Focus on research: molecular biologist Martijn Rep

Photo: Bob Bronshoff

Molecular biologist Martijn Rep started off his study of the tomato-infecting fungus Fusarium oxysporum by examining the function of secreted proteins. He later discovered that the fungus is capable of transferring entire chromosomes to similar fungi. His findings earned him a publication in Nature. The Vici grant will enable him to unravel the mechanism underlying this phenomenon.

Microscopic image of the fungus Fusarium oxysporum. The cell nuclei have been artificially marked in green. (Michielse et al. (2009) PloS Pathogens 5: 1000637)

Rep serves as university lecturer and researcher at the Swammerdam Institute for Life Sciences (SILS). In 2000, he joined the Plant Pathology group to start a study of the plant-pathogenic fungus Fusarium oxysporum. This represented an entirely new direction for Rep, who had spent the previous years specialising in yeast. He was immediately enthusiastic about the content of his new work. ‘There are plenty of labs working on yeast, but there was very little information on Fusarium available at the time. It was an unexplored area, which made the going a little rough, but I also found it incredibly challenging. I had the sense of being somewhat of a pioneer.' What's more, he adds, Fusarium is a fungus responsible for causing a broad range of diseases. ‘That means there's a direct link to day-to-day practice, which really appeals to me.'

Rep's first Fusarium projects at SILS focused on proteomics: the study of proteins. Fusarium, he explains, is a fungus that occurs in soil and penetrates plants' vascular tissue through their roots. ‘We know that water and minerals pass through the vascular tissue. Sugars and proteins are normally transported through another type of tissue: the phloem. When I started my research, a few articles had just been published on the presence of plant proteins in vascular tissue. No one really knew why they occurred there.'

Rep applied mass spectrometry to characterise the proteins in tomato stems infected with Fusarium. In addition to the plant proteins involved in defence reactions, the vascular tissue also proved to contain fungal proteins. ‘The very first fungal protein we identified proved to be a real breakthrough. That was a really lucky break.' As it turned out, the protein was both a virulence factor - a protein that promotes plant infection - and an a-virulence factor - a protein that allows the plant's immune system to recognise and subsequently fight off the fungus. Rep: ‘The second and third proteins we identified were a-virulence factors 2 and 3, respectively. Finding them in such short succession was a great experience.'

Identical genes

Photo: Bob Bronshoff

In 2007, the Cambridge (U.S.)-based Broad Institute sequenced the F. oxysprum genome. Rep: ‘It turned out that all the proteins we had identified as being involved in the infection process were located on a single chromosome. This confirmed our belief that the chromosome was extraordinary.' An remarkable phenomenon is that the fungus consists of a very large number of different strains. All the various individuals in a strain are clones with identical genetic material. Only three or four strains are capable of infecting the tomato plant. ‘The sequencing data showed that the genes which encode for the minuscule proteins that promote infection in these strains are identical: they only occur in a single form. This seemed strange, as the remaining genes show a great deal of variation between the various strains. We then found out that two of the genes for small secreted proteins are located close together. This led us to develop the theory that the chromosomes containing the pathogenic information migrate in their entirety.'

A similar chromosomal migration had been identified in another fungus, ten years prior. Rep surmised that if the same phenomenon also occurred in Fusarium, it would occur spontaneously, and might thus be reproduced in a lab environment. Accordingly, his doctoral candidate Lotje van der Does grew a Fusarium strain capable of infecting tomatoes alongside three different strains: a non-pathogenic strain, and two strains capable of infecting melons and bananas, respectively.

Transferring resistance

In order to determine whether the three recipient strains would actually receive a chromosome from the tomato-infecting fungus, Rep and his colleagues inserted markers into the various genomes. These markers make the fungus resistant to a specific chemical substance. Strains that proved resistant to two of these substances after having been merged, would thus contain a combination of two genomes. This turned out to be the case in two out of three combinations, confirming the transfer of chromosomes. Rep: ‘That was an incredibly triumphant moment.'

Two chromosomes containing pathogenic protein genes were found to be capable of migrating from one strain to another, providing additional genetic information that transformed innocent fungi into pathogens. The researchers published their findings in Nature. According to Rep, the phenomenon explains a great deal. ‘No sexual cycle had been identified for Fusarium: this left us with no explanation as to how genetic pathogenic information had found its way into the various strains.'

Fusion between hyphae (anastomosis) is probably a precondition for chromosome migration. Illustration from Tulasne and Tulasne (1863), Selecta Fungorum Carpologia.

Rep suspects that the transfer of chromosomes takes place during the fusion of hyphae from two different fungi. In order for such a fusion to succeed, the two fungi would normally have to be of the same strain. If the two are from different strains, an immune response would kick in under normal circumstances. ‘This is actually a method used to identify different strains. However, it turns out that certain chromosomes are capable of circumventing the immune system's response.'

Unravelling the process of chromosome migration

The discovery explains the genesis of new pathogenic strains in certain fungi, both in nature and in agriculture and horticulture environments, and demonstrates the high level of genetic flexibility and adaptability of fungi. The exact transfer mechanism is yet to be unravelled. However, Rep has some ideas of his own on the subject. ‘The chromosome has to have some extraordinary characteristics. Perhaps it actively suppresses the immune system's response.' He has already secured funding for the next five years of research. ‘The day after I found out the article was being published in Nature, I had an appointment for a Vici grant interview. Obviously, I mentioned the article'. Rep managed to get the grant. The discovery attracted a great deal of media attention. ‘It's good science, but that also goes for plenty of other research projects that never make the headlines. Once you get mentioned in the NRC [leading Dutch newspaper, ed], people begin to see your work in a different light. For example, my parents had always been interested in my research, but they're a bit more convinced now that I'm doing something important.'

Published by  Faculty of Science

21 August 2012