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.

Foliar resistance to Rhizoctonia solani in Arabidopsis is compromised by simultaneous loss of ethylene, jasmonate and PEN2 mediated defense pathways.

Rhizoctonia solani causes damaging yield losses on most major food crops. R. solani isolates belonging to anastomosis group 8 (AG8) are soil-borne, root-infecting pathogens with a broad host range. AG8 isolates can cause disease on wheat, canola and legumes, however Arabidopsis thaliana is heretofore thought to possess non-host resistance as A. thaliana ecotypes, including the reference strain Col-0, are resistant to AG8 infection. Using a mitochondria-targeted redox sensor (mt-roGFP2) and cell death staining, we demonstrate that both AG8 and a host isolate (AG2-1) of R. solani are able to infect A. thaliana roots. Above ground tissue of A. thaliana was found to be resistant to AG8 but not AG2. Genetic analysis revealed that ethylene, jasmonate and PENETRATION2-mediated defense pathways work together to provide resistance to AG8 in the leaves which subsequently enable tolerance of root infections. Overall, we demonstrate a significant difference in defense capabilities of above and below ground tissue in providing resistance to R. solani AG8 in Arabidopsis.

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.

Improved prediction of fungal effector proteins from secretomes with EffectorP 2.0.

Plant-pathogenic fungi secrete effector proteins to facilitate infection. We describe extensive improvements to EffectorP, the first machine learning classifier for fungal effector prediction. EffectorP 2.0 is now trained on a larger set of effectors and utilizes a different approach based on an ensemble of classifiers trained on different subsets of negative data, offering different views on classification. EffectorP 2.0 achieves an accuracy of 89%, compared with 82% for EffectorP 1.0 and 59.8% for a small size classifier. Important features for effector prediction appear to be protein size, protein net charge as well as the amino acids serine and cysteine. EffectorP 2.0 decreases the number of predicted effectors in secretomes of fungal plant symbionts and saprophytes by 40% when compared with EffectorP 1.0. However, EffectorP 1.0 retains value, and combining EffectorP 1.0 and 2.0 results in a stringent classifier with a low false positive rate of 9%. EffectorP 2.0 predicts significant enrichments of effectors in 12 of 13 sets of infection-induced proteins from diverse fungal pathogens, whereas a small cysteine-rich classifier detects enrichment in only seven of 13. EffectorP 2.0 will fast track the prioritization of high-confidence effector candidates for functional validation and aid in improving our understanding of effector biology. EffectorP 2.0 is available at http://effectorp.csiro.au.

Characterization of narrow-leaf lupin (Lupinus angustifolius L.) recombinant major allergen IgE-binding proteins and the natural ß-conglutin counterparts in sweet lupin seed species.

ß-conglutin has been identified as a major allergen for Lupinus angustifolius seeds. The aim of this study was to evaluate the binding of IgE to five recombinant ß-conglutin isoforms (rß) that we overexpressed and purified and to their natural counterparts in different lupin species and cultivars. Western blotting suggested ß-conglutins were the main proteins responsible for the IgE reactivity of the lupin species and cultivars. Newly identified polypeptides from "sweet lupin" may constitute a potential new source of primary or cross-reactive sensitization to lupin, particularly to L. albus and L. angustifolius seed proteins. Several of them exhibited qualitative and quantitative differences in IgE-binding among these species and cultivars, mainly in sera from atopic patients that react to lupin rather than peanut. IgE-binding was more consistent to recombinant ß2 than to any of the other isoforms, making this protein a potential candidate for diagnosis and immunotherapy.

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.

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.

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell.

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/.

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.

Narrow-Leafed Lupin (Lupinus angustifolius) ß1- and ß6-Conglutin Proteins Exhibit Antifungal Activity, Protecting Plants against Necrotrophic Pathogen Induced Damage from Sclerotinia sclerotiorum and Phytophthora nicotianae.

