Genome-Wide Identification and Functional Analysis of the bZIP Transcription Factor Family in Rice Bakanae Disease Pathogen, Fusarium fujikuroi

Abstract

Fungal basic leucine zipper (bZIP) proteins play a vital role in biological processes such as growth, biotic/abiotic stress responses, nutrient utilization, and invasion. In this study, genome-wide identification of bZIP genes in the fungus Fusarium fujikuroi, the pathogen of bakanae disease, was carried out. Forty-four genes encoding bZIP transcription factors (TFs) from the genome of F. fujikuroi (FfbZIP) were identified and functionally characterized. Structures, domains, and phylogenetic relationships of the sequences were analyzed by bioinformatic approaches. Based on the phylogenetic relationships with the FfbZIP proteins of eight other fungi, the bZIP genes can be divided into six groups (A-F). The additional conserved motifs have been identified and their possible functions were predicted. To analyze functions of the bZIP genes, 11 FfbZIPs were selected according to different motifs they contained and were knocked out by genetic recombination. Results of the characteristic studies revealed that these FfbZIPs were involved in oxygen stress, osmotic stress, cell wall selection pressure, cellulose utilization, cell wall penetration, and pathogenicity. In conclusion, this study enhanced understandings of the evolution and regulatory mechanism of the FfbZIPs in fungal growth, abiotic/biotic stress resistance, and pathogenicity, which could be the reference for other fungal bZIP studies.

Keywords: Fusarium fujikuroi; bZIP TF; bakanae disease; functional analysis; phylogenetic analysis.

Figure 1

Schematic diagram of the conserved domains of FfbZIP proteins.

Figure 2

Sequence alignment of FfbZIP domains.

Figure 3

Intron and exon distribution patterns in the coding sequence of FfbZIP genes.

Figure 4

Fungi morphology observation. (A) Colony morphology of FfbZIP knockout mutant strain. (B) Growth rate of FfbZIP knockout mutants versus wild type. Error bars represent means ± SEs. Some error bars were not plotted because the error bar would be shorter than the size of the symbol.

Figure 5

Oxidative stress experiment. (A) Growth of wild type and FfbZIP knockout mutants under exogenous oxygen stress. (B) Histogram of strain diameter under 0.1% H₂O₂ oxidative stress. Error bars represent means ± SEs. (C) Histogram of strain diameter under 0.25% H₂O₂ oxidative stress. Error bars represent means ± SEs. * p < 0.05, ** p < 0.01.

Figure 6

Osmotic stress experiment. (A) Growth of wild type and FfbZIP knockout mutants under NaCl osmotic stress. (B) Histogram of strain diameter under 1 mol/L NaCl osmotic pressure. Error bars represent means ± SEs. (C) Histogram of Histogram of strain diameter under 2 mol/L NaCl osmotic pressure. Error bars represent means ± SEs. * p < 0.05.

Figure 7

Cell wall selection pressure experiment. (A) Growth of wild type and FfbZIP knockout mutants under cell wall selection pressure. (B) Histogram of strain diameter under 1 mol/L E420 cell wall selection pressure. Error bars represent means ± SEs. (C) Histogram of strain diameter under 2 mol/L E420 cell wall selection pressure. Error bars represent means ± SEs, * p < 0.05, ** p < 0.01.

Figure 8

Cellulose utilization experiment. (A) Growth of wild type and FfbZIP knockout mutants in Cellulose Congo red medium. (B) Histogram of strain diameter under cellulose Congo red culture conditions. Error bars represent means ± SEs. * p < 0.05, ** p < 0.01.

Figure 8

Cellulose utilization experiment. (A) Growth of wild type and FfbZIP knockout mutants in Cellulose Congo red medium. (B) Histogram of strain diameter under cellulose Congo red culture conditions. Error bars represent means ± SEs. * p < 0.05, ** p < 0.01.

Figure 9

Determination of cellophane penetration ability of wild type and FfbZIP knockout mutants.

Figure 10

Pathogenicity observation of inoculated rice seedlings. (A) Pathogenicity alignment of FfbZIP knockout mutants and wild type. (B) Shoot length determination of rice seedlings inoculated with wild type and FfbZIP deletion mutants. CK stood for the shoot length of uninoculated rice seedlings, ** p < 0.01.