Transcriptome Analysis of Heat Shock Factor C2a Over-Expressing Wheat Roots Reveals Ferroptosis-like Cell Death in Heat Stress Recovery.

Wheat (Triticum aestivum L.) growing areas in many regions of the world are subject to heat waves which are predicted to increase in frequency because of climate change. The engineering of crop plants can be a useful strategy to mitigate heat stress-caused yield losses. Previously, we have shown that heat shock factor subclass C (TaHsfC2a-B)-overexpression significantly increased the survival of heat-stressed wheat seedlings. Although previous studies have shown that the overexpression of Hsf genes enhanced the survival of plants under heat stress, the molecular mechanisms are largely unknown. To understand the underlying molecular mechanisms involved in this response, a comparative analysis of the root transcriptomes of untransformed control and TaHsfC2a-overexpressing wheat lines by RNA-sequencing have been performed. The results of RNA-sequencing indicated that the roots of TaHsfC2a-overexpressing wheat seedlings showed lower transcripts of hydrogen peroxide-producing peroxidases, which corresponds to the reduced accumulation of hydrogen peroxide along the roots. In addition, suites of genes from iron transport and nicotianamine-related gene ontology categories showed lower transcript abundance in the roots of TaHsfC2a-overexpressing wheat roots than in the untransformed control line following heat stress, which are in accordance with the reduction in iron accumulation in the roots of transgenic plants under heat stress. Overall, these results suggested the existence of ferroptosis-like cell death under heat stress in wheat roots, and that TaHsfC2a is a key player in this mechanism. To date, this is the first evidence to show that a Hsf gene plays a key role in ferroptosis under heat stress in plants. In future, the role of Hsf genes could be further studied on ferroptosis in plants to identify root-based marker genes to screen for heat-tolerant genotypes.

Transcriptome analysis reveals infection strategies employed by Fusarium graminearum as a root pathogen.

The fungal pathogen Fusarium graminearum (Fg) infects both heads and roots of cereal crops causing several economically important diseases such as head blight, seedling blight, crown rot and root rot. Trichothecene mycotoxins such as deoxynivalenol (DON), a well-known virulence factor, produced by Fg during disease development is also an important health concern. Although how Fg infects above-ground tissues is relatively well studied, very little is known about molecular processes employed by the pathogen during below-ground infection. Also unknown is the role of DON during root infection. In the present study, we analyzed the transcriptome of Fg during root infection of the model cereal Brachypodium distachyon (Bd). We also compared our Fg transcriptome data obtained during Bd root infection with those reported during wheat head infection. These analyses suggested that both shared and unique infection strategies were employed by the pathogen during colonization of different host tissues. Several metabolite biosynthesis genes induced in Fg during root infection could be linked to phytohormone production, implying that the pathogen likely interferes with root specific defenses. In addition, to understand the role of DON in Fg root infection, we analyzed the transcriptome of the DON deficient Tri5 mutant. These analyses showed that the absence of DON had a significant effect on fungal transcriptional responses. Although DON was produced in infected roots, this mycotoxin did not act as a Fg virulence factor during root infection. Our results reveal new mechanistic insights into the below-ground strategies employed by Fg that may benefit the development of new genetic tools to combat this important cereal pathogen.

Adaptive defence and sensing responses of host plant roots to fungal pathogen attack revealed by transcriptome and metabolome analyses.

Plant root-produced constitutive and inducible defences inhibit pathogenic microorganisms within roots and in the rhizosphere. However, regulatory mechanisms underlying host responses during root-pathogen interactions are largely unexplored. Using the model species Brachypodium distachyon (Bd), we studied transcriptional and metabolic responses altered in Bd roots following challenge with Fusarium graminearum (Fg), a fungal pathogen that causes diseases in diverse organs of cereal crops. Shared gene expression patterns were found between Bd roots and spikes during Fg infection associated with the mycotoxin deoxynivalenol (DON). Overexpression of BdMYB78, an up-regulated transcription factor, significantly increased root resistance during Fg infection. We show that Bd roots recognize encroaching Fg prior to physical contact by altering transcription of genes associated with multiple cellular processes such as reactive oxygen species and cell development. These changes coincide with altered levels of secreted host metabolites detected by an untargeted metabolomic approach. The secretion of Bd metabolites was suppressed by Fg as enhanced levels of defence-associated metabolites were found in roots during pre-contact with a Fg mutant defective in host perception and the ability to cause disease. Our results help to understand root defence strategies employed by plants, with potential implications for improving the resistance of cereal crops to soil pathogens.

