Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat.

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both 'selfish' and 'altruistic' characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed.

A pan-genome and chromosome-length reference genome of narrow-leafed lupin (Lupinus angustifolius) reveals genomic diversity and insights into key industry and biological traits.

Narrow-leafed lupin (NLL; Lupinus angustifolius) is a key rotational crop for sustainable farming systems, whose grain is high in protein content. It is a gluten-free, non-genetically modified, alternative protein source to soybean (Glycine max) and as such has gained interest as a human food ingredient. Here, we present a chromosome-length reference genome for the species and a pan-genome assembly comprising 55 NLL lines, including Australian and European cultivars, breeding lines and wild accessions. We present the core and variable genes for the species and report on the absence of essential mycorrhizal associated genes. The genome and pan-genomes of NLL and its close relative white lupin (Lupinus albus) are compared. Furthermore, we provide additional evidence supporting LaRAP2-7 as the key alkaloid regulatory gene for NLL and demonstrate the NLL genome is underrepresented in classical NLR disease resistance genes compared to other sequenced legume species. The NLL genomic resources generated here coupled with previously generated RNA sequencing datasets provide new opportunities to fast-track lupin crop improvement.

ß-Conglutins' Unique Mobile Arm Is a Key Structural Domain Involved in Molecular Nutraceutical Properties of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Narrow-leafed lupin (NLL; Lupinus angustifolius L.) has multiple nutraceutical properties that may result from unique structural features of ß-conglutin proteins, such as the mobile arm at the N-terminal, a structural domain rich in α-helices. A similar domain has not been found in other vicilin proteins of legume species. We used affinity chromatography to purify recombinant complete and truncated (without the mobile arm domain, tß5 and tß7) forms of NLL ß5 and ß7 conglutin proteins. We then used biochemical and molecular biology techniques in ex vivo and in vitro systems to evaluate their anti-inflammatory activity and antioxidant capacity. The complete ß5 and ß7 conglutin proteins decreased pro-inflammatory mediator levels (e.g., nitric oxide), mRNA expression levels (iNOS, TNFα, IL-1ß), and the protein levels of pro-inflammatory cytokine TNF-α, interleukins (IL-1ß, IL-2, IL-6, IL-8, IL-12, IL-17, IL-27), and other mediators (INFγ, MOP, S-TNF-R1/-R2, and TWEAK), and exerted a regulatory oxidative balance effect in cells as demonstrated in glutathione, catalase, and superoxide dismutase assays. The truncated tß5 and tß7 conglutin proteins did not have these molecular effects. These results suggest that ß5 and ß7 conglutins have potential as functional food components due to their anti-inflammatory and oxidative cell state regulatory properties, and that the mobile arm of NLL ß-conglutin proteins is a key domain in the development of nutraceutical properties, making NLL ß5 and ß7 excellent innovative candidates as functional foods.

Transcription factor lineages in plant-pathogenic fungi, connecting diversity with fungal virulence.

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, yet relatively few virulence-specific regulators are characterised in detail and their evolutionary trajectories are not well understood. Hence, this study compared the full range of TFs across taxonomically-diverse fungal proteomes and classified their lineages through an orthology analysis. The primary aims were to characterise differences in the range and profile of TF lineages broadly linked to plant-host association or pathogenic lifestyles, and to better characterise the evolutionary origin and trajectory of experimentally-validated virulence regulators. We observed significantly fewer TFs among obligate, host-associated pathogens, largely attributed to contractions in several Zn2Cys6 TF-orthogroup lineages. We also present novel insight into the key virulence-regulating TFs Ste12, Pf2 and EBR1, providing evidence for their ancestral origins, expansion and/or loss. Ultimately, the analysis presented here provides both primary evidence for TF evolution in fungal phytopathogenicity, as well as a practical phylogenetic resource to guide further detailed investigation on the regulation of virulence within key pathogen lineages.

A MYC2/MYC3/MYC4-dependent transcription factor network regulates water spray-responsive gene expression and jasmonate levels.

