Inactivation of Snt2, a BAH/PHD-containing transcription factor, impairs pathogenicity and increases autophagosome abundance in Fusarium oxysporum

Abstract

The soil-borne, asexual fungus Fusarium oxysporum f.sp. melonis (FOM) is a causal agent of muskmelon wilt disease. The current study focused on the most virulent race of FOM-race 1,2. The tagged mutant D122, generated by Agrobacterium tumefaciens-mediated transformation, caused the delayed appearance of initial wilt disease symptoms, as well as a 75% reduction in pathogenicity. D122 was impaired in the gene product homologous to the Snt2-like transcription factor of Schizosaccharomyces pombe. Involvement of snt2 in the early stage of FOM pathogenesis and its requirement for host colonization were confirmed by targeted disruption followed by quantitative reverse transcription-polymerase chain reaction analysis of snt2 expression in planta. Δsnt2 mutants of FOM and Neurospora crassa exhibited similar morphological abnormalities, including a reduction in conidia production and biomass accumulation, slower vegetative growth and frequent hyphal septation. In N. crassa, snt-2 is required for sexual development, as Δsnt-2 mutants were unable to produce mature perithecia. Suppressive subtraction hybridization analysis of the D122 mutant versus wild-type isolate detected four genes (idi4, pdc, msf1, eEF1G) that were found previously in association with the target of rapamycin (TOR) kinase pathway. Expression of the autophagy-related idi4 and pdc genes was found to be up-regulated in the Δsnt2 FOM mutant. In N. crassa, disruption of snt-2 also conferred a significant over-expression of idi4.

Figure 1

Disruption of snt2 impairs the pathogenicity of Fusarium oxysporum f.sp. melonis (FOM) on muskmelon plants. One‐week‐old muskmelon seedlings were inoculated by dipping the roots in a conidial suspension (5 × 10⁵ conidia/mL) of FOM and plants were assessed for symptoms of vascular wilt disease over a 3‐week period. Means and standard errors were calculated from three independent experiments and compared using the Tukey–Kramer test (P < 0.05). Treatments: Δsnt2, targeted mutant; D122, tagged mutant; Ect.1, ectopic transformant; WT, wild‐type; Water, control.

Figure 2

SNT2 protein structure of Fusarium oxysporum f.sp. melonis (FOM). (A) The FOM Snt2 protein carries five conserved domains: a bromo‐adjacent domain (BAH) at amino acid position 205–324; three plant homeodomain Zn fingers (PHD) at positions 362–410, 752–793 and 1074–1144; and a GATA‐type Zn finger at position 925–971. (B) Comparison of the structural domains of SNT2 proteins between different fungi (BAH, rectangle; PHD, circle; GATA Zn finger, pentagon; SANT, hexagon). Sequences shown: SNT2 (YGL131C), Schizosaccharomyces pombe (SP); Snt2, Fusarium oxysporum f.sp. melonis (FOM); FOXG_01993.2, Fusarium oxysporum f.sp. lycopersici (FOL); XP_387009, Fusarium graminearum (FG); XP_001908700, Podospora anserina (PA); XP_001549253, Botrytis cinerea (BC); XP_750273, Aspergillus fumigatus (AF); XP_001584831, Sclerotinia sclerotiorum (SS); XP_716567, Candida albicans (CA); XP_761724, Ustilago maydis (UM); AAN75722 (ZNF1), Cryptococcus neoformans (CN). Similarity (percentage, in parentheses) between SNT2 of FOM and other fungi was analysed by the MatGAT program (Campanella et al., 2003).

Figure 3

Expression of snt2 during pathogenesis of Fusarium oxysporum f.sp. melonis on muskmelon plants. cDNA was synthesized from 0.5 µg of mRNA isolated from infected plants. All samples were analysed in triplicate. Transcript levels from infected plants at 2‐days post‐inoculation were used as a reference. Averaged crossing point values were normalized to the endogenous control gene β‐tubulin. Expression levels of snt2 with standard error of two biological and three technical repeats were evaluated using the REST© program (Pfaffl et al., 2002).

Figure 4

Targeted disruption of snt2 in Fusarium oxysporum f.sp. melonis (FOM). (A) The vector for targeted disruption was constructed by introducing a 2.13‐kb hygromycin B resistance cassette between the Acc65I (A) and BglII (B) sites of a 1.6‐kb fragment of FOM snt2, amplified with homFG1F/R primers and cloned into pGEM‐T Easy, creating a 3.8‐kb snt2::hphR cassette. The SdaI (Sd)/MunI (M) snt2::hphR cassette was transferred into the EcoRI/PstI binary vector pDHt in order to perform Agrobacterium‐mediated transformation. (B) Southern blot analysis using a 1.4‐kb fragment of snt2::hphR amplified with inHPH‐rev2/homFG1R, used as a probe (see A), was performed on Acc65I/Bsp1407I (Bs)‐digested genomic DNA, detecting a disruption of snt2 in one transformant (designated Δsnt2). Ectopic transformants (Ect.1 and Ect.2) with several insert copies and a wild‐type (WT) with an original snt2 copy are also shown. (C) Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of snt2 transcripts with the β‐tubulin gene used as a control. M denotes DNA size in kilobases.

Figure 5

snt2 disruption leads to abnormalities of hyphal septation in Fusarium oxysporum f. sp. melonis (FOM) and Neurospora crassa (NC). Congo red staining detected irregularities in cell wall deposition and shorter hyphal segments between septa in the Δsnt2 mutant of FOM and the Δsnt2 mutant of NC, compared with their respective wild‐type strains. Bars indicate 50 µm.

Figure 6

Morphological abnormalities observed in snt2 mutants of Fusarium oxysporum f.sp. melonis (FOM). Comparative analysis between Evans blue‐stained mycelial cells of the wild‐type isolate (A) and snt2 mutant strains detected decreased viability of the cells in mutants D122 (B) and Δsnt2 (C) of FOM (areas marked by circles). Monodansylcadaverine (MDC) staining of autophagosomes detected significant differences between the wild‐type (D) and the mutant isolates D122 (E) and Δsnt2 (F). Formation of autophagosomes in the presence of phenylmethanesulphonylfluoride (PMSF) and stained by MDC in the wild‐type isolate (G), D122 (H) and Δsnt2 (I) mutant isolates. Elimination of MDC staining in PMSF‐treated wild‐type cells (J) after addition of the specific autophagosome inhibitor 3‐methyladenine (K). Bars in A–C indicate 50 µm, whereas those in D–K indicate 10 µm.

Figure 7

snt2 disruption leads to increased cell death in Fusarium oxysporum f.sp. melonis (FOM) and Neurospora crassa (NC). Percentage of dead cells in the Δsnt2 mutants of FOM and NC, compared with their respective wild‐type (WT) isolates. Results represent the mean of three independent experiments (each with 1000 cells observed), analysed separately for each fungal species by Least Significant Difference (LSD) Tukey‐Kramer multiple comparison test (P≤ 0.05); the levels of significance are marked with different letters.