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Arbuscular mycorrhizal fungi (AMF) are prominent root symbionts with a multinucleate cytoplasm that can carry thousands of nuclei deriving from two parental strains and varying in relative abundance in a large syncytium. Here, we set out to improve our understanding of such remarkable genetics by resolving the nuclear genomes of all publicly available AMF heterokaryons using PacBio HiFi and Hi-C sequencing. We find that all AMF heterokaryons carry two sets of homologous chromosomes, where genes associated with plant colonization reside in gene-sparse, repeat-rich compartments. The co-existing nuclear genomes are phylogenetically related but differ significantly in content and epigenetics, resulting in nucleus-specific regulatory programs during mycorrhizal interactions. AMF heterokaryons carry signatures of past genetic exchange indicative of sexual reproduction, followed by clonal haplotype evolution. This work uncovers the contribution and origin of nuclear genomes present in AMF heterokaryons and opens avenues for improvement and environmental application of these strains.

Plants perceive externally produced microbe-associated molecular patterns (MAMPs) and endogenously produced danger-associated molecular patterns (DAMPs) to activate inducible immunity. While several inducible immune responses have been observed during Fusarium graminearum infection, the identity of the signaling pathways involved is only partly known. We screened 227 receptor kinase and innate immune response genes in Arabidopsis to identify nine genes with a role in F. graminearum resistance. Resistance-promoting genes included the chitin receptors LYK5 and CERK1, and the reactive oxygen species (ROS)-producing NADPH oxidase RbohF, which were required for full inducible immune responses during infection. Two of the genes identified in our screen, APEX and the PAMP-induced peptide 1 (PIP1) DAMP receptor RLK7, repressed F. graminearum resistance. Both RbohF and RLK7 were required for full chitin-induced immune responses and PIP1 precursor expression was induced by chitin and F. graminearum infection. Together, this indicates that F. graminearum resistance is mediated by MAMP and DAMP signaling pathways and that chitin-induced signaling is enhanced by PIP1 perception and ROS production.

Fungal pathogens survive harsh environments and overcome physical, temporal, and chemical barriers to colonize their hosts and reproduce. Fusarium graminearum was one of the first fungal plant pathogens for which transcriptomic tools were developed, making analysis of gene expression a cornerstone approach in studying its biology. The analysis of gene expression in diverse in vitro conditions and during infection of different cereal crops has revealed subsets of both unique and shared transcriptionally regulated genes. Together with genetic studies, these approaches have enhanced our understanding of the development and infection cycle of this economically important pathogen. Here, we will outline recent advances in transcriptional profiling during sporogenesis, spore germination, vegetative growth, and host infection. Several transcriptional regulators have been identified as essential components in these responses and the role of select transcription factors will be highlighted. Finally, we describe some of the gaps in our understanding of F. graminearum biology and how expression analysis could help to address these gaps.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the Fusarium graminearum fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type F. graminearum as compared with a Δtri5 mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including TaNFXL1, by treating tissues with DON directly. Previous work indicates that the NFXL1 ortholog represses trichothecene-induced defense responses and bacterial resistance in Arabidopsis, but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced TaNFXL1 gene expression in a susceptible wheat genotype relative to the F. graminearum–resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that TaNFXL1 represses F. graminearum resistance. Together, this suggests that targeting the TaNFXL1 gene may help to develop disease resistance in cultivated wheat.

Resistance to Fusarium graminearum, a fungal pathogen of cereals, has been associated with increased defense-associated hormonal responses in wheat. A comparison of susceptible (Roblin) and moderately resistant wheat genotypes (Wuhan 1, Nyubai) revealed transcriptional regulation of 1021 genes during infection. Only 4% of these genes were uniquely differentially expressed in the susceptible genotype including an auxin transporter, an ABC transporter, and transcription factors. Roblin displayed higher auxin accumulation during infection relative to resistant genotypes, but similar levels of salicylic acid and jasmonic acid. This indicates that the auxin pathway is one of the few susceptibility-associated pathways during F. graminearum infection.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the

