Trichothecenes and Fumonisins: Key Players in Fusarium-Cereal Ecosystem Interactions.

Fusarium fungi produce a diverse array of mycotoxic metabolites during the pathogenesis of cereals. Some, such as the trichothecenes and fumonisins, are phytotoxic, acting as non-proteinaceous effectors that facilitate disease development in cereals. Over the last few decades, we have gained some depth of understanding as to how trichothecenes and fumonisins interact with plant cells and how plants deploy mycotoxin detoxification and resistance strategies to defend themselves against the producer fungi. The cereal-mycotoxin interaction is part of a co-evolutionary dance between Fusarium and cereals, as evidenced by a trichothecene-responsive, taxonomically restricted, cereal gene competing with a fungal effector protein and enhancing tolerance to the trichothecene and resistance to DON-producing F. graminearum. But the binary fungal-plant interaction is part of a bigger ecosystem wherein other microbes and insects have been shown to interact with fungal mycotoxins, directly or indirectly through host plants. We are only beginning to unravel the extent to which trichothecenes, fumonisins and other mycotoxins play a role in fungal-ecosystem interactions. We now have tools to determine how, when and where mycotoxins impact and are impacted by the microbiome and microfauna. As more mycotoxins are described, research into their individual and synergistic toxicity and their interactions with the crop ecosystem will give insights into how we can holistically breed for and cultivate healthy crops.

Transcriptional Profiling Reveals the Wheat Defences against Fusarium Head Blight Disease Regulated by a NAC Transcription Factor.

The wheat NAC transcription factor TaNACL-D1 enhances resistance to the economically devastating Fusarium head blight (FHB) disease. The objective of this study was to decipher the alterations in gene expression, pathways and biological processes that led to enhanced resistance as a result of the constitutive expression of TaNACL-D1 in wheat. Transcriptomic analysis was used to determine the genes and processes enhanced in wheat due to TaNACL-D1 overexpression, both in the presence and absence of the causal agent of FHB, Fusarium graminearum (0- and 1-day post-treatment). The overexpression of TaNACL-D1 resulted in more pronounced transcriptional reprogramming as a response to fungal infection, leading to the enhanced expression of genes involved in detoxification, immune responses, secondary metabolism, hormone biosynthesis, and signalling. The regulation and response to JA and ABA were differentially regulated between the OE and the WT. Furthermore, the results suggest that the OE may more efficiently: (i) regulate the oxidative burst; (ii) modulate cell death; and (iii) induce both the phenylpropanoid pathway and lignin synthesis. Thus, this study provides insights into the mode of action and downstream target pathways for this novel NAC transcription factor, further validating its potential as a gene to enhance FHB resistance in wheat.

Acclimatisation of Fusarium langsethiae, F. poae and F. sporotrichioides to elevated CO2: Impact on fungal growth and mycotoxin production on oat-based media.

Oats are highly susceptible to infection by Fusarium species, especially F. langsethiae, F. poae and F. sporotrichioides which contaminate the grain with mycotoxins. Climate change is expected to affect fungal colonisation and associated mycotoxin production. The objective of this study was to examine the effect of acclimatisation to elevated CO2 on the growth and mycotoxin production capacity of these fungal species. Strains of F. langsethiae (FL; seven strains), F. poae (FP; two strains) and F. sporotrichioides (FS; one strain) were acclimatised by sub-culturing for 10 generations at either 400 or 1000 ppm CO2 under diurnal temperature conditions. At each sub-culturing, the effect of acclimatisation to elevated CO2 on (a) lag phase prior to growth, (b) growth rate on oat-based media was assessed. Additionally, the production of type A trichothecenes and related toxic secondary metabolites of sub-cultures after 1, 7 and 10 generations were assessed using LC-MS/MS qTRAP. The results showed that Fusarium strains had an increased lag time and growth rate in response to the combined effect of sub-culturing and elevated CO2 levels. T-2 + HT-2 production was affected by elevated CO2 in strain FL4 (7.1-fold increase) and a decrease in strain FL1 (2.0-fold decrease) at the first sub-culturing and FS (1.3-fold decrease) after 7 sub-cultures compared to ambient conditions. The effect of sub-culturing on T-2 + HT-2 production varied depending on the fungal strain. For strain FL4, significantly less T-2 + HT-2 toxins were produced after 10 generations (4.4-fold decrease) as compared to that under elevated CO2 conditions after one sub-culture, and no change was observed under ambient conditions. The FS strain showed significant stimulation of T-2 + HT-2 toxin production after 10 sub-cultured generations (1.1-fold increase) compared to the initial sub-culture of this strain under elevated CO2 conditions. The production of other toxic secondary metabolites was generally not impacted by elevated CO2 conditions or by sub-culture for 10 generations, with the exceptions of FL1 and FP1. FL1 produced significantly more neosolaniol after 10 generations, when compared to those after 1 and 7, regardless of the CO2 conditions. For FP1, elevated CO2 significantly triggered beauvericin production after an initial sub-culture when compared to ambient conditions at the same sub-culture stage (29-fold). FP1 acclimatisation to elevated CO2 led to a decrease of beauvericin production after 10 generations when compared to 1 (6-fold). In contrast, sub-culturing for 10 generations compared to 1 under ambient CO2 conditions resulted in an increase in this toxin (12-fold).

