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Virulence of banana wilt-causing fungal pathogen Fusarium oxysporum tropical race 4 is mediated by nitric oxide biosynthesis and accessory genes

Nature Microbiology volume 9, pages 2232–2243 (2024)Cite this article

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Abstract

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most damaging plant diseases known. Foc race 1 (R1) decimated the Gros Michel-based banana (Musa acuminata) trade, and now Foc tropical race 4 (TR4) threatens global production of its replacement, the Cavendish banana. Here population genomics revealed that all Cavendish banana-infecting Foc race 4 strains share an evolutionary origin distinct from that of R1 strains. Although TR4 lacks accessory chromosomes, it contains accessory genes at the ends of some core chromosomes that are enriched for virulence and mitochondria-related functions. Meta-transcriptomics revealed the unique induction of the entire mitochondrion-localized nitric oxide (NO) biosynthesis pathway upon TR4 infection. Empirically, we confirmed the unique induction of a NO burst in TR4, suggesting that nitrosative pressure may contribute to virulence. Targeted mutagenesis demonstrated the functional importance of fungal NO production and the accessory gene SIX4 as virulence factors.

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Fig. 1: The most virulent Foc variant (TR4) has spread from Southeast Asia to Africa and Latin America.
Fig. 2: Foc phylogenomics and population structure.
Fig. 3: Genomic comparison reveals the attachment of accessory sequences to core chromosomes and the dynamics of these accessory sequences.
Fig. 4: The mitochondrion-localized nitrate/nitrite-dependent NO biosynthesis pathway and NO detoxification-related genes are significantly induced in TR4 at 18 HPI (created with BioRender.com).
Fig. 5: Plant hormone-induced fungal NO burst in TR4.
Fig. 6: Proposed TR4–Cavendish banana interaction mode (created with BioRender.com).

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Data availability

Genome sequences and annotations have been deposited in MycoCosm at the Joint Genome Institute (https://mycocosm.jgi.doe.gov/mycocosm/home) and at NCBI under the accession number PRJNA859936. Raw RNA-seq files for samples in this study were uploaded to the NCBI Sequence Read Archive and are publicly available under BioProject PRJNA1113144. Source data are provided with this paper.

Code availability

Computer codes generated for this study are available via GitHub at https://github.com/zya067025/Fusarium-oxysporum-f.-sp.-cubense----genomics.

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Acknowledgements

The authors thank R. C. Ploetz, University of Florida, for providing Foc strains and J. Chambers, the director of the Microscopy Core Facility of the Institution for Applied Life Sciences at University of Massachusetts, for his help with the confocal microscopy experiments. Data were analysed at the Massachusetts Green High Performance Computing Center (MGHPCC). The Guangdong Science and Technology Project (2019B1515120088, C.L.), the Laboratory of Lingnan Modern Agriculture Project (NT2021004, G.Y.), the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (No. 2022KJ109, C.L.), the National Banana Industry and Technology System Project (nycytx-33) and the earmarked fund for CARS (CARS-31, G.Y.) provided financial support. This project was funded by the Natural Science Foundation (IOS-165241, L.-J.M.) and the National Research Initiative Competitive Grants Program (MAS00532 and MAS00496, L.-J.M.) from the USDA National Institute of Food and Agriculture. The work (proposal: 10.46936/10.25585/60008191, L.-J.M.) conducted by the US Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy operated under contract no. DE-AC02-05CH11231 (I.V.G). L.-J.M. is also supported by the National Eye Institute of the US National Institutes of Health under award number R01EY030150. H.Y. is also supported by the Lotta M. Crabtree Fellowship and the Constantine J. Gilgut Fellowship. The funding bodies played no role in the design of the study, the collection, analysis and interpretation of the data, or the writing of the manuscript.