The virulence and broad host range of Fusarium graminearum is associated with its ability to secrete an arsenal of phytotoxic secondary metabolites, including the regulated mycotoxins belonging to the deoxynivalenol family. The TRI genes responsible for the biosynthesis of deoxynivalenol and related compounds are usually expressed during fungal infection. However, the F. graminearum genome harbors an array of unexplored biosynthetic gene clusters that are also co-induced with the TRI genes, including the nonribosomal peptide synthetase 8 (NRPS8) gene cluster. Here, we identify two bicyclic lipopeptides, gramillin A (1) and B (2), as the biosynthetic end products of NRPS8. Structural elucidation by high-resolution LC-MS and NMR, including 1H-15N-13C HNCO and HNCA on isotopically enriched compounds, revealed that the gramillins possess a fused bicyclic structure with ring closure of the main peptide macrocycle occurring via an anhydride bond. Through targeted gene disruption, we characterized the GRA1 biosynthetic gene and its transcription factor GRA2 in the NRPS8 gene cluster. Further, we show that the gramillins are produced in planta on maize silks, promoting fungal virulence on maize but have no discernible effect on wheat head infection. Leaf infiltration of the gramillins induces cell death in maize, but not in wheat. Our results show that F. graminearum deploys the gramillins as a virulence agent in maize, but not in wheat, thus displaying host-specific adaptation.

Background: Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat. Results: In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes, TaABCC6, TaNFXL1 and TansLTP9.4 in a wheat protoplast assay. Deletions of sequences located between the two sgRNA in each gene were the most frequent editing events observed for two of the three genes. A comparative assessment of editing frequencies between a codon-optimized Cas9 for expression in algae, crCas9, and a plant codon-optimized Cas9, pcoCas9, showed more consistent results with the vector expressing pcoCas9. Editing of TaNFXL1 by co-expression of sgRNA pair was investigated in transgenic wheat plants. Given the ploidy of bread wheat, a rapid, robust and inexpensive genotyping protocol was also adapted for hexaploid genomes and shown to be a useful tool to identify homoeolog-specific editing events in wheat. Conclusions: Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of TaNFXL1 was successfully adapted., Correction to Plant Methods (2019) 15:119. In the original publication, the copyright line was incorrectly published as "© The Author(s) 2019". The corrected copyright line should read as " © Her Majesty the Queen in Right of Canada as represented by the Minister of Articulture and Agri-Food Canada 2019". The original article has been corrected.

Fusarium graminearum responds to environmental cues to modulate its growth and metabolism during wheat pathogenesis. Nitrogen limitation activates virulence-associated behaviours in F. graminearum including mycotoxin production and penetrative growth. In other filamentous fungi, nitrogen sensing is mediated by both the Target of Rapamycin (TOR) and the glutamine synthetase (GS)-dependent signaling pathways. While TOR-dependent nitrogen responses have been demonstrated in F. graminearum, the involvement of GS remains unclear. Our study indicates that both the TOR and GS signalling pathways are involved in nitrogen sensing in F. graminearum and contribute to glutamine-induced mycelial growth. However, neither pathway is required for glutamine-induced repression of the mycotoxin deoxynivalenol (DON) indicating that an additional nitrogen sensing pathway must exist. Further, two genes FgBMH1 and FgBMH2 encoding 14-3-3 proteins regulate nitrogen responses with effects on gene expression, DON production and mycelial growth. Unlike yeast, where 14-3-3s function redundantly in regulating nitrogen sensing, the 14-3-3 proteins have differing functions in F. graminearum. While both FgBMH1 and FgBMH2 regulate early glutamine-induced DON repression, only FgBMH2 is involved in regulating reproduction, virulence and glutamine-induced AreA repression. Together, our findings help to clarify the nitrogen sensing pathways in F. graminearum and highlight the involvement of 14-3-3s in the nitrogen response of filamentous fungi.

Plant protein kinases form redundant signaling pathways to perceive microbial pathogens and activate immunity. Bacterial pathogens repress cellular immune responses by secreting effectors, some of which bind and inhibit multiple host kinases. To understand how broadly bacterial effectors may bind protein kinases and the function of these kinase interactors, we first tested kinase-effector (K-E) interactions using the Pseudomonas syringae pv. tomato-tomato pathosystem. We tested interactions between five individual effectors (HopAI1, AvrPto, HopA1, HopM1, and HopAF1) and 279 tomato kinases in tomato cells. Over half of the tested kinases interacted with at least one effector, and 48% of these kinases interacted with more than three effectors, suggesting a role in the defense. Next, we characterized the role of select multi-effector-interacting kinases and revealed their roles in basal resistance, effector-triggered immunity (ETI), or programmed cell death (PCD). The immune function of several of these kinases was only detectable in the presence of effectors, suggesting that these kinases are critical when particular cell functions are perturbed or that their role is typically masked. To visualize the kinase networks underlying the cellular responses, we derived signal-specific networks. A comparison of the networks revealed a limited overlap between ETI and basal immunity networks. In addition, the basal immune network complexity increased when exposed to some of the effectors. The networks were used to successfully predict the role of a new set of kinases in basal immunity. Our work indicates the complexity of the larger kinase-based defense network and demonstrates how virulence- and avirulence-associated bacterial effectors alter sectors of the defense network.