Vicilins (7S globulins) are seed storage proteins and constitute the main protein family in legume seeds, particularly in narrow-leafed lupin (Lupinus angustifolius L.; NLL), where seven vicilin genes, called ß1- to ß7-conglutin have been identified. Vicilins are involved in germination processes supplying amino acids for seedling growth and plant development, as well as in some cases roles in plant defense and protection against pathogens. The roles of NLL ß-conglutins in plant defense are unknown. Here the potential role of five NLL ß-conglutin family members in protection against necrotrophic fungal pathogens was investigated and it was demonstrated that recombinant purified 6xHis-tagged ß1- and ß6-conglutin proteins exhibited the strongest in vitro growth inhibitory activity against a range of necrotrophic fungal pathogens compared to ß2, ß3, and ß4 conglutins. To examine activity in vivo, two representative necrotrophic pathogens, the fungus Sclerotinia sclerotiorum and oomycete Phytophthora nicotianae were used. Transient expression of ß1- and ß6-conglutin proteins in Nicotiana benthamiana leaves demonstrated in vivo growth suppression of both of these pathogens, resulting in low percentages of hyphal growth and elongation in comparison to control treated leaves. Cellular studies using ß1- and ß6-GFP fusion proteins showed these conglutins localized to the cell surface including plasmodesmata. Analysis of cellular death following S. sclerotiorum or P. nicotianae revealed both ß1- and ß6-conglutins suppressed pathogen induced cell death in planta and prevented pathogen induced suppression of the plant oxidative burst as determined by protein oxidation in infected compared to mock-inoculated leaves.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

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.

Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches.

BACKGROUND: The major proteins in lupin seeds are conglutins that have primary roles in supplying carbon, sulphur and nitrogen and energy for the germinating seedling. They fall into four families; α, ß, γ and δ. Interest in these conglutins is growing as family members have been shown to have beneficial nutritional and pharmaceutical properties. RESULTS: An in-depth transcriptome and draft genome from the narrow-leafed lupin (NLL; Lupinus angustifolius) variety, Tanjil, were examined and 16 conglutin genes were identified. Using RNAseq data sets, the structure and expression of these 16 conglutin genes were analysed across eight lupin varieties from five lupin species. Phylogenic analysis suggest that the α and γ conglutins diverged prior to lupin speciation while ß and δ members diverged both prior and after speciation. A comparison of the expression of the 16 conglutin genes was performed, and in general the conglutin genes showed similar levels of RNA expression among varieties within species, but quite distinct expression patterns between lupin species. Antibodies were generated against the specific conglutin families and immunoblot analyses were used to compare the levels of conglutin proteins in various tissues and during different stages of seed development in NLL, Tanjil, confirming the expression in the seed. This analysis showed that the conglutins were expressed highly at the mature seed stage, in all lupin species, and a range of polypeptide sizes were observed for each conglutin family. CONCLUSIONS: This study has provided substantial information on the complexity of the four conglutin families in a range of lupin species in terms of their gene structure, phylogenetic relationships as well as their relative RNA and protein abundance during seed development. The results demonstrate that the majority of the heterogeneity of conglutin polypeptides is likely to arise from post-translational modification from a limited number of precursor polypeptides rather than a large number of different genes. Overall, the results demonstrate a high degree of plasticity for conglutin expression during seed development in different lupin species.

Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors.

Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi.

BACKGROUND: Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. RESULTS: We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. CONCLUSIONS: Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms.

Interactions of Arabidopsis and M. truncatula with the same pathogens differ in dependence on ethylene and ethylene response factors.

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 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 over-expressing 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.

The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia.

