FUM1 GENE EXPRESSION AND FUMONISIN PRODUCTION OF Fusarium verticillioides MRC 826 SUBCULTURES

1. 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: lorraine.moses@mrc.ac.za

2. 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.

3. 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?

4. 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.

5. PREVIOUSANALYSES • 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.

6. 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

7. Gene expression analysis FUM 21 FUM 13 FUM 19 FUM 2 (12) FUM 18 FUM 17 FUM 15 FUM 3 (9) FUM 14 FUM 1 (5) FUM 16 FUM 11 FUM 10 FUM 7 FUM 8 FUM 6 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

8. Gene expression analysis Total RNA was isolated using Trizol Biological triplicates of each subculture of MRC 826 (DNase treatment) cDNA synthesis from mRNA templates using Reverse Transcription with oligo d(T) primers Quantitation of cDNA using Standard Curve Method AAAAA AAAAA AAAAA AAAAA AAAAA TTTTT TTTTT

9. 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

10. RESULTS

11. RESULTS

12. 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).

13. 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.

14. “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

15. 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)