Putative Rho-GDP dissociation inhibitor influences
aflatoxin production in Aspergillus flavus
Leah Beel and Michael S. Price, Ph.D.
Dept. of Biology & Chemistry, Liberty University
Project Overview
Introduction & Background
Aspergillus flavus is a
species of saprophytic
fungus that lives in the
soil. It has been known to
contaminate seed and
harvested crops and has
thus infected human and
animal hosts through food
and feed. It produces a
carcinogenic secondary
metabolite called
aflatoxin.
j
In a previous study, production of aflatoxin was tested at
different temperatures using different carbon soured, nitrogen
sourced, and pH media.
cDNA microarrays
were used to determine
which genes were up
regulated in conditions
conducive for aflatoxin
production. One of the
genes that was
identified in this study
was a putative RhoGDP dissociation
inhibitor.
Table 1. A. flavus showed stunted aflatoxin production after putative
Rho-GDP dissociation inhibitor knockout mutation.
Strain
3357-5
Afrdi1
Aflatoxin (mg/g dry weight)
20067.386
538.706
An experiment was conducted to knock out the gene Afrdi1 (or rdiA) in A.
flavus. When the gene was deleted, aflatoxin production was reduced. The
rdiA deletion strain (Afrdi1) produced approximately 37 times less aflatoxin
than the wild type strain 3357-5 (Table 1).
Research Outline
An experiment will be conducted to test the effects of
complementing an rdiA gene knockout mutation in A. flavus.
To accomplish this:
• Putative rdiA will be cloned into a phleomycin resistant
plasmid vector
• The recombinant plasmid will be used to transform A. flavus
• Protoplasts of A. flavus will be made, and the DNA vector
will be inserted
• The transformed A. flavus will be plated and tested for
aflatoxin production
Colony PCR will be
performed on sample
Vsap + I colonies to screen
for the presence of rdiA
A. flavus conidia will be
treated with enzymes and
washed with buffers to
remove cell wall material
Screen colonies
Make protoplasts
Preliminary Results
!
!
The rdiAΔ mutation in A.
flavus will be complemented
by the insertion of
pBC-phleo/ rdiA
!
!
XbaI!
and!rSAP!
pBC%phleo!
!
!
!
!
!!
!
rdiA/!Xba1!
pBC%phleo/!XbaI!
!
pBC%phleo/!rdiA!
!
!
Figure! 1. Ligation reaction between pBC-phleo and rdiA. First pBC!
phleo and
rdiA
were
digested
with
XbaI.
Then
pBC-phleo
was
treated
!
with rSAP
to remove free 3’ phosphates. Lastly, they were ligated
!
! to form a recombinant plasmid.
together
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
Figure 2. E. coli transformations. The V control plate had many
colonies, confirming that the pBC-phleo plasmid successfully delivered
into the cells. The Vsap plate had significantly fewer colonies, showing
that the rSAP treatment successfully removed 3’ phosphates at the XbaI
cut sites. The Vsap + I plate had more growth than the Vsap plate
indicating successful ligations between pBC-phleo and rdiA.
Transformed A flavus will
be plated on media then
tested for aflatoxin
production using HPLC
Transform A. flavus with
recombinant plasmid
Test for aflatoxin
production
References & Acknowledgements
1.
2.
3.
4.
M. S. Price, S. B. Conners, S. Tachdjian, R. M. Kelly, G. A. Payne,
Aflatoxin conducive and non- conducive growth conditions reveal new
gene associations with aflatoxin production. Fungal genetics and
biology: FG & B 42, 506-518 (2005)
M. S. Price, J. Yu, W. C. Nierman, H. S. Kim, B. Pritchard, C. A.
Jacobus, D. Bhatnagar, T. E. Cleveland, G. A. Payne, The aflatoxin
pathway regulator AflR induces gene transcription inside and outside of
the aflatoxin biosynthetic cluster. FEMS microbiology letters 255, 275279 (2006)
Z. He, M. S. Price, G. R. OBrian, D. R. Georgianna, G. A. Payne,
Improved protocols for functional analysis in the pathogenic fungus
Aspergillus flavus. BMC Microbiology 7, 1471-2180 (2007)
https://www.aspergillus.org.uk/content/aspergillus-flavus-30