Positive transformants were selected on the basis of auxotrophies complemented by heterolous orthologues of pyrG89 or riboB2 (e.g., complementation of pyrG89 by the pyr-4 gene from N. crassa in pRG3-AMA1 plasmins in media lacking pyrimidines), or for mutant sltB strains based on reversal of sensitivity to elevated concentrations of potassium (0.6 M KCl) in the media.
The strategy used to construct the mutant strains is based upon a previously described method [21].
After the incubation period, two mutant strains were selected based on their phenotypic characteristics, using colonies whose color and size differed from the wild type colonies.
Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates.
For SNP mapping of ku540, we used a Hawaii strain based elo-5 gk208 elo-5 gk208amutant al., 2009) to croSeamenh eto-5(gk208) ku540 alimals and determined the locus of ku540 by linkage analysis (Davis et al., 2009).
lcrF was shown to be expressed at ∼2-fold higher levels in the ΔrovA mutant compared to the wild type strain based on DNA microarray analysis (Supplementary Table S2), and this upregulation should contribute, at least partially, to the increased expression of T3SS genes in the ΔrovA mutant.
The wrky3 and wrky4 single and double mutants also responded normally to the virulent PstDC3000 strain based on the growth of the bacterial pathogen.
Applying this technique, the authors were able to distinguish the respective rpoB mutants from one other, as well as their parental strain based on fatty acid composition.
The selection of mutant strains for this analysis was based on the hypothesis that fungal extracellular vesicles derive from MVB-related pathways for exosome formation or from post-Golgi conventional secretion [9], [14], [16], [17].