Map-based cloning identifies velvet A as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads.

Although better known as a pathogen of wheat stem bases, Fusarium pseudograminearum also causes Fusarium head blight. A natural isolate of F. pseudograminearum was identified that showed severely reduced virulence towards wheat heads and a map-based cloning approach was undertaken to identify the genetic basis of this phenotype. Using a population of 95 individuals, a single locus on chromosome 1 was shown to be responsible for the low virulence. Fine mapping narrowed the region to just five possible SNPs of which one was in the F. pseudograminearum homologue of velvet A. Knockout mutants of velvet A, which were non-pathogenic towards wheat, confirmed that velvet A regulates virulence in this pathogen. The mutation in velvet A was only found in a single field isolate and the origin of the mutation is unknown.

Can natural gene drives be part of future fungal pathogen control strategies in plants?

Globally, fungal pathogens cause enormous crop losses and current control practices are not always effective, economical or environmentally sustainable. Tools enabling genetic management of wild pathogen populations could potentially solve many problems associated with plant diseases. A natural gene drive from a heterologous species can be used in the globally important cereal pathogen Fusarium graminearum to remove pathogenic traits from contained populations of the fungus. The gene drive element became fixed in a freely crossing population in only three generations. Repeat-induced point mutation (RIP), a natural genome defence mechanism in fungi that causes C to T mutations during meiosis in highly similar sequences, may be useful to recall the gene drive following release, should a failsafe mechanism be required. We propose that gene drive technology is a potential tool to control plant pathogens once its efficacy is demonstrated under natural settings.

Regulators of nitric oxide signaling triggered by host perception in a plant pathogen.

The rhizosphere interaction between plant roots or pathogenic microbes is initiated by mutual exchange of signals. However, how soil pathogens sense host signals is largely unknown. Here, we studied early molecular events associated with host recognition in Fusarium graminearum, an economically important fungal pathogen that can infect both roots and heads of cereal crops. We found that host sensing prior to physical contact with plant roots radically alters the transcriptome and triggers nitric oxide (NO) production in F. graminearum We identified an ankyrin-repeat domain containing protein (FgANK1) required for host-mediated NO production and virulence in F. graminearum In the absence of host plant, FgANK1 resides in the cytoplasm. In response to host signals, FgANK1 translocates to the nucleus and interacts with a zinc finger transcription factor (FgZC1), also required for specific binding to the nitrate reductase (NR) promoter, NO production, and virulence in F. graminearum Our results reveal mechanistic insights into host-recognition strategies employed by soil pathogens.

Ribosome profiling in plants: what is not lost in translation?

Translation is a highly dynamic cellular process whereby genetic information residing in an mRNA molecule is converted into a protein that in turn executes specific functions. However, pre-synthesized mRNA levels do not always correlate with corresponding protein levels, suggesting that translational control plays an essential role in gene regulation. A better understanding of how gene expression is regulated during translation will enable the discovery of new genes and mechanisms that control important traits in plants. Therefore, in recent years, several methods have been developed to analyse the translatome; that is, all mRNAs being actively translated at a given time, tissue, and/or developmental stage. Ribosome profiling or ribo-seq is one such technology revolutionizing our ability to analyse the translatome and in turn understand translational control of gene expression. Ribo-seq involves isolating mRNA-ribosome complexes, treating them with a RNase, and then identifying ribosome-protected mRNA regions by deep sequencing. Here, we briefly review recent ribosome profiling studies that revealed new insights into plant biology. Manipulation of novel genes identified using ribosome profiling could prove useful for increasing yield through improved biotic and abiotic stress tolerance.

Eastern Anatolian apples with a unique population structure are genetically different from Anatolian apples.

