Physiological and transcriptome analyses reveal tissue-specific responses of Leucaena plants to drought stress.

Leucaena leucocephala (Leucaena) is a leguminous tree widely cultivated in tropical and subtropical regions due to its strong environmental suitability for abiotic stresses, especially drought. However, the molecular mechanisms and key pathways involved in Leucaena's drought response require further elucidation. Here, we comparatively analyzed the physiological and early transcriptional responses of Leucaena leaves and roots under drought stress simulated by polyethylene glycol (PEG) treatments. Drought stress induced physiological changes in Leucaena seedlings, including decreases in relative water content (RWC) and increases in relative electrolyte leakage (REL), malondialdehyde (MDA), proline contents as well as antioxidant enzyme activities. In response to drought stress, 6461 and 8295 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. In both tissues, the signaling transduction pathway of plant hormones was notably the most enriched. Specifically, abscisic acid (ABA) biosynthesis and signaling related genes (NCED, PP2C, SnRK2 and ABF) were strongly upregulated particularly in leaves. The circadian rhythm, DNA replication, alpha-linolenic acid metabolism, and secondary metabolites biosynthesis related pathways were repressed in leaves, while the glycolysis/gluconeogenesis and alpha-linolenic acid metabolism and amino acid biosynthesis processes were promoted in roots. Furthermore, heterologous overexpression of Leucaena drought-inducible genes (PYL5, PP2CA, bHLH130, HSP70 and AUX22D) individually in yeast increased the tolerance to drought and heat stresses. Overall, these results deepen our understanding of the tissue-specific mechanisms of Leucaena in response to drought and provide target genes for future drought-tolerance breeding engineering in crops.

SfgA Renders Aspergillus flavus More Stable to the External Environment.

sfgA is known as a key negative transcriptional regulator gene of asexual sporulation and sterigmatocystin production in Aspergillus nidulans. However, here, we found that the homolog sfgA gene shows a broad and complex regulatory role in governing growth, conidiation, sclerotia formation, secondary metabolism, and environmental stress responses in Aspergillus flavus. When sfgA was deleted in A. flavus, the fungal growth was slowed, but the conidiation was significantly increased, and the sclerotia formation displayed different behavior at different temperatures, which increased at 30 °C but decreased at 36 °C. In addition, sfgA regulated aflatoxin biosynthesis in a complex way that was associated with the changes in cultured conditions, and the increased production of aflatoxin in the ∆sfgA mutant was associated with a decrease in sclerotia size. Furthermore, the ∆sfgA mutant exhibited sensitivity to osmotic, oxidative, and cell wall stresses but still produced dense conidia. Transcriptome data indicated that numerous development- and secondary-metabolism-related genes were expressed differently when sfgA was deleted. Additionally, we also found that sfgA functions downstream of fluG in A. flavus, which is consistent with the genetic position in FluG-mediated conidiation in A. nidulans. Collectively, sfgA plays a critical role in the development, secondary metabolism, and stress responses of A. flavus, and sfgA renders A. flavus more stable to the external environment.

Synthetic antimicrobial agents inhibit aflatoxin production.

Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.

Purification and characterization of two grandiuvarones from Desmos chinensis leaves and their antimicrobial activities.

A novel aromatic compound, grandiuvarone B (5-acetoxy-3-benzoyloxymethyl-5H-oxepin-4-one), along with a known compound grandiuvarone A (5-acetoxy-6-benzoyloxymethyl-5H-oxepin-4-one) were isolated from methanol extracts of Desmos chinensis leaves. Their structures were determined by various spectroscopic techniques including nuclear magnetic resonance (NMR), high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) and circular dichroism (CD). Grandiuvarone A and grandiuvarone B are isomers and the S configuration of grandiuvarone B was reported for the first time. We then determined their antifungal activity against Aspergillus flavus. Results revealed that grandiuvarone B exhibited better antifungal activity against A. flavus, with MIC values of 0.01 mg/mL compared to grandiuvarone A (MIC values of 0.02 mg/mL). In the presence of each active compound at 160 µg/g of aquafeed, A. flavus growth was completely inhibited. Grandiuvarone B also showed antibacterial activity against the plant pathogen Ralstonia solanacearum.

The Kinetochore Protein Spc105, a Novel Interaction Partner of LaeA, Regulates Development and Secondary Metabolism in Aspergillus flavus.

Nuclear protein LaeA is known as the global regulator of secondary metabolism in Aspergillus. LaeA connects with VeA and VelB to form a heterotrimeric complex, which coordinates fungal development and secondary metabolism. Here, we describe a new interaction partner of LaeA, the kinetochore protein Spc105, from the aflatoxin-producing fungus Aspergillus flavus. We showed that in addition to involvement in nuclear division, Spc105 is required for normal conidiophore development and sclerotia production of A. flavus. Moreover, Spc105 positively regulates the production of secondary metabolites such as aflatoxin and kojic acid, and negatively regulates the production of cyclopiazonic acid. Transcriptome analysis of the Δspc105 strain revealed that 23 backbone genes were differentially expressed, corresponding to 19 of the predicted 56 secondary metabolite gene clusters, suggesting a broad regulatory role of Spc105 in secondary metabolism. Notably, the reduced expression of laeA in our transcriptome data led to the discovery of the correlation between Spc105 and LaeA, and double mutant analysis indicated a functional interdependence between Spc105 and LaeA. Further, in vitro and in vivo protein interaction assays revealed that Spc105 interacts directly with the S-adenosylmethionine (SAM)-binding domain of LaeA, and that the leucine zipper motif in Spc105 is required for this interaction. The Spc105-LaeA interaction identified in our study indicates a cooperative interplay of distinct regulators in A. flavus, providing new insights into fungal secondary metabolism regulation networks.