The fungal necrotrophic pathogen Rhizoctonia solani is a significant constraint to a range of crops as diverse as cereals, canola, and legumes. Despite wide-ranging germplasm screens in many of these crops, no strong genetic resistance has been identified, suggesting that alternative strategies to improve resistance are required. In this study, we characterize moderate resistance to R. solani anastomosis group 8 identified in Medicago truncatula. The activity of the ethylene- and jasmonate-responsive GCC box promoter element was associated with moderate resistance, as was the induction of the B-3 subgroup of ethylene response transcription factors (ERFs). Genes of the B-1 subgroup showed no significant response to R. solani infection. Overexpression of a B-3 ERF, MtERF1-1, in Medicago roots increased resistance to R. solani as well as an oomycete root pathogen, Phytophthora medicaginis, but not root knot nematode. These results indicate that targeting specific regulators of ethylene defense may enhance resistance to an important subset of root pathogens. We also demonstrate that overexpression of MtERF1-1 enhances disease resistance without apparent impact on nodulation in the A17 background, while overexpression in sickle reduced the hypernodulation phenotype. This suggests that under normal regulation of nodulation, enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and that ethylene/ERF regulation of nodule number is distinct from the defenses regulated by B-3 ERFs. Furthermore, unlike the stunted phenotype previously described for Arabidopsis (Arabidopsis thaliana) ubiquitously overexpressing B-3 ERFs, overexpression of MtERF1-1 in M. truncatula roots did not show adverse effects on plant development.

The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress.

Plant glutathione transferases (GSTs) are induced by diverse biotic and abiotic stimuli, and are important for protecting plants against oxidative damage. We have studied the primary transcriptional stress response of the entire Arabidopsis GST family to seven stresses, including both biotic and abiotic stimuli, with a focus on early changes in gene expression. Our results indicate that individual GST genes are highly specific in their induction patterns. Furthermore, we have been able to link individual GSTs to particular stress stimuli. Using RNAi, we successfully co-silenced a group of four phi GSTs that represent some of the most highly expressed GST genes. Despite a marked reduction in total phi GST protein levels, the transgenic plants showed no reduction in GST activity as measured using the model substrate 1-chloro-2,4-dinitrobenzene (CDNB), and appeared to have surprisingly robust physical phenotypes during stress. However, analysis of metabolite pools showed oxidation of the glutathione pool in the RNAi lines, and we observed alterations in carbon and nitrogen compounds following salicylic acid and hydrogen peroxide stress treatments, indicative of oxidative modification of primary metabolism. Thus, there appears to be a high degree of functional redundancy within the Arabidopsis GST family, with extensive disruption being required to reveal the roles of phi GSTs in protection against oxidative stress.

Identification of Arabidopsis ethylene-responsive element binding factors with distinct induction kinetics after pathogen infection.

Ethylene-responsive element binding factors (ERF) proteins are plant-specific transcription factors, many of which have been linked to stress responses. We have identified four Arabidopsis ERF genes whose expression was specifically induced by avirulent and virulent strains of the bacterial pathogen Pseudomonas syringae pv tomato, with overlapping but distinct induction kinetics. However, a delay in ERF mRNA accumulation after infection with the virulent strain was observed when compared with the avirulent strain. The induction of ERF gene expression in most cases preceded the mRNA accumulation of a basic chitinase gene, a potential downstream target for one or more of these ERFs. The expression of the ERF genes was examined among different Arabidopsis tissues, in response to the signaling molecules ethylene, methyl jasmonate, and salicylic acid (SA), and in Arabidopsis mutants with decreased or enhanced susceptibility to pathogens, and significant differences were observed. For example, in seedlings, some of the ERF genes were not induced by SA in the wild-type but were SA responsive in the pad4-1 mutant, suggesting that PAD4-1, which acts upstream of SA accumulation, is also involved in repressing the SA-induced expression of specific ERF genes. The four ERF proteins were shown to contain transcriptional activation domains. These results suggest that transcriptional activation cascades involving ERF proteins may be important for plant defense to pathogen attack and that some ERF family members could be involved in the cross-talk between SA- and jasmonic acid-signaling pathways.