Transmitting silks of maize have a complex and dynamic microbiome.

In corn/maize, silks emerging from cobs capture pollen, and transmit resident sperm nuclei to eggs. There are > 20 million silks per U.S. maize acre. Fungal pathogens invade developing grain using silk channels, including Fusarium graminearum (Fg, temperate environments) and devastating carcinogen-producers (Africa/tropics). Fg contaminates cereal grains with mycotoxins, in particular Deoxynivalenol (DON), known for adverse health effects on humans and livestock. Fitness selection should promote defensive/healthy silks. Here, we report that maize silks, known as styles in other plants, possess complex and dynamic microbiomes at the critical pollen-fungal transmission interval (henceforth: transmitting style microbiome, TSM). Diverse maize genotypes were field-grown in two trial years. MiSeq 16S rRNA gene sequencing of 328 open-pollinated silk samples (healthy/Fg-infected) revealed that the TSM contains > 5000 taxa spanning the prokaryotic tree of life (47 phyla/1300 genera), including nitrogen-fixers. The TSM of silk tip tissue displayed seasonal responsiveness, but possessed a reproducible core of 7-11 MiSeq-amplicon sequence variants (ASVs) dominated by a single Pantoea MiSeq-taxon (15-26% of sequence-counts). Fg-infection collapsed TSM diversity and disturbed predicted metabolic functionality, but doubled overall microbiome size/counts, primarily by elevating 7-25 MiSeq-ASVs, suggestive of a selective microbiome response against infection. This study establishes the maize silk as a model for fundamental/applied research of plant reproductive microbiomes.

Bioactive Metabolites and Potential Mycotoxins Produced by Cordyceps Fungi: A Review of Safety.

Ascomycete Cordyceps fungi such as C.militaris, C. cicadae, and C.guangdongensis have been mass produced on artificial media either as food supplements or health additives while the byproducts of culture substrates are largely used as animal feed. The safety concerns associated with the daily consumption of Cordyceps fungi or related products are still being debated. On the one hand, the known compounds from these fungi such as adenosine analogs cordycepin and pentostatin have demonstrated different beneficial or pharmaceutical activities but also dose-dependent cytotoxicities, neurological toxicities and or toxicological effects in humans and animals. On the other hand, the possibility of mycotoxin production by Cordyceps fungi has not been completely ruled out. In contrast to a few metabolites identified, an array of biosynthetic gene clusters (BGCs) are encoded in each genome of these fungi with the potential to produce a plethora of as yet unknown secondary metabolites. Conservation analysis of BGCs suggests that mycotoxin analogs of PR-toxin and trichothecenes might be produced by Cordyceps fungi. Future elucidation of the compounds produced by these functionally unknown BGCs, and in-depth assessments of metabolite bioactivity and chemical safety, will not only facilitate the safe use of Cordyceps fungi as human food or alternative medicine, but will also benefit the use of mass production byproducts as animal feed. To corroborate the long record of use as a traditional medicine, future efforts will also benefit the exploration of Cordyceps fungi for pharmaceutical purposes.

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase.

A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21-32]. The physico-chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl-Asp(OMe)-fluoromethylketone] did not stop toxophallin-induced DNA fragmentation. Thus, toxophallin uses a caspase-independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR-based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l-amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l-amino acid oxidase.

Comparative mycotoxin profiles of Gibberella zeae populations from barley, wheat, potatoes, and sugar beets.

Gibberella zeae is one of the most devastating pathogens of barley and wheat in the United States. The fungus also infects noncereal crops, such as potatoes and sugar beets, and the genetic relationships among barley, wheat, potato, and sugar beet isolates indicate high levels of similarity. However, little is known about the toxigenic potential of G. zeae isolates from potatoes and sugar beets. A total of 336 isolates of G. zeae from barley, wheat, potatoes, and sugar beets were collected and analyzed by TRI (trichothecene biosynthesis gene)-based PCR assays. To verify the TRI-based PCR detection of genetic markers by chemical analysis, 45 representative isolates were grown in rice cultures for 28 days and 15 trichothecenes and 2 zearalenone (ZEA) analogs were quantified using gas chromatography-mass spectrometry. TRI-based PCR assays revealed that all isolates had the deoxynivalenol (DON) marker. The frequencies of isolates with the 15-acetyl-deoxynivalenol (15-ADON) marker were higher than those of isolates with the 3-acetyl-deoxynivalenol (3-ADON) marker among isolates from all four crops. Fusarium head blight (FHB)-resistant wheat cultivars had little or no influence on the diversity of isolates associated with the 3-ADON and 15-ADON markers. However, the frequency of isolates with the 3-ADON marker among isolates from the Langdon, ND, sampling site was higher than those among isolates from the Carrington and Minot, ND, sites. In chemical analyses, DON, 3-ADON, 15-ADON, b-ZEA, and ZEA were detected. All isolates produced DON (1 to 782 microg/g) and ZEA (1 to 623 microg/g). These findings may be useful for monitoring mycotoxin contamination and for formulating FHB management strategies for these crops.

Dosage suppression of the Kluyveromyces lactis zymocin by Saccharomyces cerevisiae ISR1 and UGP1.

The Kluyveromyces lactis zymocin complex kills Saccharomyces cerevisiae cells in a process that involves tRNA cleavage by its tRNAse gamma-toxin subunit. In contrast to the gamma-toxin mode of action, the early steps of the zymocin response are less well characterized. Here, we present high-dosage suppressors of zymocin that encode a putative Pkc1-related kinase (ISR1) and UDP-glucose pyrophosphorylase (UGPase) (UGP1). Anti-UGPase Western blots and GAL10 - ISR1 overexpression suggest that zymocin suppression correlates with overproduction of UGPase or Isr1. As judged from protection against exo-zymocin and unaltered sensitivity to endogenous gamma-toxin, high-copy ISR1 and UGP1 operate in early, nontarget steps of the zymocin pathway. Consistent with a recent report on in vitro phosphorylation of Isr1 and UGPase by the CDK Pho85, high-copy ISR1 and UGP1 suppression of zymocin is abolished in a pho85 null mutant lacking CDK activity of Pho85. Moreover, suppression requires UGPase enzyme activity, and ISR1 overexpression also protects against CFW, a chitin-interfering poison. Our data agree with roles for UGPase in cell wall biosynthetic processes and for Isr1 in Pkc1-related cell wall integrity. In sum, high-copy ISR1 and UGP1 cells affect early steps of the zymocin response and potentially prevent the lethal K. lactis killer complex from establishing cell surface recognition and/or contact.

Novel procedure for identification of compounds inhibitory to transcription of genes involved in mycotoxin biosynthesis.

A novel assay is described for the identification and isolation of compounds that inhibit the transcription of genes involved in mycotoxin biosynthesis. The thin-layer chromatography-based assay was used to screen plant extracts for compounds that would inhibit the expression of the beta-glucuronidase reporter gene under the control of an aflatoxin biosynthesis gene promoter in Aspergillus parasiticus. The assay was used to track purification of an inhibitory compound, cp2, from extracts of black pepper (Piper nigrum). Cp2 did not inhibit mycelial growth or the expression of the beta-tubulin gene but did inhibit aflatoxin biosynthesis at the transcriptional level. Applications of cp2 to the control of mycotoxins are discussed.