The Antioxidant Gallic Acid Inhibits Aflatoxin Formation in Aspergillus flavus by Modulating Transcription Factors FarB and CreA.

Aflatoxin biosynthesis is correlated with oxidative stress and is proposed to function as a secondary defense mechanism to redundant intracellular reactive oxygen species (ROS). We find that the antioxidant gallic acid inhibits aflatoxin formation and growth in Aspergillus flavus in a dose-dependent manner. Global expression analysis (RNA-Seq) of gallic acid-treated A. flavus showed that 0.8% (w/v) gallic acid revealed two possible routes of aflatoxin inhibition. Gallic acid significantly inhibited the expression of farB, encoding a transcription factor that participates in peroxisomal fatty acid β-oxidation, a fundamental contributor to aflatoxin production. Secondly, the carbon repression regulator encoding gene, creA, was significantly down regulated by gallic acid treatment. CreA is necessary for aflatoxin synthesis, and aflatoxin biosynthesis genes were significantly downregulated in ∆creA mutants. In addition, the results of antioxidant enzyme activities and the lipid oxidation levels coupled with RNA-Seq data of antioxidant genes indicated that gallic acid may reduce oxidative stress through the glutathione- and thioredoxin-dependent systems in A. flavus.

A cytosine methyltransferase ortholog dmtA is involved in the sensitivity of Aspergillus flavus to environmental stresses.

DNA methylation is an important epigenetic modification that depends on DNA methyltransferases (DMT). However, the filamentous fungus Aspergillus flavus has no detectable methylation, and role of a DMT homologue, DmtA, is undefined. Here we describe the role of the dmtA gene responding to changes in the environment by comparing knockout, point mutation, over-expression and wild type strains. Deletion of dmtA differentially affected conidia development in a media-dependent fashion, which suggests that dmtA plays an important role in conidiation. Furthermore, ΔdmtA strains lost the capacity to form the resistant structure, sclerotia, and alleviated sensitivity to several stress conditions, such as high osmotic pressure, hypoxia, low water activity and a high calcium concentration. We also noticed that deletion of dmtA and mutation C377S in DmtA negatively affected aflatoxin production and down regulated the expression of some early (fas-1, pksA, nor-1), middle and late (nor-A, ver-1, avnA, omtB) genes in the aflatoxin biosynthetic cluster. Finally, we found that all tested strains showed a similar phenotype when treated with 5-azacytidine. Our results indicate that the dmtA gene is important in regulation of aflatoxin biosynthesis and for A. flavus to adapt to stressful environments and for survival, although it may hold no apparent function in DNA methylation.

Coexistence of and interaction relationships between an aflatoxin-producing fungus and a bacterium.

The interactions between aflatoxin-producing fungi and bacteria have opened up a new avenue for identifying biological agents suitable for controlling aflatoxin contamination. In this study, we analysed the interactions between A. flavus and the bacterium Burkholderia gladioli M3 that coexist in rice that is naturally contaminated with A. flavus. Our results showed that a cell-free culture filtrate (CCF) and the metabolite bongkrekic acid of the M3 strain potently suppressed the mycelial growth and spore production, and then affected the production of aflatoxin of A. flavus. Bongkrekic acid secreted by the M3 strain exhibited higher antifungal activity than did analogues. The CCF of the M3 strain and its metabolite bongkrekic acid can inhibit the growth of A. flavus, but the metabolites of A. flavus, aflatoxins, exerted no inhibitory effect on the growth of the M3 strain. Furthermore, we determined that the M3 cells could use the dead mycelia of A. flavus as energy sources for reproduction, while A. flavus could not grow in a solution containing dead M3 cells. In summary, these results indicated that B. gladioli has a competitive advantage in survival when it coexists with its fungal partner A. flavus.

Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine.

Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. In this study, an RNA-Seq approach was applied to explore the mechanism of 5-AC's effect on A. flavus. We identified 240 significantly differentially expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes respectively in secondary metabolite clusters #27 and #35. These two clusters are involved in development or survival of sclerotia. GO functional enrichment analysis showed that these significantly differentially expressed genes were mainly involved in catalytic activity and proteolytic functions. The expressed transcripts of most genes in the AF biosynthetic gene cluster in A. flavus showed no significant changes after treatment with 5-AC and were expressed at low levels, and the transcription regulator genes aflR and aflS in this cluster did not show differential expression relative to the sample without 5-AC treatment. We found that the veA gene, which encodes protein bridges VelB and LaeA, decreased profoundly the expressed transcripts, and brlA, which encodes an early regulator of development, increased its transcripts in A. flavus after 5-AC treatment. Our data support a model whereby 5-AC affects development through increasing the expression of brlA by depressing the expression of veA and AF production through suppressing veA expression and dysregulating carboxypeptidase activity, which then prevents the aflatoxisomes (vesicles) from performing their normal function in AF formation. Furthermore, the suppressed veA expression weakens or even interrupts the connection between VelB and LaeA, leading to dysregulation of the expression pattern of genes involved in development and secondary metabolism in A. flavus. The RNA-seq data presented in this work were also served to improve the annotation of the A. flavus genome. This work provides a comprehensive view of the transcriptome of A. flavus responsive to 5-AC and supports the conclusion that fungal development and secondary metabolism are co-regulated.