VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection

Abstract

In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection.

Fig 1. Identification of the VdNUC-2 gene in mutants and complementation strains.

A, In mutant 6C4, the exon of VdNUC-2 is truncated by a T-DNA insertion. C, T-DNA insertional copy number was confirmed by Southern blot in 6C4. Genomic DNA was digested by HindIII and BamHI. B, Schematic of targeted deletion in mutants VdNUC-2Δ5 and VdNUC-2Δ14. D, Copy number of T-DNA insertion was also confirmed by Southern blot in the two deletion mutants. Their genomic DNA was digested by EcoRI and HindIII. E, The expression levels of VdNUC-2 in all test strains were analyzed by semi-quantitative PCR. VdNUC-2 3′ and VdNUC-2 5′ indicate the primer pairs bound at the 3′ and 5′ ends, respectively, of VdNUC-2 cDNA. β-tubulin was used as an internal reference gene. V07DF2 indicates the wild-type strain; VdNUC-2C1, VdNUC-2C6, and VdNUC-2C7 denote the three ectopic complementation strains.

Fig 2. Deduced amino acid sequence of VdNUC-2 and phylogenetic tree of VdNUC-2 with homologs from other fungal species.

A, Deduced amino acid sequence of VdNUC-2. The underlined N-terminus sequences indicate the SPX domain. The middle sequences marked by wavy lines represent the five putative ankyrin repeats. The C-terminus indicated by a dotted line shows the catalytic domain of the PI-PLCc_GDPD_SF superfamily. B, Phylogenetic relationships of VdNUC-2 with homologs from other fungal species. VdNUC-2: V. dahliae V07DF2; Vd-NUC-2: V. dahliae VdLs.17 (VDAG_00896T0); Va-NUC-2: V. alfalfae VaMs.102 (VDBG_03666T0); Ch-NUC-2: Colletotrichum higginsianum (CCF40876); Fo-NUC-2: Fusarium oxysporum f. sp. Melonis 26406 (EXK46788); Nc-NUC-2: Neurospora crassa (AAB03277); Mo-NUC-2: Magnaporthe oryzae 70–15 (XP_003709322); Bc-NUC-2: Botrytis cinerea BcDW1 (EMR88825); Sc-PHO81: Saccharomyces cerevisiae S288c (NP_011749).

Fig 3. Mutation of VdNUC-2 resulting in altered colony morphology in Pi-deficient Czapek–Dox media.

A, Colony morphologies of wild-type V. dahliae (V07DF2), T-DNA insertion mutant (6C4), two targeted deletion mutants (VdNUC-2Δ5 and VdNUC-2Δ14), and two complementation strains (VdNUC-2C6 and VdNUC-2C7) after 14 days of incubation on normal Pi (Con _Pi = 5.7 mM), low Pi (Con _Pi = 57 μM), and Pi-free Czapek–Dox (Con _Pi = 0 mM) media. B, C, and D, Colony diameters measured at indicated days after inoculation on culture media containing different Pi contents. Error bars represent standard deviation (n = 4). E, Reduced aerial hyphae growth in VdNUC-2 mutants under low Pi culture conditions. The pictures were obtained after 14 days of incubation. Bar = 1 cm.

Fig 4. Reduced conidia production in VdNUC-2 mutants cultured in low Pi Czapek–Dox liquid media.

Cz and low Pi indicate the normal (Con _Pi = 5.7 mM) and low Pi Czapek–Dox liquid media (Con _Pi = 57 μM), respectively. The data were collected two weeks after incubation. Error bars represent the standard deviation of three biological replicates.

Fig 5. Expression levels of the putative Pi transporter genes in the VdNUC-2 mutants and complementation strains.

Quantitative real-time PCR was employed to measure gene expression levels in the wild-type strain (V07), T-DNA insertion mutant (6C4), two targeted deletion mutants (VdNUC-2Δ5 and VdNUC-2Δ14), and two ectopic complementation strains (VdNUC-2C6 and VdNUC-2C7). A, Expression of three putative Pi transporter genes in the tested strains cultured on normal Czapek–Dox plates (Con _Pi = 5.7 mM). V07-VDAG_03222: inorganic phosphate transporter gene in V07DF2 corresponding to VdLs.17 homolog VDAG_03222; V07-VDAG_07583: phosphate-repressible phosphate permease gene in V07DF2 corresponding to VdLs.17 homolog VDAG_07583; V07-VDAG_03800: phosphate-repressible phosphate permease gene in V07DF2 corresponding to VdLs.17 homolog VDAG_03800. B, Expression levels of the three genes in the tested strains cultured on low Pi Czapek–Dox plates (Con _Pi = 57 μM).

