Fosp9, a Novel Secreted Protein, Is Essential for the Full Virulence of Fusarium oxysporum f. sp. cubense on Banana (Musa spp.)

Abstract

The banana vascular wilt pathogen, Fusarium oxysporum f. sp. cubense, delivers a number of different secreted proteins into host plant tissues during infection. Until now, only a few of the secreted proteins from this fungus have been shown to be virulence effectors. Here, the product of fosp9, which is a gene in this pathogen, was found to be a novel virulence effector. The fosp9 gene encodes a hypothetical 185-amino-acid protein which has a functional signal peptide but contains no known motifs or domains. The fosp9 disruptants displayed a significant reduction in producing wilt symptoms on bananas, indicating that fosp9 is essential for the full virulence of this pathogen for banana. These disruptants did not exhibit a change in either saprophytic growth or conidiation on potato dextrose agar medium, but their invasive growth in the rhizomes of banana was markedly compromised, suggesting a pivotal role for fosp9 in the colonization of banana rhizome tissues by this fungus. Live-cell imaging revealed that the Fosp9-GFP fusion protein accumulated in the apoplast of the plant cells. Moreover, transcriptome profiling revealed that a number of virulence-associated genes were differentially expressed in the fosp9 disruptant relative to the wild type. Taken together, these findings suggest that Fosp9 is a genuine effector of F. oxysporum f. sp. cubense. IMPORTANCE Fusarium wilt of bananas (also known as Panama disease), caused by the fungus F. oxysporum f. sp. cubense, is one of the most devastating banana diseases worldwide. The understanding of the molecular mechanism of its pathogenicity is very limited so far. We demonstrated that the secreted protein Fosp9 from this fungus contributes to its virulence against banana hosts and is essential for colonization of banana rhizome tissues by this fungus. In particular, Fosp9 contains no known domains or motifs and has no functionally characterized homologs, implying that it is a novel secreted effector involved in F. oxysporum f. sp. cubense-banana interactions. This work provides insight into molecular mechanisms of F. oxysporum f. sp. cubense pathogenicity, and the characterization of the fosp9 gene will facilitate development of transgenic banana and plantain strains resistant to this disease by silencing this effector gene through host-induced gene silencing or other strategies.

Keywords: Fusarium wilt; banana; colonization; effector; virulence.

FIG 1

Sequence analysis of Fosp9. (a) Deduced amino acid sequence and signal peptide (boxed) of Fosp9 protein. (b) Phylogenetic tree constructed based on amino acid sequences of Fosp9 and its homologs from other fungi using the neighbor-joining method with 1,000 bootstrap replicates. Numbers at the nodes denote the bootstrap confidence values, and numbers in parentheses are sequence identities between Fosp9 and other homologs.

FIG 2

Functional verification of the signal peptide of Fosp9. The Fosp9 signal peptide fragment was fused in frame to the invertase gene in the pSUC2 vector, resulting in the vector pSUC2-Fosp9. pSUC2-Fosp9, pSUC2-Six1a (positive control), and pSUC2-Mg87 (negative control) were used for the yeast signal sequence trap assay. (a) Normal growth of each strain on yeast extract peptone dextrose agar (YPDA) plates. (b) CMD-W (minus Trp) plates were used to select yeast strain YTK12 carrying the pSUC2 vector. (c) YPRAA medium (containing raffinose as the only carbohydrate source) was used to indicate invertase secretion. (d) Enzymatic activity test. The dye 2,3,5-triphenyltetrazolium chloride (TTC) was reduced to the insoluble red compound triphenylformazan, indicating invertase secretion.

FIG 3

Subcellular localization of Fosp9 after transient expression in N. benthamiana leaves. (a) Schematic diagram of the construct 35S:Fosp9-GFP. (b) Subcellular localization of GFP alone and Fosp9-GFP were observed by confocal microscopy at 36 h after Agrobacterium-mediated transformation of N. benthamiana, and merged GFP and bright-field images are shown. The N. benthamiana cells were plasmolyzed in 0.8 M mannitol solution. Cytoplasm (C) and nucleus (N) are indicated with white and red arrows, respectively, and the apoplasts (A) of plant cells are indicated with red arrowheads. Bars = 50 μm.

FIG 4

Characterization and vegetative growth of fosp9 disruptant and fosp9-complemented strains. (a) Southern blot of fosp9 disruptant strains (sp9-1 and sp9-4). The hygromycin resistance cassette (HPH) fragments were used as the probes. (b) Colony, mycelial, and conidial morphology of mutant and wild-type strains. Bars = 20 μm.

FIG 5

Disease symptoms and disease severity indices (DSI) of banana plantlets inoculated with wild-type and mutant strains. (a) Internal discoloration in rhizome tissues of banana plantlets inoculated with wild-type and mutant strains (red arrows). The wild-type strain B2, the fosp9 disruptants sp9-1 and sp9-4, the fosp9-complemented strain c-sp9, and the fosp9-complemented strain sp9(C80A, C151A) with mutations in two cysteine residues were inoculated into banana plantlets (Musa sp. AAA, cv. Brazilian) for 45 days. Inoculation with sterile water was used as a control (Mock). (b) DSI of banana plants inoculated with wild-type and mutant strains. The disease severity was recorded using a scale ranging from 0 (healthy plant) to 5 (dead plant). Values are means for 90 banana plants tested for each fungal strain in three independent experiments. *, P < 0.05 (Tukey test) relative to the WT.

FIG 6

GO and KEGG pathway enrichment analyses of differentially expressed genes (DEGs) in the fosp9 disruptant relative to wild-type strain. (a) GO functional annotation of DEGs. The black and gray histograms represent the numbers of DEGs enriched in cellular components and molecular function, respectively. The y axis shows the GO terms, and the x axis shows the numbers of DEGs enriched in the corresponding terms. (b) KEGG pathway enrichment analysis of DEGs. The x axis shows the number of DEGs enriched in the corresponding pathways, and the y axis shows the KEGG pathways.

FIG 7

Heat map showing the differences in gene expression levels (log₂ FPKM) of some genes in the fosp9 disruptant strain sp9-4 relative to the wild-type strain B2. Color from red to blue indicates high to low expression and log₂ FPKM value.