The enzyme phytochelatin synthase (PCS) has long been studied with regard to its role in metal(loid) detoxification in several organisms, i.e., plants, yeasts, and nematodes. It is in fact widely recognized that PCS detoxifies a number of heavy metals by catalyzing the formation of thiol-rich oligomers, namely phytochelatins, from glutathione and related peptides. However, recent investigations have highlighted other possible roles played by the PCS enzyme in the plant cell, e.g., the control of pathogen-triggered callose deposition. In order to examine novel aspects of Arabidopsis thaliana PCS1 (AtPCS1) functions and to elucidate its possible roles in the secondary metabolism, metabolomic data of A. thaliana wild-type and cad1-3 mutant were compared, the latter lacking AtPCS1. HPLC-ESI-MS analysis showed differences in the relative levels of metabolites from the glucosinolate and phenylpropanoid pathways between cad1-3 and wild-type plants. Specifically, in control (Cd-untreated) plants, higher levels of 4-methoxy-indol-3-ylmethylglucosinolate were found in cad1-3 plants vs. wild-type. Moreover, the cad1-3 mutant showed to be impaired in the deposit of callose after Cd exposure, suggesting that AtPCS1 protects the plant against the toxicity of heavy metals not only by synthesizing PCs, but also by contributing to callose deposition. In line with the contribution of callose in counteracting Cd toxicity, we found that another callose-defective mutant, pen2-1, was more sensitive to high concentrations of Cd than wild-type plants. Moreover, cad1-3 plants were more susceptible than wild-type to the hemibiotrophic bacterial pathogen Pseudomonas syringae. The metabolome also revealed differences in the relative levels of hydroxycinnamic acids and flavonols, with consequences on cell wall properties and auxin content, respectively. First, increased lignification in the cad1-3 stems was found, probably aimed at counteracting the entry of Cd into the inner tissues. Second, in cad1-3 shoots, increased relative levels of kaempferol 3,7 dirhamnoside and quercetin hexoside rhamnoside were detected. These flavonols are endogenous inhibitors of auxin transport in planta; auxin levels in both roots and shoots of the cad1-3 mutant were in fact lower than those of the wild-type. Overall, our data highlight novel aspects of AtPCS1 functions in A. thaliana.

Kinases facilitate detection of extracellular signals and set in motion cellular responses for plant adaptation and survival. Some of the energy utilized for kinase signal processing is produced through the activity of ion transporters. Additionally, the synergy between cellular ions and signal transduction influences plant response to pathogens, and their growth and development. In plants, the signaling elements that connect cell wall and membrane sensors with ion homeostasis and transport-mediated processes are largely unknown. Current research indicates that plant Integrin-Linked Kinases (ILKs), a subfamily Raf-like MAP2K Kinases, may have evolved to fulfill this role. In this review, we explore new findings on plant ILKs placing a particular focus on the connection between ILKs proteins unique structural features and ILKs functions. The ankyrin repeat motifs and the kinase domains of ILKs in Arabidopsis and land plants lineage, respectively, are analyzed and discussed as potential determinants of ILKs’ metal ion cofactor specificity and their enzymatic and interaction activities. Further, ILKs regulation through gene expression, subcellular localization, and ions and ion transporters is reviewed in the context of recent studies. Finally, using evidence from literature and interactomics databanks, we infer ILKs-dependent cellular pathways and highlight their potential in transmitting multiple types of signals originating at the interface between the cell wall and plasma membrane. cessing.

Protein kinase–driven phosphorylation constitutes the core of cellular signaling. Kinase components of signal transduction pathways are often targeted for inactivation by pathogens. The study of kinases and immune signal transduction in the model crop tomato (Solanum lycopersicum) would benefit from the availability of community-wide resources for large scale and systems-level experimentation. Here, we defined the tomato kinome and performed a comprehensive comparative analysis of the tomato kinome and 15 other plant species. We constructed a tomato kinase library (TOKN 1.0) of over 300 full-length open reading frames (ORF) cloned into a recombination-based vector. We developed a high-throughput pipeline to isolate and transform tomato protoplasts. A subset of the TOKN 1.0 library kinases were expressed in planta, were purified, and were used to generate a functional tomato protein microarray. All resources created were utilized to test known and novel associations between tomato kinases and Pseudomonas syringae DC3000 effectors in a large-scale format. Bsk7 was identified as a component of the plant immune response and a candidate effector target. These resources will enable comprehensive investigations of signaling pathways and host-pathogen interactions in tomato and other Solanaceae spp.

