evaluating-leaf-responses-to-microbial-secondary-metabolites-using

Evaluating Leaf Responses to Microbial Secondary Metabolites Using A High-Throughput Format

Microbes secrete structurally diverse secondary metabolites during plant infection, some of which are detected by plant cells, which trigger stress ...

Agriculture and Agri-Food Canada

A second structural gene required for culmorin biosynthesis in the plant pathogen Fusarium graminearum is described. Clm2 encodes a regio- and stereoselective cytochrome P450 monooxygenase for C-11 of longiborneol (1). Clm2 gene disruptants were grown in liquid culture and assessed for culmorin production via HPLC-evaporative light scattering detection. The analysis indicated a complete loss of culmorin (2) from the liquid culture of the ΔClm2 mutants. Culmorin production resumed in a ΔClm2 complementation experiment. A detailed analysis of the secondary metabolites extracted from the large-scale liquid culture of disruptant ΔClm2D20 revealed five new natural products: 3-hydroxylongiborneol (3), 5-hydroxylongiborneol (4), 12-hydroxylongiborneol (5), 15-hydroxylongiborneol (6), and 11-epi-acetylculmorin (7). The structures of the new compounds were elucidated by a combination of HRMS, 1D and 2D NMR, and X-ray crystallography.

Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

Cereal infection by the broad host range fungal pathogen Fusarium graminearum is a significant global agricultural and food safety issue due to the deposition of mycotoxins within infected grains. Methods to study the intracellular effects of mycotoxins often use the baker's yeast model system (Saccharomyces cerevisiae); however, this organism has an efficient drug export network known as the pleiotropic drug resistance (PDR) network, which consists of a family of multidrug exporters. This study describes the first study that has evaluated the potential involvement of all known or putative ATP-binding cassette (ABC) transporters from the PDR network in exporting the F. graminearum trichothecene mycotoxins deoxynivalenol (DON) and 15-acetyl-deoxynivalenol (15A-DON) from living yeast cells. We found that Pdr5p appears to be the only transporter from the PDR network capable of exporting these mycotoxins. We engineered mutants of Pdr5p at two sites previously identified as important in determining substrate specificity and inhibitor susceptibility. These results indicate that it is possible to alter inhibitor insensitivity while maintaining the ability of Pdr5p to export the mycotoxins DON and 15A-DON, which may enable the development of resistance strategies to generate more Fusarium-tolerant crop plants.

Protein engineering of Saccharomyces cerevisiae transporter Pdr5p identifies key residues that impact Fusarium mycotoxin export and resistance to inhibition.

Gibberella ear rot (GER) is one of the most economically important fungal diseases of maize in the temperate zone due to moldy grain contaminated with health threatening mycotoxins. To develop resistant genotypes and control the disease, understanding the host-pathogen interaction is essential.

Transcriptome profiling of two maize inbreds with distinct responses to Gibberella ear rot disease to identify candidate resistance genes.

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 H-N-C 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.

Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum.

Arbuscular mycorrhizal fungi (AMF) are prominent root symbionts that can carry thousands of nuclei deriving from two parental strains in a large syncytium. These co-existing genomes can also vary in abundance with changing environmental conditions. Here we assemble the nuclear genomes of all four publicly available AMF heterokaryons using PacBio high-fidelity and Hi-C sequencing. We find that the two co-existing genomes of these strains are phylogenetically related but differ in structure, content and epigenetics. We confirm that AMF heterokaryon genomes vary in relative abundance across conditions and show this can lead to nucleus-specific differences in expression during interactions with plants. Population analyses also reveal signatures of genetic exchange indicative of past events of sexual reproduction in these strains. This work uncovers the origin and contribution of two nuclear genomes in AMF heterokaryons and opens avenues for the improvement and environmental application of these strains.

Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Author Correction: Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.

A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Diverse plant species detect bacterial flagellin using leucine-rich repeat receptor-like kinases to activate defense responses and promote resistance against bacterial invasion. In barley, the impact of flagellin on inducible defense responses has not been fully described. Here, we report that the flagellin-derived flg22 peptide induces callose, reactive oxygen species (ROS) bursts, and differential expression of 2,603 genes in barley. Cultivated barley genotypes produce different amplitudes of flg22-induced ROS bursts, indicating genetic variation for microbe-associated molecular pattern responses. Association mapping revealed a region on chromosome 7 underlying variation in flg22-induced ROS bursts, which contains 12 flg22-induced genes. Genotypes with higher flg22 responses had higher induced resistance to  pv.  (), the causal organism of bacterial leaf streak. The flg22 region of  flagellin evades detection in both barley and Arabidopsis, and a single amino acid change can restore immunogenicity of the peptide. Together, this work underlines the effectiveness of the flagellin-triggered defenses in promoting barley resistance to , which evades recognition of the flg22 region in its host. [Formula: see text]Copyright © 2025 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Article	Year
Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum. Journal of natural products\| PubMed ID: 26673640	2016
Protein engineering of Saccharomyces cerevisiae transporter Pdr5p identifies key residues that impact Fusarium mycotoxin export and resistance to inhibition. MicrobiologyOpen\| PubMed ID: 27263049	2016
Transcriptome profiling of two maize inbreds with distinct responses to Gibberella ear rot disease to identify candidate resistance genes. BMC genomics\| PubMed ID: 29426290	2018
Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum. Journal of the American Chemical Society\| PubMed ID: 30395461	2018
Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions. Nature microbiology\| PubMed ID: 37884816	2023
Author Correction: Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions. Nature microbiology\| PubMed ID: 37989883	2024
A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence. Cell reports\| PubMed ID: 38970790	2024
Transcriptional Profiling and Genetic Mapping of Barley Responses to Bacterial Flagellin. Molecular plant-microbe interactions : MPMI\| PubMed ID: 40062987	2025

Whynn Bosnich

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