An innovative food system approach to diversifying protein intake: Protein-I: Shared Island sustainable healthy nutrition.

There is a need to transform our current food system if we are to feed the rapidly expanding global population while maintaining planetary health. Within the island of Ireland, there is an urgent need to diversify the foods that currently contribute to our populations' protein intake. A Shared Island Innovative Food System approach is required to achieve this in a manner that is sustainable and provides benefits to producers, consumers and other supply chain participants. The Protein-I project employs such an approach, with the paradigm focusing on production of plant food through to human health, while paying particular attention to the development of the rural bioeconomy. Using an interdisciplinary approach, the team will develop strategies to maximise sustainable plant protein production in a traceable/transparent fashion and assess the impact of changes to existing value chains and the development of new value chains for the rural economy. A smart supply chain technology solution tailored to the needs of the agri-food industry will be developed and tested. Additionally, we will co-design consumer-led approaches to diversify plant protein intake, model the impact of changes at the population level and perform human interventions to demonstrate efficacy in terms of achieving adequate nutrition and improved health. Comprehensive engagement with stakeholders is embedded throughout the whole project to embrace the multi-actor approach. Overall, the project will be a key step towards future-proofing our food system on the island of Ireland and moving towards protecting planetary and population health, within the context of a just transition.

Comprehensive analysis of pathogen-responsive wheat NAC transcription factors: new candidates for crop improvement.

Wheat NAC (TaNAC) transcription factors are important regulators of stress responses and developmental processes. This study proposes a new TaNAC nomenclature and identified defense-associated TaNACs based on the analysis of RNA-sequencing datasets of wheat tissue infected with major fungal pathogens. A total of 146 TaNACs were pathogen-responsive, of which 52 were orthologous with functionally characterized defense-associated NACs from barley, rice, and Arabidopsis, as deduced via phylogenetic analysis. Next, we focused on the phylogenetic relationship of the pathogen-responsive TaNACs and their expression profiles in healthy and diseased tissues. Three subfamilies ("a," "e," and "f") were significantly enriched in pathogen-responsive TaNACs, of which the majority were responsive to at least 2 pathogens (universal pathogen response). Uncharacterized TaNACs from subfamily "a" enriched with defense-associated NACs are promising candidates for functional characterization in pathogen defense. In general, pathogen-responsive TaNACs were expressed in at least 2 healthy organs. Lastly, we showed that the wheat NAM domain is significantly divergent in sequence in subfamilies "f," "g," and "h" based on HMMER and motif analysis. New protein motifs were identified in both the N- and C-terminal parts of TaNACs. Three of those identified in the C-terminal part were linked to pathogen responsiveness of the TaNACs and 2 were linked to expression in grain tissue. Future studies should benefit from this comprehensive in silico analysis of pathogen-responsive TaNACs as a basis for selecting the most promising candidates for functional validation and crop improvement.

Lactic Acid Bacteria as Potential Biocontrol Agents for Fusarium Head Blight Disease of Spring Barley.