The origin of the apple is known to be the Transcaucasian region. Eastern Anatolia, which is located on the migration routes from Asia to Europe, has a rich and an uncharacterized apple germplasm and the characterization of apple genetic sources from this region is important for both evolutionary studies and apple breeding. In this study, 94 M. domestica accessions originated from seven diverse regions within Eastern Anatolia were studied using 16 SSR (simple sequence repeat) loci. SSR markers we used produced high allele numbers in all loci and CH02d11 (PI: 0.059) with 18 alleles was the most informative locus. In addition, 14 identical accession groups were identified. Most likely due to self-incompatibility, relatively high levels of heterozygosity (Ho: 0.696) was found for Eastern Anatolia apples. Structure Harvester analyses of East Anatolian apple accessions showed that although each group seems to be somewhat distinct, some levels of admixture with other populations might also exist. Due to a significant gene flow between all pairs of seven apple populations, a limited (low) differentiation was found between the populations. Comparisons using 16 common SSR loci revealed that Eastern Anatolia accessions were genetically different from Anatolian accessions. In addition, based on FCA, and Nei's genetic distance analyses, Eastern Anatolian apples were found to be genetically different from the commercial apple cultivars Golden Delicious and Florina. Our results suggesting that Eastern Anatolia apple populations have a unique structure will be useful for future genetic and evolutionary studies on apples.

Fusaristatin A production negatively affects the growth and aggressiveness of the wheat pathogen Fusarium pseudograminearum.

Fusarium pseudograminearum (Fp), the causative fungal pathogen of the diseases Fusarium crown rot, is an important constraint to cereals production in many countries including Australia. Fp produces a number of secondary metabolites throughout its life cycle. One of these metabolites, the cyclic lipopeptide fusaristatin A, is encoded by a specific gene cluster containing a polyketide synthase and a three-module non-ribosomal peptide synthetase. However, a recent survey of Fp populations across Australia suggests that this cluster may only be present in a subset of isolates from Western Australia (WA). In this study, we screened 319 Fp isolates from WA and 110 Fp isolates from the Australian eastern states of New South Wales, Victoria, Queensland and South Australia to examine the distribution of this gene cluster among Australian Fp populations. The fusaristatin A gene cluster was found to be present in ~50% of Fp isolates from WA but completely absent in Fp isolates from eastern states. To determine its potential function, mutants of the fusaristatin A gene cluster were generated by disrupting the non-ribosomal peptide synthetase and polyketide synthase genes simultaneously in two different parental backgrounds. The mutants showed increased growth rates and were significantly more aggressive than their respective parental strains on wheat in crown rot pathogenicity assays. This suggested that fusaristatin A has a negative effect on fungal development and aggressiveness. The possible reasons for the geographically restricted presence of the fusaristatin A gene cluster and its role in fungal biology are discussed.

The AtHSP17.4C1 Gene Expression Is Mediated by Diverse Signals that Link Biotic and Abiotic Stress Factors with ROS and Can Be a Useful Molecular Marker for Oxidative Stress.

Reactive oxygen species (ROS) are highly controlled signaling species that are involved in regulating gene expression in response to different environmental cues. The production of heat shock proteins (HSPs) is a key strategy that plants use to defend themselves against diverse stresses, including oxidative stress. In this study, expression patterns of the Arabidopsis HSP17.4CI gene, a cytosolic class I small HSP, were systematically profiled under different abiotic, biotic and oxidative stresses. Our data show that HSP17.4CI was early and highly induced by heat, cold, salt, drought and high-light. HSP17.4CI also showed high expression levels in Arabidopsis plants infected with the biotrophic pathogen Pseudomonas syringae, but not in response to the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum. Oxidative stress treatments including H2O2 and the herbicide methyl viologen led to induction of HSP17.4CI. The plant hormones abscisic acid (ABA) and salicylic acid (SA) induced the expression of HSP17.4CI, whereas methyl jasmonate (MJ) did not affect the expression level of this gene. Furthermore, we found enhanced expression of HSP17.4CI in catalase mutant plants, which are deficient in catalase 2 activity and accumulate intracellular H2O2. Taken together, data presented here suggest that HSP17.4CI expression is regulated by various signals that connect biotic and abiotic stresses with ROS and can be used as a molecular marker for oxidative stress.

