-effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next-generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large-scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies.

One of the most stress-responsive genes encoding a mitochondrial protein in Arabidopsis (At3g50930) has been annotated as AtBCS1 (cytochrome bc1 synthase 1), but was previously functionally uncharacterised. Here, we show that the protein encoded by At3g50930 is present as a homo-multimeric protein complex on the outer mitochondrial membrane and lacks the BCS1 domain present in yeast and mammalian BCS1 proteins, with the sequence similarity restricted to the AAA ATPase domain. Thus we propose to re-annotate this protein as AtOM66 (Outer Mitochondrial membrane protein of 66 kDa). While transgenic plants with reduced AtOM66 expression appear to be phenotypically normal, AtOM66 over-expression lines have a distinct phenotype, showing strong leaf curling and reduced starch content. Analysis of mitochondrial protein content demonstrated no detectable changes in mitochondrial respiratory complex protein abundance. Consistent with the stress inducible expression pattern, over-expression lines of AtOM66 are more tolerant to drought stress but undergo stress-induced senescence earlier than wild type. Genome-wide expression analysis revealed a constitutive induction of salicylic acid-related (SA) pathogen defence and cell death genes in over-expression lines. Conversely, expression of SA marker gene PR-1 was reduced in atom66 plants, while jasmonic acid response genes PDF1.2 and VSP2 have increased transcript abundance. In agreement with the expression profile, AtOM66 over-expression plants show increased SA content, accelerated cell death rates and are more tolerant to the biotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic fungus Botrytis cinerea. In conclusion, our results demonstrate a role for AtOM66 in cell death and amplifying SA signalling.

Rhizoctonia solani is an important soil-borne necrotrophic fungal pathogen, with a broad host range and little effective resistance in crop plants. Arabidopsis is resistant to R. solani AG8 but susceptible to R. solani AG2-1. A screen of 36 Arabidopsis ecotypes and mutants affected in the auxin, camalexin, salicylic acid, abscisic acid and ethylene/jasmonic acid pathways did not reveal any variation in response to R. solani and demonstrated that resistance to AG8 was independent of these defense pathways. The Arabidopsis Affymetrix ATH1 Genome array was used to assess global gene expression changes in plants infected with AG8 and AG2-1 at seven days post-infection. While there was considerable overlap in the response, some gene families were differentially affected by AG8 or AG2-1 and included those involved in oxidative stress, cell wall associated proteins, transcription factors and heat shock protein genes. Since a substantial proportion of the gene expression changes were associated with oxidative stress responses, we analysed the role of NADPH oxidases in resistance. While single NADPH oxidase mutants had no effect, a NADPH oxidase double mutant atrbohf atrbohd resulted in an almost complete loss of resistance to AG8, suggesting that reactive oxidative species play an important role in Arabidopsis's resistance to R. solani.

The narrow-leafed lupin (Lupinus angustifolius L.) is a legume with much to offer to agriculture and human wellbeing through its adaptation to nitrogen- and phosphorus-deficient, acid, sandy soils, and production of nutritious, very low glycemic index grain with manifold health benefits. However, the industry has exploited only a small fraction of the genetic and adaptive diversity of the species, reflecting a short and fragmented domestication history. Given declining global production, unlocking the potential residing in untapped sources of genetic diversity to maximise yield and value is critical for the future of the crop. To this end, a wide range of genetic resources is under evaluation. The Australian Lupin Collection comprises almost 4600 diverse, mostly wild accessions, many of which have been genotyped using DArT (Diversity Array Technology) markers, and collection sites characterised to facilitate ecophysiology of contrasting material. Additional exotic genetic resources include recombinant inbred line and mutant populations, as well as inter-specific crosses. These resources are being used to investigate specific adaptation and genetic and molecular control of key traits, all of which will be expedited by current efforts to provide a reference genome sequence for L. angustifolius. Genetic base broadening is the current breeding focus, combining distantly related wild and domestic material with elite cultivars in double-backcrosses or topcrosses, with dramatic effects on yield. In future this will be complemented by marker-based, targeted trait introgression to improve narrow-leafed lupin adaptation, quality/value, and fit into the farming system. © CSIRO 2013.

Background: Cowpea aphid (CPA; Aphis craccivora) is the most important insect pest of cowpea and also causes significant yield losses in other legume crops including alfalfa, beans, chickpea, lentils, lupins and peanuts. In many of these crops there is no natural genetic resistance to this sap-sucking insect or resistance genes have been overcome by newly emerged CPA biotypes. Results: In this study, we screened a subset of the Medicago truncatula core collection of the South Australian Research and Development Institute (SARDI) and identified strong resistance to CPA in a M. truncatula accession SA30199, compared to all other M. truncatula accessions tested. The biology of resistance to CPA in SA30199 plants was characterised compared to the highly susceptible accession Borung and showed that resistance occurred at the level of the phloem, required an intact plant and involved a combination of antixenosis and antibiosis. Quantitative trait loci (QTL) analysis using a F 2 population (n = 150) from a cross between SA30199 and Borung revealed that resistance to CPA is controlled in part by a major quantitative trait locus (QTL) on chromosome 2, explaining 39% of the antibiosis resistance. Conclusions: The identification of strong CPA resistance in M. truncatula allows for the identification of key regulators and genes important in this model legume to give effective CPA resistance that may have relevance for other legume crops. The identified locus will also facilitate marker assisted breeding of M. truncatula for increased resistance to CPA and potentially other closely related Medicago species such as alfalfa.