Fusarium head blight (FHB) is a devastating disease encountered by spring-grown barley. Traditionally, synthetic chemicals have been used to control this disease on small grain cereals. A move toward biological control agents as part of sustainable agriculture is pertinent due to the evolutionary mechanisms employed by fungal diseases to circumvent current protection strategies. This study evaluated the effect of six lactic acid bacteria isolates on the development of FHB under in vitro and glasshouse conditions. The relative expression of Fusarium marker genes and transcription factors under Fusarium infection was examined. Dual-culture assays observed inhibition zones of up to 10 and 17% of total plate area for L. amylovorus FST 2.11 and L. brevis R2Δ, respectively. Detached leaf assays validated the antifungal activity and showed the potential of all test isolates to significantly inhibit sporulation of Fusarium culmorum and Fusarium graminearum strains. Spray inoculation of lactic acid bacteria to barley spikelets prior to Fusarium spore application significantly reduced disease severity for five candidates (P < 0.05) under glasshouse conditions. Mycotoxin analysis revealed the ability of L. amylovorus DSM20552 to significantly reduce deoxynivalenol content in spikelets (P < 0.05). A preliminary gene expression study showed the positive influence of lactic acid bacteria on the expression of important defense-related marker genes and transcription factors upon FHB. These results indicate the potential of lactic acid bacteria to be included as part of an integrated pest management strategy for the management of FHB disease. This strategy will reduce FHB severity and deoxynivalenol (DON) contamination of spring barley, leading to high acceptance in the grain market.

The impact of chitosan on the early metabolomic response of wheat to infection by Fusarium graminearum.

BACKGROUND: Chitosan has shown potential for the control of Fusarium head blight (FHB) disease caused by Fusarium graminearum. The objective of this study was to compare the effect of chitosan hydrochloride applied pre- or post-fungal inoculation on FHB and to better understand its' mode of action via an untargeted metabolomics study. RESULTS: Chitosan inhibited fungal growth in vitro and, when sprayed on the susceptible wheat cultivar Remus 24 hours pre-inoculation with F. graminearum, it significantly reduced the number of infected spikelets at 7, 14 and 21 days post-inoculation. Chitosan pre-treatment also increased the average grain weight per head, the number of grains per head and the 1000-grain weight compared to the controls sprayed with water. No significant impact of chitosan on grain yield was observed when the plants were sprayed 24 hours post-inoculation with F. graminearum, even if it did result in a reduced number of infected spikelets at every time point. An untargeted metabolomic study using UHPLC-QTOF-MS on wheat spikes revealed that spraying the spikes with both chitosan and F. graminearum activated known FHB resistance pathways (e.g. jasmonic acid). Additionally, more metabolites were up- or down-regulated when both chitosan and F. graminearum spores were sprayed on the spikes (117), as compared with chitosan (51) or F. graminearum on their own (32). This included a terpene, a terpenoid and a liminoid previously associated with FHB resistance. CONCLUSIONS: In this study we showed that chitosan hydrochloride inhibited the spore germination and hyphal development of F. graminearum in vitro, triggered wheat resistance against infection by F. graminearum when used as a pre-inoculant, and highlighted metabolites and pathways commonly and differentially affected by chitosan, the pathogen and both agents. This study provides insights into how chitosan might provide protection or stimulate wheat resistance to infection by F. graminearum. It also unveiled new putatively identified metabolites that had not been listed in previous FHB or chitosan-related metabolomic studies.

Agronomic Factors Influencing the Scale of Fusarium Mycotoxin Contamination of Oats.

Seven agronomic factors (crop season, farming system, harvest date, moisture, county, oat variety, and previous crop) were recorded for 202 oat crops grown across Ireland, and samples were analysed by LC-MS/MS for four major Fusarium mycotoxins: deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin and HT-2 toxin. Type A trichothecenes were present in 62% of crops, with 7.4% exceeding European regulatory limits. DON (6.4%) and ZEN (9.9%) occurrences were relatively infrequent, though one and three samples were measured over their set limits, respectively. Overall, the type of farming system and the previous crop were the main factors identified as significantly influencing mycotoxin prevalence or concentration. Particularly, the adherence to an organic farming system and growing oats after a previous crop of grass were found to decrease contamination by type A trichothecenes. These are important findings and may provide valuable insights for many other types of cereal crops as Europe moves towards a much greater organic-based food system.

Mitochondrial phosphate transporter and methyltransferase genes contribute to Fusarium head blight Type II disease resistance and grain development in wheat.

