Transcript Lorraine & Hester Maize Trust 2012

FUM1 GENE EXPRESSION AND FUMONISIN
PRODUCTION OF
Fusarium verticillioides MRC 826 SUBCULTURES
Lorraine M Moses*, Hester F Vismer
and Walter F O Marasas
PROMEC Unit, Medical Research Council,
Tygerberg, Cape Town, 7505
*Email: [email protected]
INTRODUCTION
• Fusarium verticillioides strain MRC 826 was isolated from maize
collected from the Transkei region in 1975.
• Maize samples contained levels up to 117mg/kg = 117 ppm
fumonisins.
MRC 826
• MRC 826 produced unsurpassed high levels
of fumonisin B (FB) – 17g/kg culture material.
• Subcultures (41) were established over time
showing varying ability to produce fumonisins.
• F. verticillioides is an endophytic pathogenic fungus of maize
and is the most important link in the complex aspects of fumonisin
contamination of maize.
• Fungal contamination risk of grains is high due to available
carbohydrates, protein, fat, oil content, etc.
• While most F. verticillioides strains from maize produce fumonisins –
strains varied in their ability to do so – numerous factors play a role.
QUESTIONS
• Why do F. verticillioides strains differ in their ability to produce
fumonisins?
• Why is there a variability in the ability of the subcultures from the
same F. verticillioides strain (MRC 826) to produce fumonisins?
MOLECULAR MECHANISMS OF
FUMONISN PRODUCTION
•
MRC 826 – unique set of clonal subcultures to study.
• Fumonisin biosynthetic gene cluster consists of 17 FUM genes.
•
Also, 8 regulatory genes play a role in fumonisin production
FCK1 positive regulator of fumonisin production and
regulates many aspects of development and metabolism,
such as conidiation.
PREVIOUS ANALYSES
• Determined if 17 FUM genes are differentially expressed or
absent in maize patty cultures at 3 weeks incubation.
• High Pressure Liquid Chromatography (HPLC) analyses to
determine concurrent production of FB.
• Resulting analysis revealed that the fungal strains of low,
medium and high FB producing groups displayed similar
expression patterns for all FUM genes.
• Results were not in conformity with the levels of FBs produced.
• Subsequent analysis of selected FUM genes and regulatory gene
FCK1 being performed with mRNA isolated at 9 time points
(day 7 – 31) with simultaneous HPLC analysis.
SCIENTIFIC OBJECTIVE
Elucidate the mechanism by which F. verticillioides produces fumonisins
by quantifying the level of expression of FUM and FCK1 genes at
various time points of incubation in clonal subculture strains of MRC
826 previously shown to have varying fumonisin levels.
METHODS
Maize patties were inoculated with fungal cultures and incubated at 25°C
in the dark for a specified number of days.
-
Gene expression analysis
-
HPLC analysis for FBs
FUM 19
FUM 18
FUM 17
FUM 16
FUM 15
FUM 14
FUM 13
FUM 2 (12)
FUM 11
FUM 10
FUM 3 (9)
FUM 8
FUM 7
FUM 6
FUM 1 (5)
FUM 21
Gene expression analysis
FUM gene cluster
MRC 826 Subcultures selected
•Based on FB levels in maize patty
cultures at 3 weeks at 25°C quantified by HPLC
•High, medium & low producers (A,
K, M, O, J, P)
Genes selected for analysis
•FUM1, FUM8, FUM21, FUM14
•FCK1 – FB regulation
•β-tub
•Efα
Housekeeping genes
Gene expression analysis
AAAAA
AAAAA
AAAAA
Biological triplicates of each
subculture of MRC 826
Total RNA was isolated
using Trizol
(DNase treatment)
AAAAA
TTTTT
AAAAA
TTTTT
Quantitation of cDNA using
Standard Curve Method
cDNA synthesis from mRNA templates
using Reverse Transcription with
oligo d(T) primers
HPLC analysis
Biological triplicates of each
subculture of MRC 826
Fumonisins were extracted from
maize patty culture material and
purified using SAX reverse phase
chromatography
Quantified fumonisin levels using HPLC
FUM1 Expression of Subcultures on Day 7
FCK1 Expression of Subcultures on Day 7
25
Relative expression levels
20
20
15
15
10
10
5
5
0
0
D7_A
D7_K
D7_M
D7_O
D7_J
D7_A
D7_P
D7_K
HPLC - FB levels on Day 7
30000
RESULTS
D7_M
25000
20000
FB1
FB2
15000
FB3
10000
Total
FB
5000
0
D7A
D7_O
MRC 826 Subcultures
MRC 826 Subcultures
FB levels in subcultures (ug/g)
Relative expression levels
25
D7K
D7M
D7O
D7J
MRC 826 Subcultures
D7P
D7_J
D7_P
FUM1 Expression of Subcultures on Day 19
50
25
FCK1 Expression of Subcultures on Day 19
40
Relative expression levels
20
35
30
15
25
20
10
15
10
5
0
5
0
D19_A
D19_K
D19_M
D19_O
D19_J
D19_P
D19_A
D19_K
MRC 826 Subcultures
D19_M
25000
RESULTS
20000
FB1
15000
FB2
10000
FB3
5000
Total
FB
0
D19A
D19_O
MRC 826 Subcultures
HPLC - FB levels on Day 19
30000
FB levels in subcultures (ug/g)
Relative expression levels
45
D19K
D19M
D19O
D19J
MRC 826 Subcultures
D19P
D19_J
D19_P
DISCUSSION
• Important to understand fungal mechanisms involved in toxin
•
•
•
•
•
production.
By determining the expression of essential FUM and regulatory
genes and the interactions between the genes that results in high
fumonisin levels, could aid in the development of an important tool
and screening method to identify samples with potentially high
mycotoxin content and other toxigenic fungi co-occurring on maize.
Research is being done in close collaboration to complement other
current ongoing MT projects.
Research results were presented at the 2011 International MycoRed
Conference - poster received 3rd prize for best poster.
First phase of the work is being prepared for scientific publication.
Knowledge can be transferred to maize producers (commercial,
small-scale and emerging farmers).
BENEFIT TO THE MAIZE INDUSTRY SECTORS
• The objectives of this project are in alignment with three of the five
strategic objectives of the Maize Trust.
• Long-term objectives are
-
to reduce natural contamination of cereal grains and development
/ exploitation of disease-resistant cultivars.
-
improve harvest quality and yield – less fungal / mycotoxin
contamination.
-
higher profitability - increased income – local and export crops.
-
healthier sustainable staple foods – better health.
• Globally competitive – disease resistance programmes.
“Keep your friends close, and your enemies closer .”
Sun-tzu - Chinese general & military strategist (~400 BC)
“The truth may not set you free, but used carefully, it can
confuse the hell out of your enemies.” Laurell K. Hamilton
Research may not set the Maize Industry free from mycotoxins,
but it can contribute a great deal to mycotoxin control
in the future
ACKNOWLEDGEMENTS
Maize Trust and Medical Research Council for funding
Support from several staff members of the PROMEC Unit
Collaboration of National (Univ Stellenbosch, Grains Crop
Institute , Pothchefstroom) as well as International Scientists
( Dr Robert Procter, Prof John Leslie)