Herbicides that mimic the natural auxin indole-3-acetic acid are widely used in weed control. One common auxin-like herbicide is dicamba, but despite its wide use, plant gene responses to dicamba have never been extensively studied. To further understand dicamba's mode of action, we utilized Arabidopsis auxin-insensitive mutants and compared their sensitivity to dicamba and the widely-studied auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The mutant axr4-2, which has disrupted auxin transport into cells, was resistant to 2,4-D but susceptible to dicamba. By comparing dicamba resistance in auxin signalling F-box receptor mutants (tir1-1, afb1, afb2, afb3, and afb5), only tir1-1 and afb5 were resistant to dicamba, and this resistance was additive in the double tir1-1/afb5 mutant. Interestingly, tir1-1 but not afb5 was resistant to 2,4-D. Whole genome analysis of dicamba-induced gene expression showed that 10 hours after application, dicamba stimulated many stress-responsive and signalling genes, including those involved in biosynthesis or signalling of auxin, ethylene, and abscisic acid (ABA), with TIR1 and AFB5 required for the dicamba-responsiveness of some genes. Research into dicamba-regulated gene expression and the selectivity of auxin receptors has provided molecular insight into dicamba-regulated signalling and could help in the development of novel herbicide resistance in crop plants.

Mitochondria are both a source of ATP and a site of reactive oxygen species (ROS) production.However, there is little information on the sites of mitochondrial ROS (mROS) production or the biological role of such mROS in plants. We provide genetic proof that mitochondrial complex II (Complex II) of the electron transport chain contributes to localized mROS that regulates plant stress and defense responses. We identify an Arabidopsis mutant in the Complex II subunit, SDH1-1, through a screen for mutants lacking GSTF8 gene expression in response to salicylic acid (SA). GSTF8 is an early stress-responsive gene whose transcription is induced by biotic and abiotic stresses, and its expression is commonly used as a marker of early stress and defense responses. Transcriptional analysis of this mutant, disrupted in stress responses 1 (dsr1), showed that it had altered SA-mediated gene expression for specific downstream stress and defense genes, and it exhibited increased susceptibility to specific fungal and bacterial pathogens. The dsr1 mutant also showed significantly reduced succinate dehydrogenase activity. Using in vivo fluorescence assays, we demonstrated that root cell ROS production occurred primarily from mitochondria and was lower in the mutant in response to SA. In addition, leaf ROS production was lower in the mutant after avirulent bacterial infection. This mutation, in a conserved region of SDH1-1, is a unique plant mitochondrial mutant that exhibits phenotypes associated with lowered mROS production. It provides critical insights into Complex II function with implications for understanding Complex II's role in mitochondrial diseases across eukaryotes.

Background: In legumes, seed storage proteins are important for the developing seedling and are an important source of protein for humans and animals. Lupinus angustifolius (L.), also known as narrow-leaf lupin (NLL) is a grain legume crop that is gaining recognition as a potential human health food as the grain is high in protein and dietary fibre, gluten-free and low in fat and starch.Results: Genes encoding the seed storage proteins of NLL were characterised by sequencing cDNA clones derived from developing seeds. Four families of seed storage proteins were identified and comprised three unique a, seven ß, two ? and four d conglutins. This study added eleven new expressed storage protein genes for the species. A comparison of the deduced amino acid sequences of NLL conglutins with those available for the storage proteins of Lupinus albus (L.), Pisum sativum (L.), Medicago truncatula (L.), Arachis hypogaea (L.) and Glycine max (L.) permitted the analysis of a phylogenetic relationships between proteins and demonstrated, in general, that the strongest conservation occurred within species. In the case of 7S globulin (ß conglutins) and 2S sulphur-rich albumin (d conglutins), the analysis suggests that gene duplication occurred after legume speciation. This contrasted with 11S globulin (a conglutin) and basic 7S (? conglutin) sequences where some of these sequences appear to have diverged prior to speciation. The most abundant NLL conglutin family was ß (56%), followed by a (24%), d (15%) and ? (6%) and the transcript levels of these genes increased 103 to 106 fold during seed development. We used the 16 NLL conglutin sequences identified here to determine that for individuals specifically allergic to lupin, all seven members of the ß conglutin family were potential allergens.Conclusion: This study has characterised 16 seed storage protein genes in NLL including 11 newly-identified members. It has helped lay the foundation for efforts to use molecular breeding approac

Microbial pathogens inflict large losses to agriculture annually and thus mechanisms of plant resistance and how to deploy them to enhance disease resistance in crops are the foci of much research interest. We recently described the important role of ethylene and Ethylene Response transcription Factors (ERFs), particularly MtERF1-1, in mediating resistance to the fungal pathogen Rhizoctonia solani in the model legume, Medicago truncatula. Previous studies on the closely related AtERF14, a master regulator of ethylene dependent defenses including other ERFs, suggested that in Arabidopsis these defenses were not essential for resistance to the same R. solani isolate but were required for resistance to another fungal pathogen, Fusarium oxysporum. Medicago plants with roots overexpressing MtERF1-1 were challenged with F. oxysporum but showed no altered resistance. These results further support a potential for divergent roles of ethylene associated defenses in different plant hosts responding to the same pathogen.