The Soil-Borne Legacy.

Plants greatly rely on their root microbiome for uptake of nutrients and protection against stresses. Recent studies have uncovered the involvement of plant stress responses in the assembly of plant-beneficial microbiomes. To facilitate durable crop production, deciphering the driving forces that shape the microbiome is crucial.

Fungal social influencers: secondary metabolites as a platform for shaping the plant-associated community.

Fungal secondary metabolites (FSMs) are capable of manipulating plant community dynamics by inhibiting or facilitating the establishment of co-habitating organisms. Although production of FSMs is not crucial for survival of the producer, their absence can indirectly impair growth and/or niche competition of these fungi on the plant. The presence of FSMs with no obvious consequence on the fitness of the producer leaves questions regarding ecological impact. This review investigates how fungi employ FSMs as a platform to mediate fungal-fungal, fungal-bacterial and fungal-animal interactions associated with the plant community. We discuss how the biological function of FSMs may indirectly benefit the producer by altering the dynamics of surrounding organisms. We introduce several instances where FSMs influence antagonistic- or alliance-driven interactions. Part of our aim is to decipher the meaning of the FSM 'language' as it is widely noted to impact the surrounding community. Here, we highlight the contribution of FSMs to plant-associated interaction networks that affect the host either broadly or in ways that may have previously been unclear.

Sexual reproduction contributes to the evolution of resistance-breaking isolates of the spinach pathogen Peronospora effusa.

Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long-read sequencing coupled with diversity assessment based on short-read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome-wide comparisons of the 26 isolates revealed that 10 isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogen's reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa-spinach interaction, thus assisting in sustainable production of spinach through knowledge-driven resistance breeding.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Seedborne Cercospora beticola Can Initiate Cercospora Leaf Spot from Sugar Beet (Beta vulgaris) Fruit Tissue.

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet "seed" consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC50 > 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

MYB72-dependent coumarin exudation shapes root microbiome assembly to promote plant health.

Plant roots nurture a tremendous diversity of microbes via exudation of photosynthetically fixed carbon sources. In turn, probiotic members of the root microbiome promote plant growth and protect the host plant against pathogens and pests. In the Arabidopsis thaliana-Pseudomonas simiae WCS417 model system the root-specific transcription factor MYB72 and the MYB72-controlled ß-glucosidase BGLU42 emerged as important regulators of beneficial rhizobacteria-induced systemic resistance (ISR) and iron-uptake responses. MYB72 regulates the biosynthesis of iron-mobilizing fluorescent phenolic compounds, after which BGLU42 activity is required for their excretion into the rhizosphere. Metabolite fingerprinting revealed the antimicrobial coumarin scopoletin as a dominant metabolite that is produced in the roots and excreted into the rhizosphere in a MYB72- and BGLU42-dependent manner. Shotgun-metagenome sequencing of root-associated microbiota of Col-0, myb72, and the scopoletin biosynthesis mutant f6'h1 showed that scopoletin selectively impacts the assembly of the microbial community in the rhizosphere. We show that scopoletin selectively inhibits the soil-borne fungal pathogens Fusarium oxysporum and Verticillium dahliae, while the growth-promoting and ISR-inducing rhizobacteria P. simiae WCS417 and Pseudomonas capeferrum WCS358 are highly tolerant of the antimicrobial effect of scopoletin. Collectively, our results demonstrate a role for coumarins in microbiome assembly and point to a scenario in which plants and probiotic rhizobacteria join forces to trigger MYB72/BGLU42-dependent scopolin production and scopoletin excretion, resulting in improved niche establishment for the microbial partner and growth and immunity benefits for the host plant.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Genome comparisons suggest an association between Ceratocystis host adaptations and effector clusters in unique transposable element families.