Fusarium head blight (FHB) is an economically important disease of wheat that results in yield loss and grain contaminated with fungal mycotoxins that are harmful to human and animal health. Herein we characterised two wheat genes involved in the FHB response in wheat: a wheat mitochondrial phosphate transporter (TaMPT) and a methyltransferase (TaSAM). Wheat has three sub-genomes (A, B, and D) and gene expression studies demonstrated that TaMPT and TaSAM homoeologs were differentially expressed in response to FHB infection and the mycotoxigenic Fusarium virulence factor deoxynivalenol (DON) in FHB resistant wheat cv. CM82036 and susceptible cv. Remus. Virus-induced gene silencing (VIGS) of either TaMPT or TaSAM enhanced the susceptibility of cv. CM82036 to FHB disease, reducing disease spread (Type II disease resistance). VIGS of TaMPT and TaSAM significantly reduced grain number and grain weight. This indicates TaSAM and TaMPT genes also contribute to grain development in wheat and adds to the increasing body of evidence linking FHB resistance genes to grain development. Hence, Fusarium responsive genes TaSAM and TaMPT warrant further study to determine their potential to enhance both disease resistance and grain development in wheat.

Analysis of the chromosomal clustering of Fusarium-responsive wheat genes uncovers new players in the defence against head blight disease.

There is increasing evidence that some functionally related, co-expressed genes cluster within eukaryotic genomes. We present a novel pipeline that delineates such eukaryotic gene clusters. Using this tool for bread wheat, we uncovered 44 clusters of genes that are responsive to the fungal pathogen Fusarium graminearum. As expected, these Fusarium-responsive gene clusters (FRGCs) included metabolic gene clusters, many of which are associated with disease resistance, but hitherto not described for wheat. However, the majority of the FRGCs are non-metabolic, many of which contain clusters of paralogues, including those implicated in plant disease responses, such as glutathione transferases, MAP kinases, and germin-like proteins. 20 of the FRGCs encode nonhomologous, non-metabolic genes (including defence-related genes). One of these clusters includes the characterised Fusarium resistance orphan gene, TaFROG. Eight of the FRGCs map within 6 FHB resistance loci. One small QTL on chromosome 7D (4.7 Mb) encodes eight Fusarium-responsive genes, five of which are within a FRGC. This study provides a new tool to identify genomic regions enriched in genes responsive to specific traits of interest and applied herein it highlighted gene families, genetic loci and biological pathways of importance in the response of wheat to disease.

A small secreted protein from Zymoseptoria tritici interacts with a wheat E3 ubiquitin ligase to promote disease.

Septoria tritici blotch (STB), caused by the ascomycete fungus Zymoseptoria tritici, is a major threat to wheat production worldwide. The Z. tritici genome encodes many small secreted proteins (ZtSSPs) that are likely to play a key role in the successful colonization of host tissues. However, few of these ZtSSPs have been functionally characterized for their role during infection. In this study, we identified and characterized a small, conserved cysteine-rich secreted effector from Z. tritici which has homologues in other plant pathogens in the Dothideomycetes. ZtSSP2 was expressed throughout Z. tritici infection in wheat, with the highest levels observed early during infection. A yeast two-hybrid assay revealed an interaction between ZtSSP2 and wheat E3 ubiquitin ligase (TaE3UBQ) in yeast, and this was further confirmed in planta using bimolecular fluorescence complementation and co-immunoprecipitation. Down-regulation of this wheat E3 ligase using virus-induced gene silencing increased the susceptibility of wheat to STB. Together, these results suggest that TaE3UBQ is likely to play a role in plant immunity to defend against Z. tritici.

Genome-wide association mapping of Fusarium langsethiae infection and mycotoxin accumulation in oat (Avena sativa L.).

Fusarium langsethiae is a symptomless pathogen of oat panicles that produces T-2 and HT-2 mycotoxins, two of the most potent trichothecenes produced by Fusarium fungi in cereals. In the last few years, the levels of these mycotoxin in oat grain has increased and the European commission have already recommended a maximum level for of 1000 µg kg-1 for unprocessed oat for human consumption. The optimal and most sustainable way of combating infection and mycotoxin contamination is by releasing resistant oat varieties. Here the objective was to determine if we could identify any genomic loci associated with either the accumulation of F. langsethiae DNA or mycotoxins in the grain. In each of two years, field trials were conducted wherein 190 spring oat varieties were inoculated with a mixture of three isolate of the pathogen. Mycotoxins were quantified using liquid chromatography-tandem mass spectrometry. Varieties were genotyped using 16,863 genotyping by sequencing markers. Genome-wide association studies associated 5 SNPs in the linkage group Mr06 with T-2 + HT-2 mycotoxin accumulation. Markers were highly correlated, and a single QTL was identified. The marker avgbs_6K_95238.1 mapped within genes showing similarity to lipase, lipase-like or lipase precursor mRNA sequences and zinc-finger proteins. These regions have previously been shown to confer a significant increase in resistance to Fusarium species.

