ChsVb, a class VII chitin synthase involved in septation, is critical for pathogenicity in Fusarium oxysporum

Abstract

A new myosin motor-like chitin synthase gene, chsVb, has been identified in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Phylogenetic analysis of the deduced amino acid sequence of the chsVb chitin synthase 2 domain (CS2) revealed that ChsVb belongs to class VII chitin synthases. The ChsVb myosin motor-like domain (MMD) is shorter than the MMD of class V chitin synthases and does not contain typical ATP-binding motifs. Targeted disrupted single (DeltachsVb) and double (DeltachsV DeltachsVb) mutants were unable to infect and colonize tomato plants or grow invasively on tomato fruit tissue. These strains were hypersensitive to compounds that interfere with fungal cell wall assembly, produced lemon-like shaped conidia, and showed swollen balloon-like structures in hyphal subapical regions, thickened walls, aberrant septa, and intrahyphal hyphae. Our results suggest that the chsVb gene is likely to function in polarized growth and confirm the critical importance of cell wall integrity in the complex infection process of this fungus.

FIG. 1.

Phylogenetic tree of 26 fungal CHS, representing seven classes of these enzymes. Sequences from the conserved CS2 amino acid domain found in all CHS were aligned using ClustalW. Numbers at the branch points represent bootstrap values based on 1,000 repeated samplings.

FIG. 2.

Organization of genes chsV and chsVb in a head-to-head configuration within Fusarium oxysporum f. sp. lycopersici genome. The MMD (striped bars) and the typical CHS CS2 domain (white bars) are indicated. The orientation of transcription is shown by arrowheads. The distance between both translational start points is 4,219 bp.

FIG. 3.

(A) Time course of chs2, chsV, and chsVb gene transcription during developmental mycelial growth of the F. oxysporum wild-type strain (2 to 24 h). (B) Expression analysis of chs2, chsV, and chsVb genes by RT-PCR during developmental stages of the mycelial growth of the ΔchsV, ΔchsVb, and ΔchsV ΔchsVb mutants for 2 to 10 h. Actin gene transcripts (act 1) were used as controls for cDNA amounts in the different samples. Genomic DNA (g) was used as a control for transcript size. Sizes of bands are in base pairs.

FIG. 4.

(A) Northern analysis of chsV and chsVb genes from F. oxysporum grown on SM with or without (w/o) 1.2 M sorbitol. Lanes: 1, wild-type strain; 2, ΔchsV; 3, ΔchsV ΔchsVb; 4, ectopic transformant; 5, ΔchsVb. (B) RT-PCR analysis of the indicated F. oxysporum chs genes. Lanes: 1, wild-type strain; 2, ΔchsV; 3, ΔchsVb; 4, ΔchsV ΔchsVb. Growth conditions were the same as those described for panel A. Size bands are in base pairs. g, genomic DNA.

FIG. 5.

(A) Phenotypes of colonies from F. oxysporum wild-type and mutant strains, showing alterations in cell wall or membrane integrity. (B) Germlings from the wild-type (wt) strain and ΔchsV, ΔchsVb, and ΔchsV ΔchsVb mutants grown on SM with or without (w/o) 1.2 M sorbitol (magnification, ×50). Arrows indicate swollen ballon-like structures. BCIP, 5-bromo-4-chloro-3-indolylphosphate; SDS, sodium dodecyl sulfate; CR, Congo red; CFW, calcofluor white.

FIG. 6.

Ultrastructural characteristics of cell walls (A) and septa (B) of the wild-type (wt) strain and the ΔchsV, ΔchsVb, and ΔchsV ΔchsVb mutants. Mutant cell walls are significantly larger than those of the wild-type strain. Arrows, plasma membrane; asterisks, skeletal layer; arrowheads, outer layers. In panel B, septa are denoted by arrows. (C) Septa from ΔchsVb and ΔchsV ΔchsVb were observed using fluorescence microscopy after staining with DAPI and CFW. Scale bars, 1 μm (A and B) and 100 μm (C).

FIG. 7.

Intrahyphal hypha-like structures detected in ΔchsVb and ΔchsV ΔchsVb mutants observed under TEM. Lower panels correspond to magnified areas boxed in the upper panels. Three layer-like structures can be observed: the plasma membrane (arrow), the outer layer (arrowhead), and the skeletal layer in between. Bars, 1 μm.

FIG. 8.

Virulence of F. oxysporum f. sp. lycopersici chs-deficient mutants on tomato plants (cv. Monika). Severity of disease symptoms was recorded at different times after inoculation by using an index ranging from 1 (healthy plant) to 5 (dead plant). Symbols refer to plants inoculated with the wild-type strain 4287 (▪), ΔchsVb (×), ΔchsV ΔchsVb (▵), the ectopic transformant (○), and the noninoculated control (⧫). Error bars indicate the standard errors from 15 plants for each treatment.

FIG. 9.

Invasive growth of F. oxysporum strains on tomato fruits at the inoculation site after 24 h of incubation at 100% relative humidity.