Investigation of the diversity of effector genes in the banana pathogen, Fusarium oxysporum f. sp. cubense, reveals evidence of horizontal gene transfer

Abstract

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana-infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole-genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1-SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.

Keywords: Fusarium oxysporum f. sp. cubense; effectors; horizontal gene transfer.

Figure 1

Best infraspecies phylogenetic tree inferred using maximum likelihood (ML) from the concatenated datasets of translation elongation factor‐1α (EF‐1α), RNA polymerase II subunit I (RPB1) and RNA polymerase II subunit II (RPB2) identified in isolates of Fusarium oxysporum used in this study. Trees with similar topologies were also inferred using maximum parsimony (MP) and Bayesian interference (BI) methods. Internal node support is indicated as ML bootstrap proportions/Bayesian probabilities/MP bootstrap proportions. The two major clades are as indicated. For each isolate, the abbreviated forma specialis and accession code as defined in Tables 3 and 4 are indicated. The isolates of F. oxysporum f. sp. cubense (Foc) are also labelled with their respective vegetative compatibility group (VCG). The isolates of Foc in clade A are shown in blue, whereas the isolates of Foc in clade B are shown in green. The other formae speciales and F. verticillioides NRRL 20956 are shown in black. Nucleotide sequences from F. verticillioides served as an outgroup to root the tree.

Figure 2

Best genealogies of the Secreted In Xylem (SIX) genes SIX1 (a), SIX2 (b), SIX6 (c), SIX7 (d), SIX8 (e), SIX9 (f), SIX10 (g) and SIX13 (h) generated using the maximum likelihood (ML) method. Trees with similar topologies were also inferred using maximum parsimony (MP) and Bayesian interference (BI) methods. Internal node support is indicated as ML bootstrap proportions/Bayesian probabilities/MP bootstrap proportions. For each external node, the abbreviated forma specialis as defined in Tables 3 and 4 in which the sequence was identified and the SIX gene homologue variant are indicated. For Fusarium oxysporum f. sp. cubense (Foc), the vegetative compatibility group (VCG) in which the SIX gene homologue was identified is also indicated. The VCGs of Foc that clustered in clade A of the infraspecies phylogeny are shown in blue, whereas the VCGs of Foc that clustered in clade B of the infraspecies phylogeny are shown in green. The sequences from other formae speciales are shown in black.

Figure 3

A visual representation of the models implemented for the topological constraint testing of the Secreted In Xylem (SIX) gene trees using an infraspecies backbone constraint model (a) and a Fusarium oxysporum f. sp. cubense (Foc) monophyletic constraint model (b). The infraspecies model constrained the SIX gene sequences identified in the vegetative compatibility groups (VCGs) of Foc and other formae speciales into the two major clades identified in the infraspecies phylogeny (Fig. 1). For each isolate, the abbreviated forma specialis and accession code as defined in Tables 3 and 4 are indicated. Under the Foc monophyletic model, the SIX gene sequences identified in the VCGs of Foc were constrained into a monophyletic clade, whereas the SIX gene sequences identified in other formae speciales were constrained into a reciprocating sister clade.