Author information

Author notes

  1. Yong Zhang

    Present address: NIH-National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA

  2. Houlin Yu

    Present address: Broad Institute of MIT and Harvard, Cambridge, MA, USA

  3. These authors contributed equally: Yong Zhang, Siwen Liu.

  4. These authors jointly supervised this work: Altus Viljoen, Chunyu Li, Li-Jun Ma.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA

    Yong Zhang, Houlin Yu, Gengtan Li & Li-Jun Ma

  2. Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of Agriculture and Rural Affairs; Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree; Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou, China

    Siwen Liu, Mengxia Zhuo, Cunwu Zuo, Ganjun Yi & Chunyu Li

  3. Guangdong Laboratory for Lingnan Modern Agriculture of Maoming sub-center, Guangdong Academy of Agricultural Sciences, Guangzhou, China

    Siwen Liu, Mengxia Zhuo, Ganjun Yi & Chunyu Li

  4. Department of Plant Pathology, Stellenbosch University, Stellenbosch, South Africa

    Diane Mostert & Altus Viljoen

  5. Plant Biology, University of Massachusetts Amherst, Amherst, MA, USA

    Houlin Yu & Katie Webster

  6. US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Sajeet Haridas & Igor V. Grigoriev

  7. Department of Plant Pathology/Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou, China

    Minhui Li

  8. Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA

    Igor V. Grigoriev

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  1. Yong Zhang
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Contributions

L.-J.M., Y.Z., G.Y., C.L., and A.V. conceived the research strategies. L.-J.M., C.L. and A.V. supervised the project. Y.Z. and L.-J.M. wrote the manuscript with input from all authors. Y.Z., S.L., M.Z., G.L. and C.Z. conducted NO detection and mutagenesis studies. Y.Z., H.Y., S.H. and I.V.G. analysed the data. Y.Z., S.L, G.L., M.Z., C.Z., K.W. and M.L. validated the experiments. D.M., C.L. and A.V. collected and provided the strains.

Corresponding authors

Correspondence to Altus Viljoen, Chunyu Li or Li-Jun Ma.

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Nature Microbiology thanks Jeffrey Coleman, Donald Gardiner and Martijn Rep for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 SNP density on chromosomes 3 and 6 of two Foc TR4 and two VCG 0121 strains.

Tracks from the inside to outside are as follows: four histogram circles of SNP distribution on chromosomes 3 and 6 for 811-VCG01213-TR4, 789-VCG01213-16-TR4, CAV2318-VCG0121 and CAV180-VCG0121; and II5 chromosomes 3 and 6 (blue) with accessory sequences (orange) as a reference. The mating-type (MAT) locus MAT1 on the core chromosomal region of chromosome 6 and Helitron transposable elements on accessory region of chromosome 3 are indicated.

Extended Data Fig. 2 Comparative meta-transcriptomics of Cavendish bananas infected with R1 (GD02) and TR4 (II5).

a, Infection stages of GFP-tagged Foc TR4 II5 in banana roots. Root materials were collected, and the infection process was monitored under a microscope after inoculation at different stages of infection. Left to right: At 18 HPI, Foc TR4 spores began to germinate and cover the surface of the root. The pathogen continued to grow, and hyphae penetrated the root. At approximately 32 HPI, many hyphae were evident in the xylem, and visible dark spots appeared on the root. At 56 HPI, there was a large increase in fungal biomass and necrotic root symptoms. b, Foc race 1 has weaker colonization capability than Foc TR4 during the infection process. Percentage of mapped reads derived from fungi and plants in the infection time course in the race 1–banana (red) and Foc TR4–banana (green) interactions. Data presented are the mean +/− standard deviations from three biological replicates at each time point.

Extended Data Fig. 3 Expression profiles of 18 fungal co-expressed gene clusters based on TR4 and R1 shared orthologs using a global hierarchical clustering algorithm.

The x-axis shows the time points of Cavendish banana infection with R1 (GD02) and TR4 (II5). The y-axis shows the Z-scores of gene expression. Color scale indicates the correlation of expression between genes and the cluster centroids. Genes with an expression correlation with a centroid lower than 0.8 are shown in gray.

Extended Data Fig. 4 Fungal functional network of Foc-C5, Foc-C7 and Foc-C14 that were significantly induced by 18 HPI in TR4 compared to R1.

a, Three fungal co-expressed gene clusters were significantly induced at 18 HPI in TR4 compared to R1. b, Foc-C5 (510 genes) was enriched for electron transfer activity (corrected P = 0.01), Foc-C7 (846 genes) for heme-copper terminal oxidase activity (corrected P = 0.03) and NADPH quinone reductase activity (corrected P = 0.01) and Foc-C14 (694 genes) for regulation of nitrogen compound metabolic process (corrected P = 0.05). Collectively, these three clusters are enriched for genes involved in NO biosynthesis and denitrification. GO annotations and genes are presented as nodes. The annotations are highlighted with different colors based on the distribution of the nodes in the network. The size of the node is proportional to the number of connections. Two-sided Benjamini-Hochberg corrected Fisher’s exact test was performed to identify enriched GO terms.