Fig 6. Virulence and infection assessment of the VdNUC-2 mutants and complementation strains.

A, Cotton seedlings infection assays. The mock (water treatment) and V07DF2 (wild-type strain) were considered as negative and positive controls, respectively. The 6C4 root dip indicated that the seedlings were uprooted carefully and then dipped in a conidial suspension of 6C4 (1 × 10⁷ conidia/ml). The seedlings were then replanted in fresh culture soil. Other inoculations (6C4, VdNUC-2Δ5, VdNUC-2Δ14, VdNUC-2C6, and VdNUC-2C7) were performed by root irrigation around the plant stem base using the conidial suspension of each strain (1 × 10⁷ conidia/ml). B, Assessment of disease development by relative disease severity index as described by Deng et al. [25] (n ≥ 36). C, Fungal regrowth examination. Image of fungal hyphae growing on cotton seedlings stem pieces infected by the wild-type strain and VdNUC-2 mutant 6C4. D, Tobacco (Nicotiana benthamiana) seedlings infection assays. All treatments were performed by root irrigation with conidial suspension (1 × 10⁷ conidia/ml). The relative disease severity indices are presented next to each treatment name with subscript characters.

Fig 7. Pathogenicity assays on cotton seedlings planted in nutrient solution.

Normal Pi refers to the ordinary Hoagland hydroponic nutrient solution (Con _Pi = 1 mM), whereas high Pi indicates the modified Hoagland nutrient solution with high Pi content (Con _Pi = 6 mM). The seedlings were separately treated by V07 (wild-type strain V07DF2) and 6C4 (T-DNA insertion mutant). A, Disease grades of inoculated plants under different treatments (percentage stacked column chart, P < 0.01, Fisher’s exact test). B, The relative disease severity index corresponds to each treatment in A. The values for the relative disease severity indices were calculated for each treatment, and the error bars indicate the standard deviations calculated from 6 to 12 replicates. Three to eight seedlings were included in each replicate.

Fig 8. Identification of VdNUC-1 targeted deletion mutants.

A, Detection of the VdNUC-1 gene in the genome of four deletion mutants by PCR. Top panel: analysis of targeted gene VdNUC-1; bottom panel: detection of hygromycin B resistance gene (HYG ^R). VdNUC-1Δ16, Δ25, Δ26, Δ27, and V07DF2 indicate the four deletion mutants and the wild-type strain, respectively. B, Expression levels of VdNUC-1 in all the test strains as analyzed by semi-quantitative PCR. C, Wild-type strain V07DF2, VdNUC-2 mutant 6C4, and the four VdNUC-1 deletion mutants grown on low Pi Czapek–Dox solid media (Con _Pi = 57 μM). D, Virulence assay of VdNUC-1 deletion mutants. The wild-type strain and VdNUC-2 mutant 6C4 were set as the controls. Three biological repetitions were conducted, and at least 13 seedlings were treated in each repetition. Pictures were obtained seven weeks after inoculation. The relative disease severity index is presented next to each treatment name with subscript characters.

Fig 9. Severe inhibition of microsclerotia formation by VdNUC-2 mutation in V. dahliae under Pi-deficient culture conditions.

Bp2, the other wild-type strain of V. dahliae; Bp2-VdNUC-2Δ10, Δ20, and Δ24 refer to the three VdNUC-2 targeted deletion mutants in Bp2. All of these mutants were cultured on normal (Con _Pi = 5.7 mM) and Pi-deficient Czapek–Dox media plates (Con _Pi = 57 μM). To investigate strains’ microsclerotial development, 5 μL of spore suspension (1 × 10⁷ spores/mL) of each strain was dripped onto the center of the plates. Pictures were taken after two weeks of incubation. Bar = 1 cm.

Fig 10. Analysis of cellular localization of the VdNUC-2–eGFP fusion protein.

A, Schematic of the overexpression module of VdNUC-2–eGFP in Ti vector. Pro-β-tubulin indicates the promoter of the β-tubulin gene. B, Cellular localization of VdNUC-2–eGFP examined by confocal fluorescent microscopy (Carl Zeiss LSM710). The white arrows indicate the cell nuclei.

Fig 11. Putative schematic of the signaling pathways controlled by VdNUC-2 in V. dahliae.

In V. dahliae, VdNUC-2, VdPREG, VdPGOV, and VdNUC-1 correspond to homologs PHO81, PHO80, PHO85, and PHO4, respectively, in S. cerevisiae. UmCDK5 is the homolog to VdPGOV and plays an important role in the pathogenicity of Ustilago maydis. Dotted lines indicate probable or unidentified interactions or pathways. The putative interacting partners are shown in gray.