Mechanical stimuli, such as wind, rain, and touch affect plant development, growth, pest resistance, and ultimately reproductive success. Using water spray to simulate rain, we demonstrate that jasmonic acid (JA) signaling plays a key role in early gene-expression changes, well before it leads to developmental changes in flowering and plant architecture. The JA-activated transcription factors MYC2/MYC3/MYC4 modulate transiently induced expression of 266 genes, most of which peak within 30 min, and control 52% of genes induced >100-fold. Chromatin immunoprecipitation-sequencing analysis indicates that MYC2 dynamically binds >1,300 promoters and trans-activation assays show that MYC2 activates these promoters. By mining our multiomic datasets, we identified a core MYC2/MYC3/MYC4-dependent "regulon" of 82 genes containing many previously unknown MYC2 targets, including transcription factors bHLH19 and ERF109 bHLH19 can in turn directly activate the ORA47 promoter, indicating that MYC2/MYC3/MYC4 initiate a hierarchical network of downstream transcription factors. Finally, we also reveal that rapid water spray-induced accumulation of JA and JA-isoleucine is directly controlled by MYC2/MYC3/MYC4 through a positive amplification loop that regulates JA-biosynthesis genes.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

A Trimethylguanosine Synthase1-like (TGS1) homologue is implicated in vernalisation and flowering time control.

KEY MESSAGE: A plant-specific Trimethylguanosine Synthase1-like homologue was identified as a candidate gene for the efl mutation in narrow-leafed lupin, which alters phenology by reducing vernalisation requirement. The vernalisation pathway is a key component of flowering time control in plants from temperate regions but is not well understood in the legume family. Here we examined vernalisation control in the temperate grain legume species, narrow-leafed lupin (Lupinus angustifolius L.), and discovered a candidate gene for an ethylene imine mutation (efl). The efl mutation changes phenology from late to mid-season flowering and additionally causes transformation from obligate to facultative vernalisation requirement. The efl locus was mapped to pseudochromosome NLL-10 in a recombinant inbred line (RIL) mapping population developed by accelerated single seed descent. Candidate genes were identified in the reference genome, and a diverse panel of narrow-leafed lupins was screened to validate mutations specific to accessions with efl. A non-synonymous SNP mutation within an S-adenosyl-L-methionine-dependent methyltransferase protein domain of a Trimethylguanosine Synthase1-like (TGS1) orthologue was identified as the candidate mutation giving rise to efl. This mutation caused substitution of an amino acid within an established motif at a position that is otherwise highly conserved in several plant families and was perfectly correlated with the efl phenotype in F2 and F6 genetic population and a panel of diverse accessions, including the original efl mutant. Expression of the TGS1 homologue did not differ between wild-type and efl genotypes, supporting altered functional activity of the gene product. This is the first time a TGS1 orthologue has been associated with vernalisation response and flowering time control in any plant species.

Ethylene Is Not Essential for R-Gene Mediated Resistance but Negatively Regulates Moderate Resistance to Some Aphids in Medicago truncatula.

Ethylene is important for plant responses to environmental factors. However, little is known about its role in aphid resistance. Several types of genetic resistance against multiple aphid species, including both moderate and strong resistance mediated by R genes, have been identified in Medicago truncatula. To investigate the potential role of ethylene, a M. truncatula ethylene- insensitive mutant, sickle, was analysed. The sickle mutant occurs in the accession A17 that has moderate resistance to Acyrthosiphon kondoi, A. pisum and Therioaphis trifolii. The sickle mutant resulted in increased antibiosis-mediated resistance against A. kondoi and T. trifolii but had no effect on A. pisum. When sickle was introduced into a genetic background carrying resistance genes, AKR (A. kondoi resistance), APR (A. pisum resistance) and TTR (T. trifolii resistance), it had no effect on the strong aphid resistance mediated by these genes, suggesting that ethylene signaling is not essential for their function. Interestingly, for the moderate aphid resistant accession, the sickle mutant delayed leaf senescence following aphid infestation and reduced the plant biomass losses caused by both A. kondoi and T. trifolii. These results suggest manipulation of the ethylene signaling pathway could provide aphid resistance and enhance plant tolerance against aphid feeding.

Identification and profiling of narrow-leafed lupin (Lupinus angustifolius) microRNAs during seed development.

BACKGROUND: Whilst information regarding small RNAs within agricultural crops is increasing, the miRNA composition of the nutritionally valuable pulse narrow-leafed lupin (Lupinus angustifolius) remains unknown. RESULTS: By conducting a genome- and transcriptome-wide survey we identified 7 Dicer-like and 16 Argonaute narrow-leafed lupin genes, which were highly homologous to their legume counterparts. We identified 43 conserved miRNAs belonging to 16 families, and 13 novel narrow-leafed lupin-specific miRNAs using high-throughput sequencing of small RNAs from foliar and root and five seed development stages. We observed up-regulation of members of the miRNA families miR167, miR399, miR156, miR319 and miR164 in narrow-leafed lupin seeds, and confirmed expression of miR156, miR166, miR164, miR1507 and miR396 using quantitative RT-PCR during five narrow-leafed lupin seed development stages. We identified potential targets for the conserved and novel miRNAs and were able to validate targets of miR399 and miR159 using 5' RLM-RACE. The conserved miRNAs are predicted to predominately target transcription factors and 93% of the conserved miRNAs originate from intergenic regions. In contrast, only 43% of the novel miRNAs originate from intergenic regions and their predicted targets were more functionally diverse. CONCLUSION: This study provides important insights into the miRNA gene regulatory networks during narrow-leafed lupin seed development.

