In a previous study, our lab showed how the devastating Tropical Race 4 (TR4) lineage of banana-infecting F. oxysporum is spreading in Mozambique, beyond its initial incursion area.

In that study, we routinely sequenced the new TR4 isolates and compared them to those already in our collection. One of the strains, called M1, was chosen almost at random for long-read sequencing. That arbitrary choice, however, turned out to be the starting point of an unexpected story about a “jumping gene” that is reshaping the genome of a clonal pathogen.

Three key sequential observations

Because TR4 threatens banana production globally, understanding its genome dynamics is essential for developing durable control strategies. Once we began examining the new genomes, the surprises appeared in quick succession. When we compared the Mozambican strains’ genomes with the TR4 reference strain (II5), M1 stood out. Two large chunks of DNA, several hundred kilobases in size, were missing from chromosomes that are usually highly conserved. In our experience, such deletions are rare, and they immediately raised the question of what exactly had been lost.

One clue came from a previous routine analysis. We knew that the genes needed to produce the toxin fusaric acid are located near one end of chromosome 3. When we saw that one of M1’s deletions was at that very end, alarm bells rang. Could this strain have lost the cluster responsible for fusaric acid production?

This possibility prompted a closer look. Although we initially assumed these were simple deletions, curiosity led us to investigate the region further. Was this really just a deletion, or had it been replaced by something else? To our surprise, both missing regions had been replaced by almost the exact same small 6 kb sequence, which is absent from the reference strain in that location. A quick BLAST search revealed that this sequence was a Helitron transposable element, previously named FoHeli1.

Helitrons, what are they (doing)?

Prior to this work, the word Helitron would have sounded, to us, like something from science fiction and it still does a little bit. In the strange world of transposable elements or jumping genes, Helitrons are somewhat unusual. They are DNA transposons that replicate through a rolling-circle mechanism. Instead of cutting themselves out and pasting into a new spot, they make a circular copy of themselves that can then be inserted elsewhere in the genome. Helitrons can be found in essentially all eukaryotes, including fungi, but have been studied predominantly in plants and animals. Another remarkable characteristic is their tendency to capture genes or gene fragments, which allows them to mobilize other sequences.

By comparing all FoHeli1 copies in M1 and other TR4 strains, we saw very little overall sequence divergence, consistent with recent jumps. Using a PCR assay that works only if FoHeli1 forms a circular intermediate, we could directly detect this circular form, supporting the idea that it remains active. In a clonal lineage that does not gain diversity through sexual reproduction, such jumping genes may be a crucial source of genetic innovation.

FoHeli1 is absent in strains most closely related to TR4, leaving its origin within the TR4 lineage an open question. One possibility is that FoHeli1 hitchhiked into TR4 on an accessory chromosome unique to this lineage. This smallest chromosome carries the highest number of FoHeli1 copies and is also missing from the closest relative that lacks FoHeli1.

In that sense, the accessory chromosome might have acted as a carrier, bringing FoHeli1 into TR4. Whether we can ever reconstruct this journey in detail remains uncertain and will require the right genomic tools.

The need for long-read genome assemblies

Because transposable elements like FoHeli1 are mobile and often consist of many (nearly) identical copies, short-read sequencing tends to collapse them into a single copy. They are like identical puzzle pieces: you can count them, but you cannot place them in their correct spots.

Long-read assemblies, in contrast, can span entire transposable element copies and their flanking regions, revealing their precise positions and the structural changes they might cause, such as in M1. In our case, we hypothesize that the large deletions arose from recombination between two similar FoHeli1 elements. However, the transient nature of these events and the limited number of long-read assemblies make it difficult to reconstruct the exact sequence of events.

Impact and future prospects

Altogether, what began as a routine comparison of genome assemblies became a case study of how a single transposable element can reshape the genome of a clonal pathogen. The likely ongoing activity of FoHeli1 suggests that what we see now may be only a snapshot, a tip of the iceberg of potential structural variation within TR4.

The loss of fusaric acid production in M1 also challenges our assumptions about this toxin. If a highly virulent TR4 strain can do without this generally conserved toxin, under at least some conditions, we may need to reconsider its role in disease.

Looking ahead, broader long-read sequencing of TR4 isolates could reveal the extent of FoHeli1-driven changes. An experimental evolution approach, such as artificially inducing Helitron activity in a naïve strain and tracking the genomic consequences, could provide a controlled view of how such elements generate diversity.

For us, this project was a reminder that even routine comparisons of a clonal pathogen can reveal unexpected stories. Sometimes, picking a strain at random is enough to stumble upon a transposable element you had never heard of before.