Specialized metabolites as mediators for plant–fungus crosstalk and their evolving roles

Abstract

Plants, fungi, and bacteria produce numerous natural products with bioactive properties essential for ecological adaptation. Because of their chemical complexity, these natural products have been adapted for diverse applications in industry. The discovery of their biosynthetic pathways has been accelerated due to improved ‘omics’ approaches, metabolic engineering, and the availability of genetic manipulation techniques. Ongoing research into these metabolites is not only resolving the enzymatic diversity underlying their biosynthesis, but also delving into the physiological and mechanistic basis of their modes of action. This review highlights progress made in the elucidation of biosynthetic pathways and biological roles of specialized metabolites, focusing on some that play important roles at the interface of plant–fungus interactions.

Introduction

Plants produce a multitude of specialized metabolites with important roles in response to environmental stressors. As a society we have adapted these diverse chemicals for a host of applications in industry and health care. Intensive research to improve our understanding of these specialized metabolites provides the opportunity not only to develop methods for their sustainable production but also to potentially manipulate plant metabolism to enhance plant adaptation to biotic and abiotic stresses.

The world’s major food crops face large fungal-induced losses that threaten food security [1]. The study of specialized metabolites involved in plant–fungus interactions and their modes of action could lead to novel strategies to control fungal diseases and to prevent contamination of food crops by fungal toxins. Specialized metabolites play a key role in the interaction of an organism with its surroundings [2]. Like their plant hosts, pathogens such as fungi also produce metabolites to aid host colonization and survival. The complex interaction between plants and fungi involves the exchange of various small molecules. For example, the pathogenic fungi attack plants with toxins, and in response plants secrete protective defense compounds for their protection [3]. Here, we review recent progress in deciphering specialized metabolite biosynthesis, both in plants and fungi, and discuss how this opens avenues to interrogate biological function. Finally, we highlight how this information is paving the way for more detailed studies of the plant–fungus interface.

Plant-specialized metabolites as primary mediators of defense response

From genes to biosynthetic pathways

Terpenes are a large and diverse group of specialized metabolites, derived from 5-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), including over 80,000 known compounds. A simplified terpenoid biosynthetic pathway consists of the scaffold-forming terpene synthases followed by decorating enzymes, predominantly cytochromes P450 (Figure 1). In cereal crops such as rice (Oryza sativa), maize (Zea mays), and wheat (Triticum sativa), terpenoids were originally discovered as defense response molecules, but recent evidence points to alternate roles for these compounds in response to a variety of biotic and abiotic stressors [4, 5]. Here, we use the diversification of terpenes in Poaceae to highlight their biosynthesis and progress to deciphering their functional roles in plants.

Figure 1: Multiple roles of diterpenoids in rice.

(A) An overview of diterpenoid biosynthesis. (B) The multiple (emerging) biological roles of the diterpenoids.

In rice, diterpenes including momilactones, oryzalexins, and phytocassanes were initially identified as part of the defense response to infection by the rice blast fungus Magnaporthe oryzae [4, 6]. A substantial amount of work has contributed to the identification of enzymes and the reconstruction of the biosynthetic pathways involved in the biosynthesis of these diterpenes [7–13]. Deciphering the metabolic diversity has been aided by advances in genome sequencing, enzyme discovery, and metabolic engineering. Similarly, in maize, the terpene-based compounds dolabralexins, kauralexins, and zealexins have been identified [14–17]. These compounds have demonstrated antifungal activity toward Fusarium graminearumand Fusarium verticilloides. Recently, detailed work has uncovered an intricate metabolic grid of pathways essential to the maize chemical defense system and innate immunity [18]. For a more detailed description of the biosynthesis of terpenoids in rice and maize, the interested reader is referred to recent reviews [5, 19].

Linking biosynthetic pathways to molecular mechanisms and physiological roles

Work to elucidate the biosynthetic pathways of defense compounds has highlighted the importance of genomic advances in associating genes to molecule and synthetic biology approaches to functionally characterize candidate genes [20, 21]. These approaches have been important not only for deciphering the diversity and evolutionary origins of the enzymes involved but also for facilitating interrogation of their physiological roles. For example, in rice, genetic tools enabled the knockout of OsKSL4 that blocks the production of momilactones [22]. The knockout plants showed no significant difference in their susceptibility to infection, but growth assays revealed that momilactones were allelochemicals rather than being involved in defense (Figure 1). More recently, cps mutants (scaffold-forming genes of diterpenes – Figure 1) derived through CRISPR/Cas9 suggest that the rice diterpenes play a role in stomatal closure [23]. The implication of the plant diterpenes in stomatal closure suggests that these compounds function in water use efficiency or in establishing alternative routes for the control of microbial entry. Future work to explore the mechanistic basis of these specialized metabolites may involve more detailed investigation at the cellular level. In maize the terpenoids—dolabralexins, kauralexins, and zealexins—accumulate under pathogen attack by Fusarium spp., indicating a role in defense [14, 18, 20]. These compounds have also been suggested to be involved in responses to abiotic stresses including drought and below ground communication [24, 25].

Genetic approaches have extended the physiological functions of terpenoids beyond their originally discovered role as defense compounds. The diterpenoids in cereal crops were initially identified as a response to fungal infection, but recent research suggests that these metabolites have multiple roles. The field of plant metabolism is actively discovering new chemistry, more fully understanding the established functions of the metabolites, and defining alternate functions for the already described molecules. We are still struggling to comprehend the diversity of plant metabolism. Kosmaczet al. highlighted the recurring theme of multiple roles for primary metabolites [26]. Huang et al. identified triterpene metabolites that influence the composition of the microbiota in addition to their role in defense [27]. These examples focus on terpene metabolism, and a similar trend toward multiple roles has been observed for other classes of specialized metabolites, including glucosinolates and phenolics.