Transcriptome analysis reveals molecular mechanisms of sclerotial development in the rice sheath blight pathogen Rhizoctonia solani AG1-IA.

Rhizoctonia solani AG1-IA is a soil-borne necrotrophic pathogen that causes devastating rice sheath blight disease in rice-growing regions worldwide. Sclerotia play an important role in the life cycle of R. solani AG1-IA. In this study, RNA sequencing was used to investigate the transcriptomic dynamics of sclerotial development (SD) of R. solani AG1-IA. Gene ontology and pathway enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to investigate the functions and pathways of differentially expressed genes (DEGs). Six cDNA libraries were generated, and more than 300 million clean reads were obtained and assembled into 15,100 unigenes. In total, 12,575 differentially expressed genes were identified and 34.62% (4353) were significantly differentially expressed with a FDR ≤ 0.01 and |log2Ratio| ≥ 1, which were enriched into eight profiles using Short Time-series Expression Miner. Furthermore, KEGG and gene ontology analyses suggest the DEGs were significantly enriched in several biological processes and pathways, including binding and catalytic functions, biosynthesis of ribosomes, and other biological functions. Further annotation of the DEGs using the Clusters of Orthologous Groups (COG) database found most DEGs were involved in amino acid transport and metabolism, as well as energy production and conversion. Furthermore, DEGs relevant to SD of R. solani AG1-IA were involved in secondary metabolite biosynthesis, melanin biosynthesis, ubiquitin processes, autophagy, and reactive oxygen species metabolism. The gene expression profiles of 10 randomly selected DEGs were validated by quantitative real-time reverse transcription PCR and were consistent with the dynamics in transcript abundance identified by RNA sequencing. The data provide a high-resolution map of gene expression during SD, a key process contributing to the pathogenicity of this devastating pathogen. In addition, this study provides a useful resource for further studies on the genomics of R. solani AG1-IA and other Rhizoctonia species.

INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow-leafed lupin (Lupinus angustifolius L.).

Narrow-leafed lupin (Lupinus angustifolius L.) cultivation was transformed by 2 dominant vernalization-insensitive, early flowering time loci known as Ku and Julius (Jul), which allowed expansion into shorter season environments. However, reliance on these loci has limited genetic and phenotypic diversity for environmental adaptation in cultivated lupin. We recently predicted that a 1,423-bp deletion in the cis-regulatory region of LanFTc1, a FLOWERING LOCUS T (FT) homologue, derepressed expression of LanFTc1 and was the underlying cause of the Ku phenotype. Here, we surveyed diverse germplasm for LanFTc1 cis-regulatory variation and identified 2 further deletions of 1,208 and 5,162 bp in the 5' regulatory region, which overlap the 1,423-bp deletion. Additionally, we confirmed that no other polymorphisms were perfectly associated with vernalization responsiveness. Phenotyping and gene expression analyses revealed that Jul accessions possessed the 5,162-bp deletion and that the Jul and Ku deletions were equally capable of removing vernalization requirement and up-regulating gene expression. The 1,208-bp deletion was associated with intermediate phenology, vernalization responsiveness, and gene expression and therefore may be useful for expanding agronomic adaptation of lupin. This insertion/deletion series may also help resolve how the vernalization response is mediated at the molecular level in legumes.

Additive and epistatic interactions between AKR and AIN loci conferring bluegreen aphid resistance and hypersensitivity in Medicago truncatula.