Ceratocystis fimbriata is a host specific fungal pathogen of sweet potato (Ipomoea batatas). The closely related species, C. manginecans, is an important pathogen of trees (e.g. Acacia mangium and Mangifera indica) but has never been isolated from tuber crops. The genetic factors that determine the host range and host specificity of these species have not been determined. The aim of this study was to compare the genomes of C. fimbriata and C. manginecans in order to identify species-specific genetic differences that could be associated with host specificity. This included whole-genome alignments as well as comparisons of gene content and transposable elements (TEs). The genomes of the two species were found to be very similar, sharing similar catalogues of CAZymes, peptidases and lipases. However, the genomes of the two species also varied, harbouring species-specific genes (e.g. small secreted effectors, nutrient processing proteins and stress response proteins). A portion of the TEs identified (17%) had a unique distribution in each species. Transposable elements appeared to have played a prominent role in the divergence of the two species because they were strongly associated with chromosomal translocations and inversions as well as with unique genomic regions containing species-specific genes. Two large effector clusters, with unique TEs in each species, were identified. These effectors displayed non-synonymous mutations and deletions, conserved within a species, and could serve as mutational hot-spots for the development of host specificity in the two species.

The Age of Coumarins in Plant-Microbe Interactions.

Coumarins are a family of plant-derived secondary metabolites that are produced via the phenylpropanoid pathway. In the past decade, coumarins have emerged as iron-mobilizing compounds that are secreted by plant roots and aid in iron uptake from iron-deprived soils. Members of the coumarin family are found in many plant species. Besides their role in iron uptake, coumarins have been extensively studied for their potential to fight infections in both plants and animals. Coumarin activities range from antimicrobial and antiviral to anticoagulant and anticancer. In recent years, studies in the model plant species tobacco and Arabidopsis have significantly increased our understanding of coumarin biosynthesis, accumulation, secretion, chemical modification and their modes of action against plant pathogens. Here, we review current knowledge on coumarins in different plant species. We focus on simple coumarins and provide an overview on their biosynthesis and role in environmental stress responses, with special attention for the recently discovered semiochemical role of coumarins in aboveground and belowground plant-microbe interactions and the assembly of the root microbiome.

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi.

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.

Molecular dialogue between arbuscular mycorrhizal fungi and the nonhost plant Arabidopsis thaliana switches from initial detection to antagonism.

Approximately 29% of all vascular plant species are unable to establish an arbuscular mycorrhizal (AM) symbiosis. Despite this, AM fungi (Rhizophagus spp.) are enriched in the root microbiome of the nonhost Arabidopsis thaliana, and Arabidopsis roots become colonized when AM networks nurtured by host plants are available. Here, we investigated the nonhost-AM fungus interaction by analyzing transcriptional changes in Rhizophagus, Arabidopsis and the host plant Medicago truncatula while growing in the same mycorrhizal network. In early interaction stages, Rhizophagus activated the Arabidopsis strigolactone biosynthesis genes CCD7 and CCD8, suggesting that detection of AM fungi is not completely impaired. However, in colonized Arabidopsis roots, fungal nutrient transporter genes GintPT, GintAMT2, GintMST2 and GintMST4, essential for AM symbiosis, were not activated. RNA-seq transcriptome analysis pointed to activation of costly defenses in colonized Arabidopsis roots. Moreover, Rhizophagus colonization caused a 50% reduction in shoot biomass, but also led to enhanced systemic immunity against Botrytis cinerea. This suggests that early signaling between AM fungi and Arabidopsis is not completely impaired and that incompatibility appears at later interaction stages. Moreover, Rhizophagus-mediated defenses coincide with reduced Arabidopsis growth, but also with systemic disease resistance, highlighting the multifunctional role of AM fungi in host and nonhost interactions.

Microbial small molecules - weapons of plant subversion.

Covering: up to 2018 Plants live in close association with a myriad of microbes that are generally harmless. However, the minority of microbes that are pathogens can severely impact crop quality and yield, thereby endangering food security. By contrast, beneficial microbes provide plants with important services, such as enhanced nutrient uptake and protection against pests and diseases. Like pathogens, beneficial microbes can modulate host immunity to efficiently colonize the nutrient-rich niches within and around the roots and aerial tissues of a plant, a phenomenon mirroring the establishment of commensal microbes in the human gut. Numerous ingenious mechanisms have been described by which pathogenic and beneficial microbes in the plant microbiome communicate with their host, including the delivery of immune-suppressive effector proteins and the production of phytohormones, toxins and other bioactive molecules. Plants signal to their associated microbes via exudation of photosynthetically fixed carbon sources, quorum-sensing mimicry molecules and selective secondary metabolites such as strigolactones and flavonoids. Molecular communication thus forms an integral part of the establishment of both beneficial and pathogenic plant-microbe relations. Here, we review the current knowledge on microbe-derived small molecules that can act as signalling compounds to stimulate plant growth and health by beneficial microbes on the one hand, but also as weapons for plant invasion by pathogens on the other. As an exemplary case, we used comparative genomics to assess the small molecule biosynthetic capabilities of the Pseudomonas genus; a genus rich in both plant pathogenic and beneficial microbes. We highlight the biosynthetic potential of individual microbial genomes and the population at large, providing evidence for the hypothesis that the distinction between detrimental and beneficial microbes is increasingly fading. Knowledge on the biosynthesis and molecular activity of microbial small molecules will aid in the development of successful biological agents boosting crop resiliency in a sustainable manner and could also provide scientific routes to pathogen inhibition or eradication.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen.

Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms.

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction.

Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing.

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

The heterothallic sugarbeet pathogen Cercospora beticola contains exon fragments of both MAT genes that are homogenized by concerted evolution.

Dothideomycetes is one of the most ecologically diverse and economically important classes of fungi. Sexual reproduction in this group is governed by mating type (MAT) genes at the MAT1 locus. Self-sterile (heterothallic) species contain one of two genes at MAT1 (MAT1-1-1 or MAT1-2-1) and only isolates of opposite mating type are sexually compatible. In contrast, self-fertile (homothallic) species contain both MAT genes at MAT1. Knowledge of the reproductive capacities of plant pathogens are of particular interest because recombining populations tend to be more difficult to manage in agricultural settings. In this study, we sequenced MAT1 in the heterothallic Dothideomycete fungus Cercospora beticola to gain insight into the reproductive capabilities of this important plant pathogen. In addition to the expected MAT gene at MAT1, each isolate contained fragments of both MAT1-1-1 and MAT1-2-1 at ostensibly random loci across the genome. When MAT fragments from each locus were manually assembled, they reconstituted MAT1-1-1 and MAT1-2-1 exons with high identity, suggesting a retroposition event occurred in a homothallic ancestor in which both MAT genes were fused. The genome sequences of related taxa revealed that MAT gene fragment pattern of Cercospora zeae-maydis was analogous to C. beticola. In contrast, the genome of more distantly related Mycosphaerella graminicola did not contain MAT fragments. Although fragments occurred in syntenic regions of the C. beticola and C. zeae-maydis genomes, each MAT fragment was more closely related to the intact MAT gene of the same species. Taken together, these data suggest MAT genes fragmented after divergence of M. graminicola from the remaining taxa, and concerted evolution functioned to homogenize MAT fragments and MAT genes in each species.

Seed tuber imprinting shapes the next-generation potato microbiome.

BACKGROUND: Potato seed tubers are colonized and inhabited by soil-borne microbes, that can affect the performance of the emerging daughter plant in the next season. In this study, we investigated the intergenerational inheritance of microbiota from seed tubers to next-season daughter plants under field condition by amplicon sequencing of bacterial and fungal microbiota associated with tubers and roots, and tracked the microbial transmission from different seed tuber compartments to sprouts. RESULTS: We observed that field of production and potato genotype significantly (P < 0.01) affected the composition of the seed tuber microbiome and that these differences persisted during winter storage of the seed tubers. Remarkably, when seed tubers from different production fields were planted in a single trial field, the microbiomes of daughter tubers and roots of the emerging plants could still be distinguished (P < 0.01) according to the production field of the seed tuber. Surprisingly, we found little vertical inheritance of field-unique microbes from the seed tuber to the daughter tubers and roots, constituting less than 0.2% of their respective microbial communities. However, under controlled conditions, around 98% of the sprout microbiome was found to originate from the seed tuber and had retained their field-specific patterns. CONCLUSIONS: The field of production shapes the microbiome of seed tubers, emerging potato plants and even the microbiome of newly formed daughter tubers. Different compartments of seed tubers harbor distinct microbiomes. Both bacteria and fungi on seed tubers have the potential of being vertically transmitted to the sprouts, and the sprout subsequently promotes proliferation of a select number of microbes from the seed tuber. Recognizing the role of plant microbiomes in plant health, the initial microbiome of seed tubers specifically or planting materials in general is an overlooked trait. Elucidating the relative importance of the initial microbiome and the mechanisms by which the origin of planting materials affect microbiome assembly will pave the way for the development of microbiome-based predictive models that may predict the quality of seed tuber lots, ultimately facilitating microbiome-improved potato cultivation.