Aline Koch

Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species

Significance We demonstrate that host-induced gene silencing (HIGS) targeting the fungal sterol 14α-demethylase (CYP51) genes restricts Fusarium infection in plants. Fusarium diseases have a significant impact not only on global grain production, but also on food safety because of grain contamination with mycotoxins. We capitalized on the knowledge that demethylation inhibitor fungicides target cytochrome P450 lanosterol C-14α-demethylase. In Fusarium graminearum (Fg), this enzyme is encoded by three paralogous genes. Transgenic Arabidopsis and barley expressing a double-stranded RNA targeting all three CYP51 genes exhibited complete immunity to Fg. Our results provide proof-of-concept that HIGS of the CYP51 genes is an effective strategy for controlling Fusarium, demonstrating that HIGS is a powerful tool, which could revolutionize crop plant protection. Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.

New wind in the sails: improving the agronomic value of crop plants through RNAi‐mediated gene silencing

RNA interference (RNAi) has emerged as a powerful genetic tool for scientific research over the past several years. It has been utilized not only in fundamental research for the assessment of gene function, but also in various fields of applied research, such as human and veterinary medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content. In addition, the demonstration that agricultural pests, such as insects and nematodes, can be killed by exogenously supplied RNAi targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNAi signals generated in planta. Indeed, recent evidence argues that this strategy, called host-induced gene silencing (HIGS), is effective against sucking insects and nematodes; it also has been shown to compromise the growth and development of pathogenic fungi, as well as bacteria and viruses, on their plant hosts. Here, we review recent studies that reveal the enormous potential RNAi strategies hold not only for improving the nutritive value and safety of the food supply, but also for providing an environmentally friendly mechanism for plant protection.

Conserved nematode signalling molecules elicit plant defenses and pathogen resistance

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery.

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley—Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14α-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy.

Lucimycin, an antifungal peptide from the therapeutic maggot of the common green bottle fly Lucilia sericata

Abstract We report the identification, cloning, heterologous expression and functional characterization of a novel antifungal peptide named lucimycin from the common green bottle fly Lucilia sericata. The lucimycin cDNA was isolated from a library of genes induced during the innate immune response in L. sericata larvae, which are used as therapeutic maggots. The peptide comprises 77 amino acid residues with a molecular mass of 8.2 kDa and a pI of 6.6. It is predicted to contain a zinc-binding motif and to form a random coil, lacking β-sheets or other secondary structures. Lucimycin was active against fungi from the phyla Ascomycota, Basidiomycota and Zygomycota, in addition to the oomycete Phytophtora parasitica, but it was inactive against bacteria. A mutant version of lucimycin, lacking the four C-terminal amino acid residues, displayed 40-fold lower activity. The activity of lucimycin against a number of highly-destructive plant pathogens could be exploited to produce transgenic crops that are resistant against fungal diseases.

The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens

Barley microrchidia ATPases bind to DNA, have endonuclease activity, and regulate plant immunity. MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other’s function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

MORC Proteins: Novel Players in Plant and Animal Health

Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5 fold. MORC proteins in plants were first discovered via a genetic screen for Arabidopsis mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and transposable element repression. Emerging data suggest that MORC proteins also participate in pathogen-induced chromatin remodeling and epigenetic gene regulation. In addition, biochemical analyses recently demonstrated that plant MORCs have topoisomerase II (topo II)-like DNA modifying activities that may be important for their function. Interestingly, animal MORC proteins exhibit many parallels with their plant counterparts, as they have been implicated in disease development and gene silencing. In addition, human MORCs, like plant MORCs, bind salicylic acid and this inhibits some of their topo II-like activities. In this review, we will focus primarily on plant MORCs, although relevant comparisons with animal MORCs will be provided.

RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 α-Demethylase

RNA-based disease control has shown great potential for controlling pest and diseases in crop plants. While delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants, which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Focusing on agronomic important barley - Fusarium spec. pathosystems, we have sought for alternative strategies to apply dsRNAs for fungal control. Recently, we have demonstrated that a spray application of a long noncoding dsRNA termed CYP3RNA, which targets the three fungal Cytochrome P450 lanosterol C-14α-demethylase genes FgCYP51A, FgCYP51B, and FgCYP51C, inhibits Fusarium graminearum (Fg) on barley leaves (Koch et al., PLoS Pathogens, 12, e1005901, 2016). Here we show that another Fusarium species, F. culmorum (Fc), also is sensitive to CYP51-derived dsRNAs. Treating Fc with various dsRNAs targeting the genes FcCYP51A, FcCYP51B and FcCYP51C was destructive to the fungus and resulted in growth retardation in in vitro cultures. We discuss important consequences of this finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential for novel plant protection strategies.