Aphids, including the bluegreen aphid (BGA; Acyrthosiphon kondoi), are important pests in agriculture. Two BGA resistance genes have been identified in the model legume Medicago truncatula, namely AKR (Acyrthosiphon kondoi resistance) and AIN (Acyrthosiphon induced necrosis). In this study, progeny derived from a cross between a resistant accession named Jester and a highly susceptible accession named A20 were used to study the interaction between the AKR and AIN loci with respect to BGA performance and plant response to BGA infestation. These studies demonstrated that AKR and AIN have additive effects on the BGA resistance phenotype. However, AKR exerts dominant suppression epistasis on AIN-controlled macroscopic necrotic lesions. Nevertheless, both AKR and AIN condition production of H2O2 at the BGA feeding site. Electrical penetration graph analysis demonstrated that AKR prevents phloem sap ingestion, irrespective of the presence of AIN. Similarly, the jasmonic acid defense signaling pathway is recruited by AKR, irrespective of AIN. This research identifies an enhancement of aphid resistance through gene stacking, and insights into the interaction of distinct resistance genes against insect pests.

ApoplastP: prediction of effectors and plant proteins in the apoplast using machine learning.

The plant apoplast is integral to intercellular signalling, transport and plant-pathogen interactions. Plant pathogens deliver effectors both into the apoplast and inside host cells, but no computational method currently exists to discriminate between these localizations. We present ApoplastP, the first method for predicting whether an effector or plant protein localizes to the apoplast. ApoplastP uncovers features of apoplastic localization common to both effectors and plant proteins, namely depletion in glutamic acid, acidic amino acids and charged amino acids and enrichment in small amino acids. ApoplastP predicts apoplastic localization in effectors with a sensitivity of 75% and a false positive rate of 5%, improving the accuracy of cysteine-rich classifiers by > 13%. ApoplastP does not depend on the presence of a signal peptide and correctly predicts the localization of unconventionally secreted proteins. The secretomes of fungal saprophytes as well as necrotrophic, hemibiotrophic and extracellular fungal pathogens are enriched for predicted apoplastic proteins. Rust pathogens have low proportions of predicted apoplastic proteins, but these are highly enriched for predicted effectors. ApoplastP pioneers apoplastic localization prediction using machine learning. It will facilitate functional studies and will be valuable for predicting if an effector localizes to the apoplast or if it enters plant cells.

Salicylic Acid-Dependent Plant Stress Signaling via Mitochondrial Succinate Dehydrogenase.

Mitochondria are known for their role in ATP production and generation of reactive oxygen species, but little is known about the mechanism of their early involvement in plant stress signaling. The role of mitochondrial succinate dehydrogenase (SDH) in salicylic acid (SA) signaling was analyzed using two mutants: disrupted in stress response1 (dsr1), which is a point mutation in SDH1 identified in a loss of SA signaling screen, and a knockdown mutant (sdhaf2) for SDH assembly factor 2 that is required for FAD insertion into SDH1. Both mutants showed strongly decreased SA-inducible stress promoter responses and low SDH maximum capacity compared to wild type, while dsr1 also showed low succinate affinity, low catalytic efficiency, and increased resistance to SDH competitive inhibitors. The SA-induced promoter responses could be partially rescued in sdhaf2, but not in dsr1, by supplementing the plant growth media with succinate. Kinetic characterization showed that low concentrations of either SA or ubiquinone binding site inhibitors increased SDH activity and induced mitochondrial H2O2 production. Both dsr1 and sdhaf2 showed lower rates of SA-dependent H2O2 production in vitro in line with their low SA-dependent stress signaling responses in vivo. This provides quantitative and kinetic evidence that SA acts at or near the ubiquinone binding site of SDH to stimulate activity and contributes to plant stress signaling by increased rates of mitochondrial H2O2 production, leading to part of the SA-dependent transcriptional response in plant cells.

Characterization of the genetic factors affecting quinolizidine alkaloid biosynthesis and its response to abiotic stress in narrow-leafed lupin (Lupinus angustifolius L.).

Quinolizidine alkaloids (QAs) are toxic secondary metabolites that complicate the end use of narrow-leafed lupin (NLL; Lupinus angustifolius L.) grain, as levels sometimes exceed the industry limit for its use as a food and feed source. The genotypic and environmental influences on QA production in NLL are poorly understood. Here, the expression of QA biosynthetic genes was analysed in vegetative and reproductive tissues of bitter (high QA) and sweet (low QA) accessions. It was demonstrated that sweet accessions are characterized by lower QA biosynthetic gene expression exclusively in leaf and stem tissues than bitter NLL, consistent with the hypothesis that QAs are predominantly produced in aerial tissues and transported to seeds, rather than synthesized within the seed itself. This analysis informed our identification of additional candidate genes involved in QA biosynthesis. Drought and temperature stress are two major abiotic stresses that often occur during NLL pod set. Hence, we assessed the effect of drought, increased temperature, and their combination, on QA production in three sweet NLL cultivars. A cultivar-specific response to drought and temperature in grain QA levels was observed, including the identification of a cultivar where alkaloid levels did not change with these stress treatments.