Ferroptosis: Yet Another Way to Die.

Cell death is one of the most fundamental biological processes operating in multicellular organisms. Recent research highlighted here [Distéfano et al. (J. Cell Biol. 2017:216;463-476) and Dangol et al. (Plant Cell 2019:31;189-209)] revealed an iron- and ROS-dependent cell death phenomenon called ferroptosis in plants. Features distinguishing ferroptosis from other cell death events and how ferroptosis can be exploited to improve plant performance are discussed.

Selection is required for efficient Cas9-mediated genome editing in Fusarium graminearum.

Genome engineering using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nucleases, such as Cas9 (CRISPR-associated protein 9), are revolutionising molecular biology. In this study, we established a Cas9-based genome editing system in Fusarium graminearum, a highly destructive fungal pathogen of cereal crops. Although the molecular toolkit of F. graminearum is well developed compared to other fungi, Cas9-mediated engineering offers a number of potential benefits, such as the ability to create marker free mutants in this species. Here we have used a codon-optimised Cas9 nuclease and dual ribozyme-based expression of a single guide RNA (sgRNA) to induce mutations. Cas9-mediated mutations were identified through a fungicide resistance-based phenotypic screen, which selects for null mutations in the FgOs1 gene encoding an osmosensor histidine kinase. In the absence of selection, however, mutations were identified at very low frequency. Examination of the mutant alleles identified suggests that, a microhomology-mediated end joining (MMEJ) DNA repair pathway is likely to be the predominant process involved in erroneous repairing of Cas9-induced double-stranded breaks in F. graminearum.

A high-resolution genetic map of the cereal crown rot pathogen Fusarium pseudograminearum provides a near-complete genome assembly.

Fusarium pseudograminearum is an important pathogen of wheat and barley, particularly in semi-arid environments. Previous genome assemblies for this organism were based entirely on short read data and are highly fragmented. In this work, a genetic map of F. pseudograminearum has been constructed for the first time based on a mapping population of 178 individuals. The genetic map, together with long read scaffolding of a short read-based genome assembly, was used to give a near-complete assembly of the four F. pseudograminearum chromosomes. Large regions of synteny between F. pseudograminearum and F. graminearum, the related pathogen that is the primary causal agent of cereal head blight disease, were previously proposed in the core conserved genome, but the construction of a genetic map to order and orient contigs is critical to the validation of synteny and the placing of species-specific regions. Indeed, our comparative analyses of the genomes of these two related pathogens suggest that rearrangements in the F. pseudograminearum genome have occurred in the chromosome ends. One of these rearrangements includes the transposition of an entire gene cluster involved in the detoxification of the benzoxazolinone (BOA) class of plant phytoalexins. This work provides an important genomic and genetic resource for F. pseudograminearum, which is less well characterized than F. graminearum. In addition, this study provides new insights into a better understanding of the sexual reproduction process in F. pseudograminearum, which informs us of the potential of this pathogen to evolve.

Transcriptomics of cereal-Fusarium graminearum interactions: what we have learned so far.

The ascomycete fungal pathogen Fusarium graminearum causes the globally important Fusarium head blight (FHB) disease on cereal hosts, such as wheat and barley. In addition to reducing grain yield, infection by this pathogen causes major quality losses. In particular, the contamination of food and feed with the F. graminearum trichothecene toxin deoxynivalenol (DON) can have many adverse short- and long-term effects on human and animal health. During the last decade, the interaction between F. graminearum and both cereal and model hosts has been extensively studied through transcriptomic analyses. In this review, we present an overview of how such analyses have advanced our understanding of this economically important plant-microbe interaction. From a host point of view, the transcriptomes of FHB-resistant and FHB-susceptible cereal genotypes, including near-isogenic lines (NILs) that differ by the presence or absence of quantitative trait loci (QTLs), have been studied to understand the mechanisms of disease resistance afforded by such QTLs. Transcriptomic analyses employed to dissect host responses to DON have facilitated the identification of the genes involved in toxin detoxification and disease resistance. From the pathogen point of view, the transcriptome of F. graminearum during pathogenic vs. saprophytic growth, or when infecting different cereal hosts or different tissues of the same host, have been studied. In addition, comparative transcriptomic analyses of F. graminearum knock-out mutants with altered virulence have provided new insights into pathogenicity-related processes. The F. graminearum transcriptomic data generated over the years are now being exploited to build a systems level understanding of the biology of this pathogen, with an ultimate aim of developing effective and sustainable disease prevention strategies.

Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: recent progress and future prospects.

Diseases caused by Fusarium pathogens inflict major yield and quality losses on many economically important plant species worldwide, including cereals. Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is a cereal disease that occurs in many arid and semi-arid cropping regions of the world. In recent years, this disease has become more prevalent, in part as a result of the adoption of moisture-preserving cultural practices, such as minimum tillage and stubble retention. In this pathogen profile, we present a brief overview of recent research efforts that have not only advanced our understanding of the interactions between F. pseudograminearum and cereal hosts, but have also provided new disease management options. For instance, significant progress has been made in the genetic characterization of pathogen populations, the development of new tools for disease prediction, and the identification and pyramiding of loci that confer quantitative resistance to FCR in wheat and barley. In addition, transcriptome analyses have revealed new insights into the processes involved in host defence. Significant progress has also been made in understanding the mechanistic details of the F. pseudograminearum infection process. The sequencing and comparative analyses of the F. pseudograminearum genome have revealed novel virulence factors, possibly acquired through horizontal gene transfer. In addition, a conserved pathogen gene cluster involved in the degradation of wheat defence compounds has been identified, and a role for the trichothecene toxin deoxynivalenol (DON) in pathogen virulence has been reported. Overall, a better understanding of cereal host-F. pseudograminearum interactions will lead to the development of new control options for this increasingly important disease problem. Taxonomy: Fusarium pseudograminearum O'Donnell & Aoki; Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Sordariomycetes; Subclass Hypocreomycetidae; Order Hypocreales; Family Nectriaceae; Genus Fusarium. Disease symptoms: Fusarium crown rot caused by F. pseudograminearum is also known as crown rot, foot rot and root rot. Infected seedlings can die before or after emergence. If infected seedlings survive, typical disease symptoms are browning of the coleoptile, subcrown internode, lower leaf sheaths and adjacent stems and nodal tissues; this browning can become evident within a few weeks after planting or throughout plant development. Infected plants may develop white heads with no or shrivelled grains. Disease symptoms are exacerbated under water limitation. Identification and detection: Fusarium pseudograminearum macroconidia usually contain three to five septa (22-60.5 × 2.5-5.5 µm). On potato dextrose agar (PDA), aerial mycelia appear floccose and reddish white, with red or reddish-brown reverse pigmentation. Diagnostic polymerase chain reaction (PCR) tests based on the amplification of the gene encoding translation elongation factor-1a (TEF-1a) have been developed for molecular identification. Host range: All major winter cereals can be colonized by F. pseudograminearum. However, the main impact of this pathogen is on bread (Triticum aestivum L.) and durum (Triticum turgidum L. spp. durum (Dest.)) wheat and barley (Hordeum vulgare L.). Oats (Avena sativa L.) can be infected, but show little or no disease symptoms. In addition, the pathogen has been isolated from various other grass genera, such as Phalaris, Agropyron and Bromus, which may occur as common weeds. Useful websites: https://nt.ars-grin.gov/fungaldatabases/; http://plantpath.psu.edu/facilities/fusarium-research-center; https://nt.ars-grin.gov/fungaldatabases/; http://www.speciesfungorum.org/Names/Names.asp.

A Highly Efficient and Reproducible Fusarium spp. Inoculation Method for Brachypodium distachyon.

Fusarium spp. are devastating fungal pathogens which cause significant losses in many cereal crops like wheat, maize, and barley. Genetic improvement of disease resistance requires an improved understanding of defense-associated processes operating in the host in response to an attack by Fusarium spp. Brachypodium distachyon is emerging as a model where host-cereal-infecting pathogen interactions can be studied conveniently. However, this requires developing an efficient infection assay that facilitates quick screening of germplasm (e.g., mutant lines). Here, we provide an efficient and reproducible Fusarium infection assay for Brachypodium. We believe this method will help further develop Brachypodium as a model for genetic improvement of disease resistance in cereals against Fusarium pathogens.

