Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is well-known as the causal agent of Fusarium wilt of banana and is one of the most destructive phytopathogens for banana plants. The molecular mechanisms underlying Foc TR4 virulence remain elusive. Here, we demonstrate that a cerato-platanin (CP) protein, FocCP1, functions as a virulence factor that is required by Foc TR4 for penetration and full virulence. The FocCP1 gene was expressed in every condition studied, showing a high transcript level in planta at the early stage of infection. Infiltration of the recombinant FocCP1 protein induced significant cell death and upregulated defence-related gene expression. FocCP1 knock-out strains showed a significant decrease in aerial growth rather than aqueous growth, which is reminiscent of hydrophobins. Furthermore, deletion of FocCP1 significantly reduced virulence and dramatically reduced infective growth in banana roots, likely resulting from a defective penetration ability. Taken together, the results of this study provide novel insight into the function of the recently identified FocCP1 as a virulence factor in Foc TR4.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, the most devastating disease on a banana plant. The genome of Foc TR4 encodes many candidate effector proteins. However, little is known about the functions of these effector proteins on their contributions to disease development and Foc TR4 virulence. Here, we discovered a secreted metalloprotease, FocM35_1, which is an essential virulence effector of Foc TR4. FocM35_1 was highly upregulated during the early stages of Foc TR4 infection progress in bananas. The FocM35_1 knockout mutant compromised the virulence of Foc TR4. FocM35_1 could interact with the banana chitinase MaChiA, and it decreased banana chitinase activity. FocM35_1 induced cell death in Nicotiana benthamiana while suppressing the INF1-induced hypersensitive response (HR), and its predicted enzymatic site was required for lesion formation and the suppression to INF1-induced HR on N. benthamiana leaves. Importantly, treatment of banana leaves with recombinant FocM35_1 accelerates Foc TR4 infection. Collectively, our study provides evidence that metalloprotease effector FocM35 seems to contribute to pathogen virulence by inhibiting the host immunity.

Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is one of the most destructive diseases for banana. For their risk assessment and hazard characterization, it is vital to quickly determine the virulence of Foc isolates. However, this usually takes weeks or months using banana plant assays, which demands a better approach to speed up the process with reliable results. Foc produces various mycotoxins, such as fusaric acid (FSA), beauvericin (BEA), and enniatins (ENs) to facilitate their infection. In this study, we developed a linear regression model to predict Foc virulence using the production levels of the three mycotoxins. We collected data of 40 Foc isolates from 20 vegetative compatibility groups (VCGs), including their mycotoxin profiles (LC-MS) and their plant disease index (PDI) values on Pisang Awak plantlets in greenhouse. A linear regression model was trained from the collected data using FSA, BEA and ENs as predictor variables and PDI values as the response variable. Linearity test statistics showed this model meets all linearity assumptions. We used all data to predict PDI with high fitness of the model (coefficient of determination (R2 = 0.906) and adjust coefficient (R2adj = 0.898)) indicating a strong predictive power of the model. In summary, we developed a linear regression model useful for the prediction of Foc virulence on banana plants from the quantification of mycotoxins in Foc strains, which will facilitate quick determination of virulence in newly isolated Foc emerging Fusarium wilt of banana epidemics threatening banana plantations worldwide.

Fusaric acid (FA) is an important secondary metabolite of many Fusarium species and involved in the wilt symptoms caused in banana by Fusarium oxysporum f. sp. cubense (Foc). To investigate the evolution characteristics of the 12 Foc FA biosynthetic genes (FUB), coding sequences of the 12 FUB genes and three housekeeping genes, EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II), were subjected to genetic diversity analysis, phylogenetic analysis, recombination detection, and selective pressure analysis. The results of selective pressure analysis showed that the 15 genes were mainly subjected to negative selection. However, a significantly higher number of silent mutations, which could not be simply explained by selective pressure difference, were observed in the 12 FUB genes in Foc than in the three housekeeping genes. Infraspecies phylogeny and recombination detection analysis showed that significantly more horizontal gene transfer (HGT) events (normalized) had occurred in the FUB genes than in the three housekeeping genes. In addition, many of these events involved outgroup isolates and significantly increased the genetic diversity of FUB genes in Foc. The infraspecies phylogenetic analysis suggested that the polyphyletic phylogeny proposed for Foc requires further discussion, and the divergence of race 1, race 4, and the common ancestor of several F. oxysporum (Fo) isolates pathogenic to nonbanana plants should have diverged over a short period. Finally, our results suggest that the FUB genes in Fo should have benefited from HGT to gain a relatively high genetic diversity to respond to different host plants and environments despite mainly being subject to negative selection.