Ero1‐Pdi1 module‐catalysed dimerization of a nucleotide sugar transporter, FonNst2, regulates virulence of Fusarium oxysporum on watermelon

Abstract

Fusarium oxysporum f. sp. niveum (Fon) is a soilborne fungus causing vascular Fusarium wilt on watermelon; however, the molecular network regulating Fon virulence remains to be elucidated. Here, we report the function and mechanism of nucleotide sugar transporters (Nsts) in Fon. Fon genome harbours nine FonNst genes with distinct functions in vegetative growth, asexual production, cell wall stress response and virulence. FonNst2 and FonNst3 are required for full virulence of Fon on watermelon and FonNst2 is mainly involved in fungal colonization of the plant tissues. FonNst2 and FonNst3 form homo-or hetero-dimers but function independently in Fon virulence. FonNst2, which has UDP-galactose transporter activity in yeast, interacts with FonEro1 and FonPdi1, both of which are required for full virulence of Fon. FonNst2, FonPdi1 and FonEro1 target to endoplasmic reticulum (ER) and are essential for ER homeostasis and function. FonEro1-FonPdi1 module catalyses the dimerization of FonNst2, which is critical for Fon virulence. Undimerized FonNst2 is unstable and degraded via ER-associated protein degradation in vivo. These data demonstrate that FonEro1-FonPdi1 module-catalysed dimerization of FonNst2 is critical for Fon virulence on watermelon and provide new insights into the regulation of virulence in plant fungal pathogens via disulfide bond formation of key pathogenicity factors.

The SUMOylation Pathway Components Are Required for Vegetative Growth, Asexual Development, Cytotoxic Responses, and Programmed Cell Death Events in Fusarium oxysporum f. sp. niveum

“…The SUMOylation pathway is critical for stress responses in fungi, including M. oryzae [ 8 , 44 ]. To test whether the SUMOylation pathway has a role in the maintenance of Fon cell wall integrity, we analyzed the effect of three cell wall-perturbing agents, including CR, CFW, and NaCl [ 33 ], on the mycelial growth of the WT, deletion mutants, and complementation strains. When grown on PDA supplemented with 0.2 mg/mL CR, 0.1 mg/mL CFW or 0.5 M NaCl, mycelial growth of the deletion mutants was significantly inhibited compared to WT ( Figure 5 A–D).…”

“…To generate the gene replacement constructs, the 5′ and 3′ flanking sequences of the SUMOylation pathway genes were amplified from Fon genomic DNA and fused with the 1349 bp hygromycin gene using the double-joint PCR method. The obtained gene knockout fragments were directly transformed into WT protoplasts [ 32 , 33 ]. The resulting transformants were screened with 100 µg/mL hygromycin B, and were further validated by polymerase chain reaction (PCR), reverse transcriptase (RT)-quantitative PCR, and Southern blotting assays.…”

“…The Fon strains, including WT, deletion mutants, and complementation strains, were grown on PDA or MM supplemented with/without various stress-inducing compounds, and colony sizes were measured [ 33 ]. For the conidiation assays, the strains were incubated on MM or PDA at 26 °C for 7 d, and the conidia were collected and counted as previously described [ 33 ]. The conidiation data were then normalized by dividing the conidia number by the colony area, and presented as the conidia per cm 2 .…”

“…After incubation for 6 or 12 h at 26 °C, microscopic examination of at least 100 randomly selected spores per field was carried out to determine the spore germination status. Stress sensitivity was assessed by determining the growth inhibition rate, as previously described [ 33 ]. The chemicals or enzymes used and their sources are as follows: Congo red (CR; Sigma-Aldrich, St. Louis, MO, USA), Calcofluor white (CFW; Sigma-Aldrich), Camptothecin (CPT; Sigma-Aldrich), Methyl methane sulfonate (MMS; Sigma-Aldrich), 4-Nitroquinoline (4-NQ; Sigma-Aldrich), Hydroxyurea (HU; Sigma-Aldrich), Calcium chloride (CaCl 2 ; Sigma-Aldrich), Sodium chloride (NaCl; Sinopharm Chemical, Shanghai, China), Iron chloride (FeCl 3 ; Sinopharm Chemical), Zinc chloride (ZnCl 2 ; Sinopharm Chemical), Copper chloride (CuCl 2 ; Sinopharm Chemical), Driselase (Sigma-Aldrich), Lysozyme (Shanghai Ryon BioTech, Shangha, China), and Cellulase (Shanghai Ryon BioTech).…”

Palmitoyl Transferase FonPAT2-Catalyzed Palmitoylation of the FonAP-2 Complex Is Essential for Growth, Development, Stress Response, and Virulence in Fusarium oxysporum f. sp. niveum

“…niveum ( Fon ), is one of the most devastating fungal diseases ( 43 ). It was previously found that an effector, FonSIX6, a CCR4-Not complex subunit, FonNOT2, and a nucleotide sugar transporter, FonNst2, are essential for virulence in Fon ( 44 – 46 ). The present study aimed to investigate the involvement of protein palmitoylation in Fon virulence and revealed that FonPAT1, FonPAT2, and FonPAT4 are required for virulence in Fon .…”