Fig 1.
Reference maps from 2-DE analysis of conidial proteins from F. oxysporum f. sp conglutinans.
Proteins were separated by using 24 cm, pH 4–7 IPG strips for IEF and followed by 12.5% SDS-PAGE. The gels were stained by GAP method, which used CBB-G250, ammonium sulfate, and phosphoric acid. A: Gel of conidia proteins from Foc grown in PDB. B: The four proteins (spots 1, 2, 3 and 4), named Foc-SIX1 with high abundance in Foc, were indicated by arrows and numbers and listed in Table 1; Figure B is the partial enlarged details of the rectangular box in Figure A.
Fig 2.
Phylogenetic analysis of Foc-SIX1 by using neighbor-joining method.
The tree was generated based on alignments of full amino acid sequences of homologs using MEGA 6 with 1000 bootstraps.
Table 1.
Identification of the four proteins indicated in Fig 1 from the conidia of Foc.
Fig 3.
The expression of SIX1 gene in mycelia, conidia and infected cabbage roots at 7 dpi.
Mycelia and conidia were obtained from Foc grown in PDB for 48 h. Gene expression levels were relative to the internal controls EF-1α and β-tubulin. Averages of four biological replicates were shown with the standard error.
Fig 4.
Construction and verification of deletion mutants Foc-ΔSIX1.
A: Foc-SIX1 gene deletion and replacement with an intact selectable marker gene (hph) by homologous recombination. B: Confirmation of the correct deletion by PCR. Numbers 1, 2, 3 and 4 represent the four primer pairs that specify the SIX1 gene, hph gene, and the correct upstream and downstream homologous recombination, respectively. C: Southern hybridization analysis of wild type isolate and mutants Foc-ΔSIX1. The genomic DNAs from the wild type Foc isolate and deletion mutants Foc-ΔSIX1 were digested with Sal I. A fragment amplified from upstream of the target gene was used as probe.
Fig 5.
Functional analysis of the SIX1 gene in Foc.
A: Disease symptoms of cabbage seedlings inoculated with wild type isolate Foc and their mutants Foc-ΔSIX1 were shown at 8 dpi, 10 dpi and 11 dpi. B: Disease index of cabbage seedlings inoculated with wild type isolate Foc and mutants Foc-ΔSIX1 from 8 to 13 days after inoculation. The average of three replicates was shown with standard error of the mean. Each value in the figure was an average of three independent biological replicates. Each replicate included more than 30 seedlings for each isolate.
Fig 6.
Construction, verification, and virulence analysis of complementation mutants Foc-ΔSIX1::Foc-SIX1.
A: Foc-ΔSIX1 mutants were complemented by transformation with approximately 3.0 kb of Foc-SIX1 spanning from -1838 bp (promoter relative to start codon) to 1132 bp (ORF and 3’ UTR). B: The SIX1 gene, hph gene and Neo gene fragments were amplified by PCR to confirm the correct complementation mutants. C: Disease symptoms of cabbage seedlings inoculated with wild type isolate Foc, deletion mutants Foc-ΔSIX1, and complementation mutants Foc-ΔSIX1::Foc-SIX1 were shown at 14 dpi. Similar results were obtained in three independent biological replicates.
Fig 7.
Construction, verification, and virulence analysis of transformants Foc-ΔSIX1::Fol-SIX1.
A: Foc-ΔSIX1 mutants were complemented by transformation with approximately 2.0 kb of Fol-SIX1 spanning from -832 bp (promoter relative to start codon) to 1091 bp (ORF and 3’ UTR). B: The Fol-SIX1 gene and Neo gene fragments were amplified by PCR to confirm the correct complementation mutants. C: Disease symptoms of cabbage seedlings inoculated with wild type isolate Foc, deletion mutants Foc-ΔSIX1 (D1 and D2), complementation mutants (C2 and C3) and transformants Foc-ΔSIX1::Fol-SIX1 (T1 and T2) were shown at 18 dpi. Similar results were obtained in three independent biological replicates.