Transcriptome analysis of Brachypodium during fungal pathogen infection reveals both shared and distinct defense responses with wheat.

Fusarium crown rot (FCR) of wheat and barley, predominantly caused by the fungal pathogen Fusarium pseudograminearum, is a disease of economic significance. The quantitative nature of FCR resistance within cultivated wheat germplasm has significantly limited breeding efforts to enhanced FCR resistance in wheat. In this study, we characterized the molecular responses of Brachypodium distachyon (Brachypodium hereafter) to F. pseudograminearum infection using RNA-seq to determine whether Brachypodium can be exploited as a model system towards better understanding of F. pseudograminearum-wheat interaction. The transcriptional response to infection in Brachypodium was strikingly similar to that previously reported in wheat, both in shared expression patterns of wheat homologs of Brachypodium genes and functional overlap revealed through comparative gene ontology analysis in both species. Metabolites produced by various biosynthetic pathways induced in both wheat and Brachypodium were quantified, revealing a high degree of overlap between these two species in metabolic response to infection but also showed Brachypodium does not produce certain defence-related metabolites found in wheat. Functional analyses of candidate genes identified in this study will improve our understanding of resistance mechanisms and may lead to the development of new strategies to protect cereal crops from pathogen infection.

Emerging Roles and Landscape of Translating mRNAs in Plants.

Plants use a wide range of mechanisms to adapt to different environmental stresses. One of the earliest responses displayed under stress is rapid alterations in stress responsive gene expression that has been extensively analyzed through expression profiling such as microarrays and RNA-sequencing. Recently, expression profiling has been complemented with proteome analyses to establish a link between transcriptional and the corresponding translational changes. However, proteome profiling approaches have their own technical limitations. More recently, ribosome-associated mRNA profiling has emerged as an alternative and a robust way of identifying translating mRNAs, which are a set of mRNAs associated with ribosomes and more likely to contribute to proteome abundance. In this article, we briefly review recent studies that examined the processes affecting the abundance of translating mRNAs, their regulation during plant development and tolerance to stress conditions and plant factors affecting the selection of translating mRNA pools. This review also highlights recent findings revealing differential roles of alternatively spliced mRNAs and their translational control during stress adaptation. Overall, better understanding of processes involved in the regulation of translating mRNAs has obvious implications for improvement of stress tolerance in plants.

DNA-Demethylase Regulated Genes Show Methylation-Independent Spatiotemporal Expression Patterns.

Recent research has indicated that a subset of defense-related genes is downregulated in the Arabidopsis DNA demethylase triple mutant rdd (ros1 dml2 dml3) resulting in increased susceptibility to the fungal pathogen Fusarium oxysporum. In rdd plants these downregulated genes contain hypermethylated transposable element sequences (TE) in their promoters, suggesting that this methylation represses gene expression in the mutant and that these sequences are actively demethylated in wild-type plants to maintain gene expression. In this study, the tissue-specific and pathogen-inducible expression patterns of rdd-downregulated genes were investigated and the individual role of ROS1, DML2, and DML3 demethylases in these spatiotemporal regulation patterns was determined. Large differences in defense gene expression were observed between pathogen-infected and uninfected tissues and between root and shoot tissues in both WT and rdd plants, however, only subtle changes in promoter TE methylation patterns occurred. Therefore, while TE hypermethylation caused decreased gene expression in rdd plants it did not dramatically effect spatiotemporal gene regulation, suggesting that this latter regulation is largely methylation independent. Analysis of ros1-3, dml2-1, and dml3-1 single gene mutant lines showed that promoter TE hypermethylation and defense-related gene repression was predominantly, but not exclusively, due to loss of ROS1 activity. These data demonstrate that DNA demethylation of TE sequences, largely by ROS1, promotes defense-related gene expression but does not control spatiotemporal expression in Arabidopsis. Summary: Ros1-mediated DNA demethylation of promoter transposable elements is essential for activation of defense-related gene expression in response to fungal infection in Arabidopsis thaliana.