An important extension of engineering the pathways is not only understanding the enzymatic steps of the pathways but also being able to increase the production of chemicals such as the momilactones, which are potent allelochemicals and have the potential for use as herbicides [28].

Fungus-specialized metabolites and their effects on plantdefense responses

Like plants, fungi produce several specialized metabolites that aid attachment to their host plant and adapt to the dynamic environmental conditions inside and outside the host cells. Among the specialized metabolites synthesized by fungi, terpenoids, polyketides, and non-ribosomal peptides are known to play major roles as toxins and as agents to manipulate host cellular systems. Just as recent research in plants has led from the biosynthetic pathways of specialized metabolites to their physiological roles, a similar path is visualized for fungi.

The best-studied specialized metabolites in plant pathogenic fungi are those that result in virulence or in the production of harmful mycotoxins, for example, the trichothecenes, fusarins, and fumonisins of Fusarium species (Figure 2). Also well-known is the production of the hormone gibberellic acid by Fusarium fujikuroi,the causal agent of rice “foolish seedling” disease, which is also an early example of a fungus using phytohormones to hijack its plant host [29].

Figure 2: Chemical structures of phytopathogen metabolites that facilitate virulence.

(A) Phytohormones essential for plant growth and development that are used by fungal pathogens to manipulate their hosts. (B) Toxins produced by fungal organisms to ensure virulence. (C) Recent examples of specialized metabolites involved in virulence. Higginsianin B is a terpenoid involved in modulating the plant (Arabidopsis) JA pathway, and fusaoctaxin A, a linear non-ribosomal octapeptide that facilitates invasion of wheat through adjacent cell movement.

Despite extensive characterization of specialized metabolites in Fusarium species, recent findings highlight the continued importance of deciphering the products of unknown biosynthetic gene clusters, such as the discovery of an octapeptide, fusaoctaxin A, in Fusarium graminearum that contributes to virulence in wheat [30]. A key challenge to this approach is that genes are not detectable under standard laboratory conditions. The F. Graminearum fusaoctaxin A biosynthetic gene cluster was identified using in planta coexpression analysis [31]. Subsequent fluorescence imaging and infection assays using deletion mutants showed that fusaoctaxin A facilitates cell-to-cell invasion in wheat [30]. Deletion of this cluster hampered the ability of the pathogen to infect wheat, and complementation studies using the synthetic compound were able to restore cell-to-cell invasion.

Modulation of plant phytohormones

In plant–fungus pathogenic interactions, the fungi produce metabolites that not only function as toxic chemicals but also hijack and regulate plant systems by mimicking phytohormones (Figure 2a) that are essential for regulating plant growth, development, and productivity [32, 33].

In addition to producing phytohormone mimics, a fungal pathogen has recently been found to use a terpenoid to manipulate a phytohormone pathway. Colletotrichum higginsianum is a pathogen that causes anthracnose disease in plants of the Brassicaceae family. As a hemibiotrophic fungus, C. higginsianum first establishes a biotrophic interaction by evading the plant’s defense mechanisms and later switches to a necrotrophic phase, in which it kills the host cells and feeds on them. The role of a terpenoid in manipulating the host defense response was discovered through a forward chemical genetics approach, using Arabidopsis thaliana and C. higginsianum as the plant–pathogen pair [34, 35]. Briefly, a library of partially purified fractions from ΔcclA mutant strains of C. higginsianum, overexpressing terpenoids, was used to test for inhibitory effects on jasmonic acid (JA)–induced defense responses. The transgenic Arabidopsis lines expressing a GUS reporter gene fused to JA-responsive promoters were treated with the fractions, and methyl jasmonate was used to induce JA signaling.

Fluorometric quantification was used to detect the inhibitory effect of fractions containing the terpenoid higginsianin B on the JA pathway. Further analysis revealed that higginsianin B represses the JA pathway by preventing methyl jasmonate–induced degradation of the host protein JAZ, a repressor of JA responses. The terpenoid also inhibited an auxin-mediated developmental signaling pathway [34]. Overall, this study used a forward chemical genetics approach to show that terpenoids can also act as effectors to modulate phytohormone signaling and manipulate host defense responses.

The work on fusaoctaxin A and higginsianin B demonstrates the power of combining biosynthetic discovery, chemical genetics, biological complementation, and fluorescence studies to determine the mechanisms by which specialized metabolites function as virulence factors [30, 34].

Conclusions

Immense progress is being made towards understanding the biosynthesis and biological roles of specialized metabolites in the plants and their fungal pathogens. A number of studies provide in planta evidence of the role of specialized metabolites in plant defense responses and fungal control, but more intriguing is the emergence of multiple roles for these metabolites. These studies highlight the complexity of the plant metabolome and reveal how its dynamics are balanced in response to environmental cues. Relatively little is known about the fungal counterpart. The study of specialized metabolism in fungal organisms faces additional challenges in identifying appropriate conditions for the expression of genes encoding these metabolites as well as detecting the presence of cryptic genes.

On both sides of plant–fungus interactions, there is limited knowledge about the biological roles and modes of action of specialized metabolites. A main challenge in the field of host–microbe interactions is to discriminate between metabolites produced by the plant versus the pathogen. Further complexity is added by detoxification (toxin degradation) during the host–pathogen interaction. However, it is becoming increasingly feasible to study metabolites at the plant–pathogen interface. Continued work in synthetic biology and the integration of proteomics, transcriptomics, and metabolomics as well as cell biology techniques will be essential for decoding the relationship between the host and the pathogen.