Vegetative hyphal fusion is not essential for plant infection by Fusarium oxysporum

Abstract

Vegetative hyphal fusion (VHF) is a ubiquitous phenomenon in filamentous fungi whose biological role is poorly understood. In Neurospora crassa, the mitogen-activated protein kinase (MAPK) Mak-2 and the WW domain protein So are required for efficient VHF. A MAPK orthologous to Mak-2, Fmk1, was previously shown to be essential for root penetration and pathogenicity of the vascular wilt fungus Fusarium oxysporum. Here we took a genetic approach to test two hypotheses, that (i) VHF and plant infection have signaling mechanisms in common and (ii) VHF is required for efficient plant infection. F. oxysporum mutants lacking either Fmk1 or Fso1, an orthologue of N. crassa So, were impaired in the fusion of vegetative hyphae and microconidial germ tubes. Deltafmk1 Deltafso1 double mutants exhibited a more severe fusion phenotype than either single mutant, indicating that the two components function in distinct pathways. Both Deltafso1 and Deltafmk1 strains were impaired in the formation of hyphal networks on the root surface, a process associated with extensive VHF. The Deltafso1 mutants exhibited slightly reduced virulence in tomato fruit infection assays but, in contrast to Deltafmk1 strains, were still able to perform functions associated with invasive growth, such as secretion of pectinolytic enzymes or penetration of cellophane sheets, and to infect tomato plants. Thus, although VHF per se is not essential for plant infection, both processes have some signaling components in common, suggesting an evolutionary relationship between the underlying cellular mechanisms.

FIG. 1.

Fmk1 and Fso1 are required for vegetative complementation of nitrate utilization deficiency in F. oxysporum. Non-nitrate-utilizing nit1 mutants were obtained from different genetic backgrounds (wild type, Δfmk1, Δfso1, and an ectopic transformant with the fso1 knockout construct) and inoculated, 1 cm from a nitM mutant, onto MM with nitrate as the sole nitrogen source. Vigorous hyphal growth in the region of contact between colonies denotes the presence of vegetative complementation through heterokaryon formation.

FIG. 2.

Vegetative fusion between hyphae and microconidial germ tubes of F. oxysporum. Hyphae of the indicated strains from subperipheral regions of colonies grown on solid MM (A) or microconidia germinated in liquid MM (B) were imaged by the Nomarski technique. Fusion events in the wild type (wt) and points of contact without fusion in the Δfso1 strain are indicated by arrows. In panel A, details of the regions marked by asterisks are shown below the general view. Bar, 10 μm.

FIG. 3.

VHF affects fungal development and conidiation. The indicated strains were grown for 35 days on potato dextrose broth in submerged culture with shaking. (A) The entire culture was transferred to a petri dish and photographed. Note the presence of large mycelial aggregates in the wild-type (wt) and Δfso1+fso1 strains. (B) Concentrations of microconidia in the culture supernatants of the different strains. Error bars indicate standard deviations from three samples. The experiment was performed twice with similar results.

FIG. 4.

(A) VHF is required for efficient adhesion and root colonization. Roots of tomato seedlings were immersed for 24 h in microconidial suspensions of the indicated strains, washed by vigorous shaking in water, and observed in a stereomicroscope. Adhering fungal mycelium is visible as a white mass covering lateral roots. wt, wild type. (B) VHF during colonization of tomato roots by F. oxysporum wild-type strain 4287. Upper left, hyphal aggregates on the root surface (indicated by arrows). Right, F. oxysporum germlings undergoing anastomosis on a tomato root surface. An example of a hyphal fusion bridge is indicated by an arrow. Lower left, detail of the fusion bridge.

FIG. 5.

Fmk1 and Fso1 contribute differentially to invasive growth. Microconidial suspensions of the indicated strains were used for the different phenotypic assays. (A) Clear halo production on polygalacturonic acid (PGA)-containing plates for visualization of extracellular polygalacturonase activity (visible as a dark halo underneath the fungal colony). (B) Penetration of cellophane sheets. Colonies were grown for 4 days on a plate with MM covered by a cellophane sheet (before), and then the cellophane with the colony was removed and the plate was incubated for 1 additional day (after). (C) Invasive growth on tomato fruits inoculated with the indicated concentrations of microconidia and incubated for 4 or 6 days at 28°C. wt, wild type.

FIG. 6.

Fso1 is not required for virulence of F. oxysporum on tomato plants. The graph shows the incidence of Fusarium wilt on tomato plants (cultivar Monica) inoculated with the indicated strains. Severity of disease symptoms was recorded at different times after inoculation, with indices ranging from 1 (healthy plant) to 5 (dead plant). Error bars represent the standard deviations calculated from 20 plants. wt, wild type.

FIG. 7.

VHF and pathogenicity have some signaling mechanisms in common. The Fmk1 MAPK cascade regulates both VHF and virulence-related functions such as adhesion and invasive growth. By contrast, Fso1 functions in a distinct pathway which is essential for VHF but contributes only marginally to virulence, indicating that VHF is not essential for pathogenicity on plants.