Metatranscriptomic Comparison of Endophytic and Pathogenic \u3cem\u3eFusarium\u3c/em\u3e-Arabidopsis Interactions Reveals Plant Transcriptional Plasticity

Plants are continuously exposed to beneficial and pathogenic microbes, but how plants recognize and respond to friends versus foes remains poorly understood. Here, we compared the molecular response of Arabidopsis thaliana independently challenged with a Fusarium oxysporum endophyte Fo47 versus a pathogen Fo5176. These two F. oxysporum strains share a core genome of about 46 Mb, in addition to 1,229 and 5,415 unique accessory genes. Metatranscriptomic data reveal a shared pattern of expression for most plant genes (about 80%) in responding to both fungal inoculums at all timepoints from 12 to 96 h postinoculation (HPI). However, the distinct responding genes depict transcriptional plasticity, as the pathogenic interaction activates plant stress responses and suppresses functions related to plant growth and development, while the endophytic interaction attenuates host immunity but activates plant nitrogen assimilation. The differences in reprogramming of the plant transcriptome are most obvious in 12 HPI, the earliest timepoint sampled, and are linked to accessory genes in both fungal genomes. Collectively, our results indicate that the A. thaliana and F. oxysporum interaction displays both transcriptome conservation and plasticity in the early stages of infection, providing insights into the fine-tuning of gene regulation underlying plant differential responses to fungal endophytes and pathogens

Examining the transcriptional response in wheat Fhb1 near-isogenic lines to Fusarium graminearum infection and deoxynivalenol treatment

Citation: Hofstad, A. N., Nussbaumer, T., Akhunov, E., Shin, S., Kugler, K. G., Kistler, H. C., . . . Muehlbauer, G. J. (2016). Examining the transcriptional response in wheat Fhb1 near-isogenic lines to Fusarium graminearum infection and deoxynivalenol treatment. Plant Genome, 9(1). https://doi.org/10.3835/plantgenome2015.05.0032Fusarium head blight (FHB) is a disease caused predominantly by the fungal pathogen Fusarium graminearum that affects wheat and other small-grain cereals and can lead to severe yield loss and reduction in grain quality. Trichothecene mycotoxins, such as deoxynivalenol (DON), accumulate during infection and increase pathogen virulence and decrease grain quality. The Fhb1 locus on wheat chromosome 3BS confers Type II resistance to FHB and resistance to the spread of infection on the spike and has been associated with resistance to DON accumulation. To gain a better genetic understanding of the functional role of Fhb1 and resistance or susceptibility to FHB, we examined DON and ergosterol accumulation, FHB resistance, and the whole-genome transcriptomic response using RNA-seq in a near-isogenic line (NIL) pair carrying the resistant and susceptible alleles for Fhb1 during F. graminearum infection and DON treatment. Our results provide a gene expression atlas for the resistant and susceptible wheat–F. graminearum interaction. The DON concentration and transcriptomic results show that the rachis is a key location for conferring Type II resistance. In addition, the wheat transcriptome analysis revealed a set of Fhb1-responsive genes that may play a role in resistance and a set of DON-responsive genes that may play a role in trichothecene resistance. Transcriptomic results from the pathogen show that the F. graminearum genome responds differently to the host level of resistance. The results of this study extend our understanding of host and pathogen responses in the wheat–F. graminearum interaction. © Crop Science Society of America

The Wor1-like Protein Fgp1 Regulates Pathogenicity, Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein

Functional Analysis of the Kinome of the Wheat Scab Fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study

Model of trichothecene biosynthesis and export in <i>F.graminearum</i>.

<p>Within the nucleus, transcription factors Tri6p and Tri10p positively regulate transcription of genes involved in trichothecene (DON) biosynthesis and tolerance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063077#pone.0063077-Seong1" target="_blank">[22]</a>. The genes for enzymes in the isoprenoid biosynthesis pathway, including HMG CoA reductase (<i>Hmr1</i>), are constitutively expressed but are up-regulated during DON synthesis. DON biosynthesis induction leads to a shift in Hmr1p targeting from the ER to the toxisome. The vesicular toxisome is the site of trichothecene biosynthetic enzymes Tri1p and Tri4p, proteins that also are up-regulated under the control of Tri10p and Tri6p during DON biosynthesis. Also regulated by these transcription factors is the DHA14 protein Tri12p that confers a level of tolerance to DON <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063077#pone.0063077-Menke1" target="_blank">[23]</a>. Motility of Tri12p::GFP labeled vesicles appears to be dependent upon F-actin and results in fusion with the vacuole and the plasma membrane <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063077#pone.0063077-Menke1" target="_blank">[23]</a>. DON synthesis therefore may be sequestered within the toxisome and export of toxic products may be facilitated by trafficking of Tri12p::GFP labeled vesicles.</p

Fgp1 mediates expression of genes in several gene clusters.

<p>Histograms represent mean relative expression levels from three replicate experiments. Error bars represent standard deviation. A) Expression means for genes from the <i>TRI</i> cluster in wild type PH-1 grown in putrescine (black bars) or during wheat head infection (dark gray bars) and in a <i>FGP1</i> deletion strain grown in putrescine (white bars) or during wheat head infection (light gray bars). B) Expression means for genes from the butenolide biosynthetic cluster in wild type PH-1 during wheat head infection (black bars) and in a <i>FGP1</i> deletion strain during wheat head infection (white bars). C) Expression means for genes from the aurofusarin biosynthetic cluster in wild type PH-1 grown in putrescine medium (black bars) or for a <i>FGP1</i> deletion strain grown in putrescine medium (white bars). D) Expression means for genes from the NPS8 cluster in wild type PH-1 grown in putrescine medium (black bars) or a <i>FGP1</i> deletion strain grown in putrescine medium (white bars).</p

Nanoscale enrichment of the cytosolic enzyme trichodiene synthase near reorganized endoplasmic reticulum in Fusarium graminearum

Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms

<i>FGP1</i> is required for pathogenicity and trichothecene accumulation but <i>FGP2</i> is not.

<p>A) Average number of diseased spikelets of 30 plants after mock inoculated with H₂O, inoculated with four independent <i>FGP1</i> deletion mutants, with wild type PH-1 or with four independent <i>FGP1</i> complemented transformants. Wheat heads were point inoculated and disease spread through adjacent spikelets was enumerated after 14 days. Error bars indicate standard deviation. B) Photographs from wild type inoculated wheat head (lower head on the left), wheat heads inoculated with the four <i>FGP1</i> deletion mutants (upper four heads on the left) and wheat heads inoculated with the four <i>FGP1</i> complemented transformants (four heads on the right). Arrows indicate the inoculated spikelet in each head photographed. C) DON and 15-ADON concentrations in the inoculated spikelet 14 days after inoculation with H₂O, four independent <i>FGP1</i> deletion mutants, wild type PH-1 or four <i>FGP1</i> complemented transformants. Asterisks mean no toxin was detected. D) Average number of diseased spikelets of 30 plants after mock inoculated with H₂O or inoculated with four independent <i>FGP2</i> deletion mutants or wild type PH-1. Wheat heads were point inoculated and spread through adjacent spikelets was enumerated after 14 days. Error bars indicate standard deviation. E) DON and 15-ADON concentrations in the inoculated spikelet 14 days after inoculation with H₂O, four independent <i>FGP2</i> deletion mutants or wild type PH-1. Asterisk means no toxin was detected.</p