Extended Data Fig. 5 qRT-PCR of banana phytoglobin genes.

qRT-PCR confirming that all three genes were significantly induced in Cavendish banana at 18 HPI with TR4 strain II5 compared with R1 strain GD02. Data are shown as box plots of three biological replicates (n=3) with the interquartile range as the upper and lower confines of the box and the median as a solid line within the box. Our RNA-seq data identified one banana phytoglobin gene, Ma02_g10610, as one of the top 10 most highly induced plant genes. Asterisks indicate significantly induced phytoglobin gene expression (* = P < 0.05, Student’s t test) in Foc TR4–inoculated Cavendish banana. All statistical tests carried out were two-sided.

Source data

Extended Data Fig. 6 Expression profiles of 24 plant-expressed gene clusters.

The x-axis represents the time point of Cavendish banana infection with R1 (GD02) and TR4 (II5). The y-axis shows the Z-score of gene expression. Color scale indicates the correlation of expression between genes and the cluster centroids. Genes with a correlation of expression with a centroid < 0.8 are shown in gray.

Extended Data Fig. 7 Mutants of fungal genes involved in the fungal NO biosynthesis pathway have reduced fungal virulence and NO production.

a, The ΔGene_2699 and ΔGene_9725 mutants were generated in the Foc TR4 II5 WT background using the standard one-step gene replacement strategy69. The complemented strains ΔGene_2699-C (complemented FocΔGene_2699 strain) and ΔGene_9725-C (complemented FocΔGene_9725 strain) were generated using whole genes and their native promoters combined with the neomycin resistance gene. Cavendish banana plantlets with four or five leaves were inoculated with mock (H2O) or treated soil samples harboring conidia from different strains (5,000 conidia/g soil). The disease index (DI) distribution (top panel; see Methods) was calculated among 20 inoculated plants based on the DI from 0–4: 0, no symptoms; 1, some brown spots in the inner rhizome; 2, <25% of the inner rhizome showing browning; 3, up to 75% of the inner rhizome showing browning; and 4, 100% of the inner rhizome and pseudostem dark brown (considered dead). The cross sections were sampled from inoculated plantlets (bottom panel) at 30 days post inoculation. The extent of discoloration is correlated with the DI distribution shown in the top panel. b, Analysis of NO production by TR4 WT, ΔGene_2699 and ΔGene_9725 mutants with NO-sensitive fluorescent dye DAR-4M-AM as a probe; NO production was measured at 6 hours after the JA stimulation. Data are shown as box plots with the interquartile range as the upper and lower confines of the box and the median as a solid line within the box. Different letters indicate significant differences according to one-way ANOVA (P < 0.05). Each experiment included three biological replicates and was repeated independently at least three times.

Source data

Extended Data Fig. 8 Phylogeny of expressed, accessory transcription factor (TF) genes encoding the GAL4-like Zn2-C6 binuclear cluster DNA-binding domain.

The phylogenetic tree (generated using the maximum-likelihood method) reveals that the GAL4-like domain–containing TFs that expanded in TR4 reside in three major clades. Clade III comprises 26 GAL4-like TFs that are phylogenetically close to FgZC1 (FGSG_05068), a transcription factor known to play an important role in host-mediated fungal NO production and virulence in F. graminearum. In addition to these expressed accessory genes, this analysis included functionally characterized genes with a GAL4-like domain. Ab, Alternaria brassicicola; Cl, Colletotrichum lindemuthianum; Fg, Fusarium graminearum; Fo, Fusarium oxysporum; Fv, Fusarium verticillioides; Ff, Fusarium fujikuroi; Mo, Magnaporthe oryzae; Vd, Verticillium dahliae.

Extended Data Fig. 9 Functional analysis of the fungal effector genes SIX1a and SIX4, which are present in the accessory region of chromosome 3 and were significantly induced upon host infection.