Insights into the resistance of a synthetically-derived wheat to Septoria tritici blotch disease: less is more.

BACKGROUND: Little is known about the initial, symptomless (latent) phase of the devastating wheat disease Septoria tritici blotch. However, speculations as to its impact on fungal success and disease severity in the field have suggested that a long latent phase is beneficial to the host and can reduce inoculum build up in the field over a growing season. The winter wheat cultivar Stigg is derived from a synthetic hexaploid wheat and contains introgressions from wild tetraploid wheat Triticum turgidum subsp. dicoccoides, which contribute to cv. Stigg's exceptional STB resistance, hallmarked by a long latent phase. We compared the early transcriptomic response to Zymoseptoria tritici of cv. Stigg to a susceptible wheat cultivar, to elucidate the mechanisms of and differences in pathogen recognition and disease response in these two hosts. RESULTS: The STB-susceptible cultivar Longbow responds to Z. tritici infection with a stress response, including activation of hormone-responsive transcription factors, post translational modifications, and response to oxidative stress. The activation of key genes associated with these pathways in cv. Longbow was independently observed in a second susceptible wheat cultivar based on an independent gene expression study. By comparison, cv. Stigg is apathetic in response to STB, and appears to fail to activate a range of defence pathways that cv. Longbow employs. Stigg also displays some evidence of sub-genome bias in its response to Z. tritici infection, whereas the susceptible cv. Longbow shows even distribution of Z. tritici responsive genes across the three wheat sub-genomes. CONCLUSIONS: We identify a suite of disease response genes that are involved in early pathogen response in susceptible wheat cultivars that may ultimately lead to susceptibility. In comparison, we hypothesise that rather than an active defence response to stave off disease progression, cv. Stigg's defence strategy is molecular lethargy, or a lower-amplitude of pathogen recognition that may stem from cv. Stigg's wild wheat-derived ancestry. Overall, we present insights into cv. Stigg's exceptional resistance to STB, and present key biological processes for further characterisation in this pathosystem.

Taxonomically Restricted Wheat Genes Interact With Small Secreted Fungal Proteins and Enhance Resistance to Septoria Tritici Blotch Disease.

Understanding the nuances of host/pathogen interactions are paramount if we wish to effectively control cereal diseases. In the case of the wheat/Zymoseptoria tritici interaction that leads to Septoria tritici blotch (STB) disease, a 10,000-year-old conflict has led to considerable armaments being developed on both sides which are not reflected in conventional model systems. Taxonomically restricted genes (TRGs) have evolved in wheat to better allow it to cope with stress caused by fungal pathogens, and Z. tritici has evolved specialized effectors which allow it to manipulate its' host. A microarray focused on the latent phase response of a resistant wheat cultivar (cv. Stigg) and susceptible wheat cultivar (cv. Gallant) to Z. tritici infection was mined for TRGs within the Poaceae. From this analysis, we identified two TRGs that were significantly upregulated in response to Z. tritici infection, Septoria-responsive TRG6 and 7 (TaSRTRG6 and TaSRTRG7). Virus induced silencing of these genes resulted in an increased susceptibility to STB disease in cvs. Gallant and Stigg, and significantly so in the latter (2.5-fold increase in STB disease). In silico and localization studies categorized TaSRTRG6 as a secreted protein and TaSRTRG7 as an intracellular protein. Yeast two-hybrid analysis and biofluorescent complementation studies demonstrated that both TaSRTRG6 and TaSRTRG7 can interact with small proteins secreted by Z. tritici (potential effector candidates). Thus we conclude that TRGs are an important part of the wheat-Z. tritici co-evolution story and potential candidates for modulating STB resistance.

Wheat Encodes Small, Secreted Proteins That Contribute to Resistance to Septoria Tritici Blotch.