Cytosolic glutamine synthetase (GS1) is responsible for the primary assimilation of ammonia, and a role in nitrogen (N) remobilization is implicated from its vascular localization and enhanced expression during senescence. This paper tested the hypothesis that overexpression (OX) of GS1 in rice improves utilization N use efficiency (UtE = spikelet yield/shoot N content). Three GS1 OX lines were identified using activity assays and quantitative polymerase chain reaction. Physiological analysis of the OX lines, as well as azygous and wild-type (Wt) controls, was conducted with mature plants after growth under varying nitrate conditions (non-limiting N, limiting N, transfer from non-limiting N to limiting N at panicle emergence) and growth environments (growth chamber vs greenhouse). Overall, OX lines did not differ from azygous controls in vegetative yield or shoot N content. In two of the three growth trials (i.e. the growth chamber trials) harvest index, N harvest index (spikelet N content/shoot N content) and UtE were generally enhanced in the OX lines relative to their azygous controls. These characteristics were highly correlated with percent spikelets filled and spikelet number. Thus, N partitioning in rice during grain filling could be altered by GS1 OX, resulting in improved UtE. Unfortunately, GS OX did not result in more efficient use of N under limiting N than under non-limiting N, and is therefore unlikely to result in the use of less N under field conditions. Transformation effects significantly hindered the productivity of the OX lines, but backcrossing to the Wt should overcome this.

There is considerable confusion about N use efficiency (NUE) in the plant literature. We would like to propose the simple and ubiquitous definitions described by Good et al. (2004) as a starting point for studies of NUE. Based on this terminology, there is evidence from breeding programs for variation in both uptake efficiency (UpE) and utilization efficiency (UtE). Molecular physiology studies typically address mechanisms for improving NUE, but often do not calculate NUE or even acquire appropriate data for calculating NUE. Herein, we report in detail on recent studies involving molecular approaches for improving NUE, and calculate changes in NUE where possible. The evidence suggests that there is potential for improving usage index and UpE in dicots and UpE and UtE in monocots by overexpressing enzymes for N assimilation, specifically glutamine synthetase 1, glutamate synthase, and alanine aminotransferase. If decreased fertilizer-N input and improved NUE are truly goals of the plant biology community, researchers are encouraged to (i) consider the use of both wild type and azygous controls, (ii) compare general NUE (on the basis of grain or biomass yield per unit of applied N) of overexpression mutants and controls at both limiting and non-limiting N levels, (iii) select an appropriate type of specific NUE for assessing the physiological mechanisms involved (uptake versus internal utilization), and (iv) confirm promising results under field conditions.

The balance between accumulation of stress-induced polyamines and reactive oxygen species (ROS) is arguably a critical factor in plant tolerance to salt stress. Polyamines are compounds, which accumulate in plants under salt stress and help maintain cellular ROS homeostasis. In this review we first outline the role of polyamines in mediating salt stress responses through their modulation of redox homeostasis. The two proposed roles of polyamines in regulating ROS – as antioxidative molecules and source of ROS synthesis – are discussed and exemplified with recent studies. Second, the proposed function of polyamines as modulators of ion transport is discussed in the context of plant salt stress. Finally, we highlight the apparent connection between polyamine accumulation and programmed cell death induction during stress. Thus polyamines have a complex functional role in regulating cellular signaling and metabolism during stress. By focusing future efforts on how polyamine accumulation and turnover is regulated, research in this area may provide novel targets for developing stress tolerance.

Comprehensive analysis of protein kinases and cellular signaling pathways requires the identification of kinase substrates and interaction partners using large-scale amenable approaches. Here, we describe our methods for producing plant protein microarrays (PMAs) and discuss various parameters critical to the quality of PMAs. Next, we describe methods for detecting protein-protein interactions and kinase activity including auto-phosphorylation and substrate phosphorylation. We have provided a short video demonstrating how to conduct an interaction assay and how to properly handle a protein microarray. Finally, a set of analytical methods are presented as a bioinformatics pipeline for the acquisition of PMA data and for selecting PMA candidates using statistical testing. The experimental and analytical protocols described here outline the steps to produce and utilize PMAs to analyze signaling networks.

A Report on the Plant Genomes & Biotechnology: From Genes to Networks meeting, held at the Cold Spring Harbor Laboratories, USA, December 4–7, 2013.