The ΔSIX1a and ΔSIX4 mutants were generated in the Foc TR4 II5 WT background using the standard one-step gene replacement strategy69. The complemented strains ΔSIX1a-C (complemented FocΔSIX1a strain) and ΔSIX4-C (complemented FocΔSIX4 strain) were generated using whole effector genes and their native promoters combined with the neomycin resistance gene. Cavendish banana plantlets with four or five leaves were inoculated with mock (H2O) or treated soil samples harboring conidia from different stains (5,000 conidia/g soil). The disease index (DI) distribution (top panel; see Methods) was calculated among 20 inoculated plants based on the DI from 0–−4: 0, no symptoms; 1, some brown spots in the inner rhizome; 2, <25% of the inner rhizome showing browning; 3, up to 75% of the inner rhizome showing browning; and 4, 100% of the inner rhizome and pseudostem dark brown (considered dead). The cross sections were sampled from inoculated plantlets (bottom panel) at 30 days post inoculation. The extent of discoloration is correlated with the DI distribution shown in the top panel.

Extended Data Fig. 10 Generation of gene mutants.

a, Schematic representation of the disruption strategy for SIX1a, SIX4, Gene_2699 and Gene_9725. Primer locations are marked with arrows. b, Plasmid map of pYF11. Restriction enzyme cutting sites are indicated. Features including antibiotic resistant genes and promoters are shown.

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

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Supplementary Tables 1–19

Supplementary Table 1 Fusarium oxysporum isolates used in this study, provided by Altus Vijoen (Stellenbosch University, South Africa), Randy C. Ploetz (University of Florida, USA) and Chunyu Li (Fruit Tree Research Institute, GDAAS, China). Supplementary Table 2 Summary of pathogenicity assay tests on banana cultivars with Foc TR4, STR4, VCG 0121 and race 1. Supplementary Table 3 II5 chromosomal accessory sequence size comparing to Fol 4287. Supplementary Table 4 Copy numbers of Helitrons by normalized read mappings. Supplementary Table 5 Genome statisitics of the de novo assembled Fusarium oxysporum f. sp. cubense strains. Supplementary Table 6 Heatmap of shared chromosomal accessory sequence size between II5 and other Foc strains. Supplementary Table 7 Statistics of the RNA-seq data and reads mapped to the reference genome II5 at different inoculation time points. Supplementary Table 8 Clusters of co-expressed banana and fungal genes. Supplementary Table 9 Presence/absence of 2,050 genes of three fungal gene clusters (Foc-C5, Foc-C7 and Foc-C14) in race 4 strains (six TR4, two VCG 0121 and two STR4). Supplementary Table 10 Expression of NO biosynthesis, denitrification and electron transport chain complex genes during infection. Supplementary Table 11 Top 20 expressed banana genes during host–pathogen interaction. Supplementary Table 12 Expressions of genes in plant-C22. Supplementary Table 13 Gene Ontology enrichment of 1,562 AS genes. Supplementary Table 14 GO enrichement of expressed 842 accessory sequence genes. Supplementary Table 15 Annotation for accessory genes enriched in obsolete electron transport. Supplementary Table 16 A list of selected F. oxysporum genomes for pan-geneme analysis. Supplementary Table 17 A list of 25 Foc-only gene familes and 189 Foc-race 4-only gene familes. Supplementary Table 18 Expression of fungal effectors in Foc TR4 accessory regions. Supplementary Table 19 PCR primers used in this study.

Source data

Source Data Fig. 1

Latitude and longitude of locations where strains were isolated.

Source Data Fig. 4

Expressions of genes involved in NO biosynthesis and denitrification.

Source Data Fig. 5

Raw fluorescence intensity of TR4 and R1 strains treated with/without MJ.

Source Data Extended Data Fig./Table 5

Relative expression of banana phytoglobin genes.

Source Data Extended Data Fig./Table 7

Raw fluorescence intensity of WT TR4 strain, gene mutants and their complement strains.

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Zhang, Y., Liu, S., Mostert, D. et al. Virulence of banana wilt-causing fungal pathogen Fusarium oxysporum tropical race 4 is mediated by nitric oxide biosynthesis and accessory genes. Nat Microbiol 9, 2232–2243 (2024). https://doi.org/10.1038/s41564-024-01779-7

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