skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Silencing of the Transmembrane Transporter (swnT) Gene of the Fungus Slafractonia leguminicola Results in a Reduction of Mycotoxin Transport
Slafractonia leguminicola infects red clover and other legumes, causing black patch disease. This pathogenic fungus also produces two mycotoxins, slaframine and swainsonine, that are toxic to livestock grazing on clover hay or pasture infested with S. leguminicola. Swainsonine toxicosis causes locoism, while slaframine causes slobbers syndrome. The mechanism of toxin secretion by S. leguminicola is poorly understood. The aim of this research was to investigate the role of a putative transmembrane transporter, SwnT, in mycotoxin transport. The swnT gene was silenced by RNA interference using the silencing vector Psilent1, which included inverted repeat transgenes of swnT. This resulted in a significant reduction of swnT transcript levels compared with the controls. Silencing caused a decline in the active efflux of toxins from the mycelia to the media, as shown by LC–MS analysis. Transformants in which swnT was silenced showed higher concentrations of both toxins in the mycelia compared with the concentrations in the media. These transformants exhibited a visibly distinct phenotype with much thicker and shorter mycelia than in the wild type. These transformants were also unable to infect detached clover leaves, unlike the controls, suggesting that SwnT function may play an important role in pathogenesis in addition to mycotoxin transport. This research demonstrates the importance of this transporter to the secretion of mycotoxins for this phytopathogenic fungus.  more » « less
Award ID(s):
2027806
PAR ID:
10478123
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
MDPI
Date Published:
Journal Name:
Journal of Fungi
Volume:
9
Issue:
3
ISSN:
2309-608X
Page Range / eLocation ID:
370
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. de_Paula, Renato G; Silva, Roberto N (Ed.)
    The fungal plant pathogen Slafractonia leguminicola produces two mycotoxins that affect animals: slaframine, which causes slobbers, and swainsonine, which causes locoism. Slafractonia leguminicola contains the swainsonine-associated orthologous gene clusters, “SWN”, which include a multifunctional swnK gene (NRPS-PKS hybrid), swnH1 and swnH2 (nonheme iron dioxygenase genes), swnN and swnR (reductase genes), and swnT (transmembrane transporter). In addition to these genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2), are found in S. leguminicola. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points, and expression pattern of the SWN genes were analyzed using RT-qPCR. The concentrations of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography–mass spectrometry. The timing of gene expression was similar in cultured fungus and inoculated leaves and agreed with our proposed biosynthetic pathway. Substantially more swainsonine was produced than slaframine during time course studies. 
    more » « less
  2. Abstract In Cameroon, dietary staples are frequently contaminated with diverse toxic fungal metabolites, known as mycotoxins. Aflatoxins and fumonisins are a public health concern, particularly concerning cancer and/or early life stunting. Mycotoxin mixtures are predicted from food measures; and this study reports the levels and frequencies of urinary mycotoxin biomarkers in Cameroonian adults. A single first void urine sample was collected from 89 adults from Yaoundé, Cameroon. Urine samples were tested for eight distinct mycotoxins using measures of both parent compounds and/or their metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Altogether, seven distinct mycotoxins, aflatoxin, fumonisin, deoxynivalenol, zearalenone, nivalenol, ochratoxin A, and citrinin, (or their metabolites) were observed in urine samples. At least one mycotoxin was detected in all of the urine samples, 87 (98%) of which were above the limit of quantitation. Aflatoxin M1was detected in 42% (n.d.-0.21 μg/l) of samples of which about a quarter additionally contained fumonisin B1. Of the remaining toxins deoxynivalenol (78%), zearalenone (99%), ochratoxin A (95%), nivalenol (53%), and citrinin (87%) were present in the samples. Alternariol was not detected in any sample. Mixtures of mycotoxins in the samples were frequently observed with 64 samples (72%) containing more than five mycotoxin exposure biomarkers. Estimates of intake exceeded the TDIs for fumonisin B1(n = 4), deoxynivalenol (n = 1) and zearalenone (n = 2), no TDI is set for aflatoxin. This study reveals frequent co-exposure of Cameroonian individuals to a complex mixture of toxic and carcinogenic mycotoxins, with mixtures of aflatoxin and fumonisin being a particular priority from a public health standpoint. 
    more » « less
  3. Mitchell, Aaron P. (Ed.)
    Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A . fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A . fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A . fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis -thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A . nidulans . However, the A . nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A . fumigatus and A . nidulans , two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A . fumigatus and A . nidulans . However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A . fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae , was also essential for virulence and GT biosynthesis in A . fumigatus , and for GT protection in A . fumigatus , A . nidulans , and A . oryzae . KojR regulates rglT , gliT , gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species. 
    more » « less
  4. Hydrogel microsphere media allows for postsynthetic purification of single-walled carbon nanotubes (SWNTs), affording characterization and application of their unique (n,m) chirality-dependent properties. This work reports the characterization of five hydrogel resins, Sephacryl S-100, S-200, S-300, S-400, and S-500, and the implementation of each as a SWNT purification medium. The physiochemical properties of each resin were explored spectroscopically through elemental analyses and with both light and electron microscopy. Both surface porosity and hydrogel swelling ratio were found to increase as the concentration of component allyl dextran (aDEX) decreased, each with an increasing Sephacryl S-number. Conversely, invariant properties included a hydrogel microsphere size distribution and concentrations of components methylenebisacrylamide and ammonium persulfate. When employed within gel-based SWNT purification schemes in overloading conditions, Sephacryl formulations of larger S-number adsorbed fewer SWNTs, but the chirality dependence of SWNT adsorption and elution was approximately consistent across all resins. In underloading conditions, approximately one-third of introduced SWNTs passed through each resin unabsorbed, while the resins showed varying chirality-dependent adsorption efficiencies. These observations collectively identify aDEX-rich gel regions as being responsible for SWNT purification, along with a SWNT-exclusive parameter other than chirality (speculated as length) that convolutes the effectiveness of gel-based single-chirality purification. 
    more » « less
  5. Maize is an important food and fuel crop globally. Ear rots, caused by fungal pathogens, are some of the most detrimental maize diseases, due to reduced grain yield and the production of harmful mycotoxins. Mycotoxins are naturally occurring toxins produced by certain fungal species that can cause acute and chronic health issues in humans and animals that consume mycotoxin-contaminated grain. Pathogens can infect the developing ear through silks, or through wounds in the ears produced by pests. Plants naturally develop genetic resistance to pathogens. The maize genes involved in resistance to the pathogen may be different, depending on whether the ear was infected via silks or wounds. To differentiate between these two forms of resistance, natural infections can be reproduced by injecting inoculum through the silk channel, or by producing wounds using a needle, and introducing inoculum directly onto developing ears. Our protocol describes a technique used to inoculate developing maize ears withFusarium graminearum, one of the fungal species that causes ear rot. We describe both silk channel and side needle inoculation techniques. Our protocol uses a backpack inoculator for both methods of infection, allowing for high-throughput inoculations, which are necessary for large field experiments. After harvest, the ears are visually rated on a percentage of disease scale. The protocol results in quantitative data that can be used for research on elucidating genetic resistance to fungal pathogens to assist breeding selections, and to understand plant–pathogen interactions of ear rots in maize. 
    more » « less