Exploring the genetic and adaptive diversity of a pan-Mediterranean crop wild relative: narrow-leafed lupin.

KEY MESSAGE: This first pan-Mediterranean analysis of genetic diversity in wild narrow-leafed lupin revealed strong East-West genetic differentiation of populations, an historic eastward migration, and signatures of genetic adaptation to climatic variables. Most grain crops suffer from a narrow genetic base, which limits their potential for adapting to new challenges such as increased stresses associated with climate change. Plant breeders are returning to the wild ancestors of crops and their close relatives to broaden the genetic base of their crops. Understanding the genetic adaptation of these wild relatives will help plant breeders most effectively use available wild diversity. Here, we took narrow-leafed lupin (Lupinus angustifolius L.) as a model to understand adaptation in a wild crop ancestor. A set of 142 wild accessions of narrow-leafed lupin from across the Mediterranean basin were subjected to genotyping-by-sequencing using Diversity Arrays Technology. Phylogenetic, linkage disequilibrium and demographic analyses were employed to explore the history of narrow-leafed lupin within the Mediterranean region. We found strong genetic differentiation between accessions from the western and eastern Mediterranean, evidence of an historic West to East migration, and that eastern Mediterranean narrow-leafed lupin experienced a severe and recent genetic bottleneck. We showed that these two populations differ for flowering time as a result of local adaptation, with the West flowering late while the East flowers early. A genome-wide association study identified single nucleotide polymorphism markers associated with climatic adaptation. Resolving the origin of wild narrow-leafed lupin and how its migration has induced adaptation to specific regions of the Mediterranean serves as a useful resource not only for developing narrow-leafed lupin cultivars with greater resilience to a changing climate, but also as a model which can be applied to other legumes.

Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts.

Rhizoctonia solaniis an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about howR. solanicauses disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility toR. solaniwhen expressed inNicotiana benthamiana In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806.

Ethylene Signaling Is Important for Isoflavonoid-Mediated Resistance to Rhizoctonia solani in Roots of Medicago truncatula.

The root-infecting necrotrophic fungal pathogen Rhizoctoniasolani causes significant disease to all the world's major food crops. As a model for pathogenesis of legumes, we have examined the interaction of R. solani AG8 with Medicago truncatula. RNAseq analysis of the moderately resistant M. truncatula accession A17 and highly susceptible sickle (skl) mutant (defective in ethylene sensing) identified major early transcriptional reprogramming in A17. Responses specific to A17 included components of ethylene signaling, reactive oxygen species metabolism, and consistent upregulation of the isoflavonoid biosynthesis pathway. Mass spectrometry revealed accumulation of the isoflavonoid-related compounds liquiritigenin, formononetin, medicarpin, and biochanin A in A17. Overexpression of an isoflavone synthase in M. truncatula roots increased isoflavonoid accumulation and resistance to R. solani. Addition of exogenous medicarpin suggested this phytoalexin may be one of several isoflavonoids required to contribute to resistance to R. solani. Together, these results provide evidence for the role of ethylene-mediated accumulation of isoflavonoids during defense against root pathogens in legumes. The involvement of ethylene signaling and isoflavonoids in the regulation of both symbiont-legume and pathogen-legume interactions in the same tissue may suggest tight regulation of these responses are required in the root tissue.

A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into plant-microbe interactions and legume evolution.

Lupins are important grain legume crops that form a critical part of sustainable farming systems, reducing fertilizer use and providing disease breaks. It has a basal phylogenetic position relative to other crop and model legumes and a high speciation rate. Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is gaining popularity as a health food, which is high in protein and dietary fibre but low in starch and gluten-free. We report the draft genome assembly (609 Mb) of NLL cultivar Tanjil, which has captured >98% of the gene content, sequences of additional lines and a dense genetic map. Lupins are unique among legumes and differ from most other land plants in that they do not form mycorrhizal associations. Remarkably, we find that NLL has lost all mycorrhiza-specific genes, but has retained genes commonly required for mycorrhization and nodulation. In addition, the genome also provided candidate genes for key disease resistance and domestication traits. We also find evidence of a whole-genome triplication at around 25 million years ago in the genistoid lineage leading to Lupinus. Our results will support detailed studies of legume evolution and accelerate lupin breeding programmes.

Genome sequencing and comparative genomics of the broad host-range pathogen Rhizoctonia solani AG8.

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level "hypermutation" of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens.