Silencing of the Transmembrane Transporter (swnT) Gene of the Fungus Slafractonia leguminicola Results in a Reduction of Mycotoxin Transport

Abstract

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.

Analysis of the Mycotoxin Levels and Expression Pattern of SWN Genes at Different Time Points in the Fungus Slafractonia leguminicola

“…Previous work showed that silencing of swnT had a significant effect on S. leguminicola growth and pathogenicity and effected secretion of both swainsonine and slaframine [38]. This differed significantly from knockouts of swnT in Metarhizium, which caused no change in growth of the fungus and no change in secretion.…”

“…Standards and sample extracts were analyzed by HPLC-HRMS using the following instrumentation [38]. A Q-Exactive quadrupole/orbitrap high-resolution mass spectrometer (Thermo Scientific) equipped with a heated electrospray ion source (HEIS) and coupled to an Ultimate 3000 HPLC (Thermo Scientific) was used for analyses.…”

“…Metarhizium sp. and S. leguminicola secrete swainsonine into media (both plates and liquid culture) [38]. Cultures of S. leguminicola and Metarhizium grow much more rapidly than those of locoweed endophytes, which rarely fill a plate even after a month of growth [39].…”

“…In addition, S. leguminicola produces both swainsonine and slaframine in infected plants [38]. Swainsonine can be found in locoweeds containing A. oxytropis, A. cinerea, and A. fulva in all aerial portions of the plant, with highest levels in the seeds [40].…”

“…Silencing of swnT in S. leguminicola caused decreased movement of swainsonine and slaframine from mycelia into the culture media [38]. Transformants grew poorly and were unable to infect clover leaves.…”