During plant-pathogen interactions, pathogens secrete many rapidly evolving, small secreted proteins (SSPs) that can modify plant defense and permit pathogens to colonize plant tissue. The fungal pathogen Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most important foliar diseases of wheat, globally. Z. tritici is a strictly apoplastic pathogen that can secrete numerous proteins into the apoplast of wheat leaves to promote infection. We sought to determine if, during STB infection, wheat also secretes small proteins into the apoplast to mediate the recognition of pathogen proteins and/or induce defense responses. To explore this, we developed an SSP-discovery pipeline to identify small, secreted proteins from wheat genomic data. Using this pipeline, we identified 6,998 SSPs, representing 2.3% of all proteins encoded by the wheat genome. We then mined a microarray dataset, detailing a resistant and susceptible host response to STB, and identified 141 Z. tritici- responsive SSPs, representing 4.7% of all proteins encoded by Z. tritici - responsive genes. We demonstrate that a subset of these SSPs have a functional signal peptide and can interact with Z. tritici SSPs. Transiently silencing two of these wheat SSPs using virus-induced gene silencing (VIGS) shows an increase in susceptibility to STB, confirming their role in defense against Z. tritici.

Quantification of In Planta Zymoseptoria tritici Progression Through Different Infection Phases and Related Association with Components of Aggressiveness.

In planta growth of Zymoseptoria tritici, causal agent of Septoria tritici blotch of wheat, during the infection process has remained an understudied topic due to the long symptomless latent period before the emergence of fruiting bodies. In this study, we attempted to understand the relationship between in planta growth of Z. tritici relative to the primary components of aggressiveness, i.e., latent period and pycnidia coverage in regard to contrasting host resistance. We tested isolates collected from Ireland against the susceptible cultivar Gallant and cultivar Stigg, which has strong partial resistance. A clear isolate-host interaction effect (F = 3.018; P = 0.005, and F = 6.008; P < 0.001) for latent period and pycnidia coverage, respectively, was identified. Furthermore, during the early infection phase of latency from 5 to 11 days postinoculation (dpi), in planta growth rate of fungal biomass was significantly (F = 30.06; P < 0.001) more affected by host resistance than isolate specificity (F = 1.27; P = 0.27), indicating the importance of host resistance in the early infection phase. In planta Z. tritici growth rates in cultivar Gallant spiked between 11 and 16 dpi followed by a continuous fall onward, whereas in cultivar Stigg it was slowly progressive in nature. From correlation and regression analysis, we found that the in planta growth rate preceding the average latent period of cultivar Gallant has more influence on latency duration and pycnidia production. Likewise, correlation between component of aggressiveness and in planta growth rate of pathogen supports our understanding of aggressiveness to be driven by the pathogen's multiplication capacity within host tissue.

The wheat SnRK1α family and its contribution to Fusarium toxin tolerance.

Deoxynivalenol (DON) is a mycotoxin produced by phytopathogenic Fusarium fungi in cereal grain and plays a role as a disease virulence factor. TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) enhances wheat resistance to DON and it interacts with a sucrose non-fermenting-1 (SNF1)-related protein kinase 1 catalytic subunit α (SnRK1α). This protein kinase family is central integrator of stress and energy signalling, regulating plant metabolism and growth. Little is known regarding the role of SnRK1α in the biotic stress response, especially in wheat. In this study, 15 wheat (Triticum aestivum) SnRK1α genes (TaSnRK1αs) belonging to four homoeologous groups were identified in the wheat genome. TaSnRK1αs are expressed ubiquitously in all organs and developmental stages apart from two members predominantly detected in grain. While DON treatment had either no effect or downregulated the transcription of TaSnRK1αs, it increased both the kinase activity associated with SnRK1α and the level of active (phosphorylated) SnRK1α. Down-regulation of two TaSnRK1αs homoeolog groups using virus induced gene silencing (VIGS) increased the DON-induced damage of wheat spikelets. Thus, we demonstrate that TaSnRK1αs contribute positively to wheat tolerance of DON and conclude that this gene family may provide useful tools for the improvement of crop biotic stress resistance.

Insights into the transcriptomic response of the plant engineering bacterium Ensifer adhaerens OV14 during transformation.

The ability to engineer plant genomes has been primarily driven by the soil bacterium Agrobacterium tumefaciens but recently the potential of alternative rhizobia such as Rhizobium etli and Ensifer adhaerens OV14, the latter of which supports Ensifer Mediated Transformation (EMT) has been reported. Surprisingly, a knowledge deficit exists in regards to understanding the whole genome processes underway in plant transforming bacteria, irrespective of the species. To begin to address the issue, we undertook a temporal RNAseq-based profiling study of E. adhaerens OV14 in the presence/absence of Arabidopsis thaliana tissues. Following co-cultivation with root tissues, 2333 differentially expressed genes (DEGs) were noted. Meta-analysis of the RNAseq data sets identified a clear shift from plasmid-derived gene expression to chromosomal-based transcription within the early stages of bacterium-plant co-cultivation. During this time, the number of differentially expressed prokaryotic genes increased steadily out to 7 days co-cultivation, a time at which optimum rates of transformation were observed. Gene ontology evaluations indicated a role for both chromosomal and plasmid-based gene families linked specifically with quorum sensing, flagellin production and biofilm formation in the process of EMT. Transcriptional evaluation of vir genes, housed on the pCAMBIA 5105 plasmid in E. adhaerens OV14 confirmed the ability of E. adhaerens OV14 to perceive and activate its transcriptome in response to the presence of 200 µM of acetosyringone. Significantly, this is the first study to characterise the whole transcriptomic response of a plant engineering bacterium in the presence of plant tissues and provides a novel insight into prokaryotic genetic processes that support T-DNA transfer.

Serpins: Genome-Wide Characterisation and Expression Analysis of the Serine Protease Inhibitor Family in Triticum aestivum.

The serine protease inhibitor (serpin) gene family is the largest family of protease inhibitors. Serine protease inhibitors have an active, but under-characterized, role in grain development and defense against pathogen attack in cereal crops. By exploiting publicly available genomic, transcriptomic and proteomic data for wheat (Triticum aestivum), we have identified and annotated the entire 'serpinome' of wheat and constructed a high-quality and robust phylogenetic tree of the gene family, identifying paralogous and homeologous clades from the hexaploid wheat genome, including the Serpin-Z group that have been well characterized in barley. Using publicly available RNAseq data (http://www.wheat-expression.com/), expression profiles of the wheat serpins were explored across a variety of tissues from the developing grain, spikelet and spike. We show that the SERPIN-Z clade, among others, are highly expressed during grain development, and that there is homeologous and paralogous functional redundancy in this gene family. Further to their role in grain development, serpins play an important but under-explored role in response to fungal pathogens. Using 13 RNAseq datasets of wheat tissues infected by fungal pathogens, we identified 37 serpins with a significant disease response. The majority of the disease-responsive serpins were upregulated by Fusarium graminearum, a destructive fungal pathogen that attacks the spike and developing grain of wheat. As serpins are ubiquitous in wheat grain, the genes encoding serpins may be linked to grain development, with their disease response a result of pleiotropy.

A wheat NAC interacts with an orphan protein and enhances resistance to Fusarium head blight disease.

Taxonomically-restricted orphan genes play an important role in environmental adaptation, as recently demonstrated by the fact that the Pooideae-specific orphan TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) enhanced wheat resistance to the economically devastating Fusarium head blight (FHB) disease. Like most orphan genes, little is known about the cellular function of the encoded protein TaFROG, other than it interacts with the central stress regulator TaSnRK1α. Here, we functionally characterized a wheat (T. aestivum) NAC-like transcription factor TaNACL-D1 that interacts with TaFROG and investigated its' role in FHB using studies to assess motif analyses, yeast transactivation, protein-protein interaction, gene expression and the disease response of wheat lines overexpressing TaNACL-D1. TaNACL-D1 is a Poaceae-divergent NAC transcription factor that encodes a Triticeae-specific protein C-terminal region with transcriptional activity and a nuclear localisation signal. The TaNACL-D1/TaFROG interaction was detected in yeast and confirmed in planta, within the nucleus. Analysis of multi-protein interactions indicated that TaFROG could form simultaneously distinct protein complexes with TaNACL-D1 and TaSnRK1α in planta. TaNACL-D1 and TaFROG are co-expressed as an early response to both the causal fungal agent of FHB, Fusarium graminearum and its virulence factor deoxynivalenol (DON). Wheat lines overexpressing TaNACL-D1 were more resistant to FHB disease than wild type plants. Thus, we conclude that the orphan protein TaFROG interacts with TaNACL-D1, a NAC transcription factor that forms part of the disease response evolved within the Triticeae.