Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

Authors: Palencia, Edwin; Glenn, Anthony - Tony; Hinton, Dorothy; Bacon, Charles

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2013
Publication Date: 7/27/2013
Publication URL: http://handle.nal.usda.gov/10113/58197
Citation: Palencia, E.R., Glenn, A.E., Hinton, D.M., Bacon, C.W. 2013. Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants. Journal of Microbiological Methods. 94(2013):381-389. doi: 10.1016/j.mimet.2013.07.016.

Interpretive Summary: A group of common fungi that was viewed in the past as being rather non-hazardous to human and livestock was studied to determine their ability to produce two mycotoxins. These fungi are referred to as the black Aspergillus species since they are dark pigmented which contrast them with other species of this genus. We demonstrated earlier that this group consists of several cryptic species and now we have examined them for their ability to produce mycotoxins. This work highlights our approach to modify this group with marker fluorescent protein so that we can follow these transformed strains in specific crops. We report here a successful technique that transformed these strains so that they emit either yellow or red fluorescence that can be used to detect them in and among tissue of hosts such as peanuts and corn. The transformation frequency was very high making this technique much better than earlier reports and this is the first transformation of species of this group using this technique.

Technical Abstract: Aspergillus niger and A. carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli-maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP1) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.