The FomYjeF Protein Influences the Sporulation and Virulence of Fusarium oxysporum f. sp. momordicae

Abstract

Fusarium oxysporum causes vascular wilt in more than 100 plant species, resulting in massive economic losses. A deep understanding of the mechanisms of pathogenicity and symptom induction by this fungus is necessary to control crop wilt. The YjeF protein has been proven to function in cellular metabolism damage-repair in Escherichia coli and to play an important role in Edc3 (enhancer of the mRNA decapping 3) function in Candida albicans, but no studies have been reported on related functions in plant pathogenic fungi. In this work, we report how the FomYjeF gene in F. oxysporum f. sp. momordicae contributes to conidia production and virulence. The deletion of the FomYjeF gene displayed a highly improved capacity for macroconidia production, and it was shown to be involved in carbendazim's associated stress pathway. Meanwhile, this gene caused a significant increase in virulence in bitter gourd plants with a higher disease severity index and enhanced the accumulation of glutathione peroxidase and the ability to degrade hydrogen peroxide in F. oxysporum. These findings reveal that FomYjeF affects virulence by influencing the amount of spore formation and the ROS (reactive oxygen species) pathway of F. oxysporum f. sp. momordicae. Taken together, our study shows that the FomYjeF gene affects sporulation, mycelial growth, pathogenicity, and ROS accumulation in F. oxysporum. The results of this study provide a novel insight into the function of FomYjeF participation in the pathogenicity of F. oxysporum f. sp. momordicae.

Keywords: Fusarium oxysporum; YjeF protein; sporulation; virulence.

Figure 1

Sequence analysis of FomYjeF. (A) The phylogenetic tree was constructed based on amino acid sequences of FomYjeF and other fungi and bacteria using the neighbor-joining method with 1000 bootstrap replicates. The numbers at the nodes of the phylograms denote the bootstrap confidence values. (B) Phylogenetic analysis of conserved domains was structured based on the fungi and bacteria of the phylogenetic tree. (C) The everyday expression level of FomYjeF was determined by RT-PCR. Actin was a reference gene. D1–D5 represented the 5 day transcription level of FomYjeF in SD-1. The FomYjeF-IN-F/R and Actin-F/R primer sets were used.

Figure 2

Vegetative growth, mycelial growth, and conidia production of wild-type SD-1, FomYjeF deletion, and FomYjeF-complemented strains. (A) Wild-type SD-1, FomYjeF-KO-2, and FomYjeF-CO-2 were cultured on PDA plates at 28 °C in dark for 5 days. (B) Growth rates. (C) Hyphae of wild-type SD-1 and mutant strains observed by EM. Bars:20µm. (D) Macroconidia production of wild-type SD-1 and all mutants were counted in per square centimeter after culturing on PDA for 4 days (See Figure S2 for the schematic diagram of sampling points). * over the histogram indicates standard error of the mean (SEM) at p < 0.05 (two-way ANOVA test). ns indicates no significant difference. Error bars represent the SEM of three biological replicates with three technical replicates.

Figure 3

The abiotic stress and carbendazim response of mycelial growth in wild-type SD-1, FomYjeF deletion, and FomYjeF-complemented strains. (A) Colonies of SD-1, FomYjeF-KO-2, and FomYjeF-CO-2 were cultured on a PDA medium amended with 0.05% SDS, 0.05% Congo red, and 1.5 M sorbitol. (B) Colonies of SD-1, FomYjeF-KO-2, and FomYjeF-CO-2 were cultured on a PDA medium amended with 0.7 µg/mL, 0.9 µg/mL, and 1.1 µg/mL carbendazim. (C) The inhibition rate of mycelial growth of carbendazim. All Petri dishes were incubated at 28 °C for 5 days. * over the histogram indicates standard error of the mean (SEM) at p < 0.05 (two-way ANOVA test). ns indicates no significant difference. Error bars represent the SEM of three biological replicates with three technical replicates.

Figure 4

FomYjeF regulated the pathogenicity of F. oxysporum f. sp. momordicae. (A) The wild-type strain SD-1, FomYjeF-KO-2, and FomYjeF-CO-2 were inoculated to bitter gourd plantlets at the two-leaf stage for 28 days. As the control, MOCK was inoculated with sterile water. (B) Yellow symptoms in the leaf veins of bitter gourd. (C) Disease severity index (DSI) of bitter gourd plants inoculated with SD-1 and mutant strains. The disease severity was recorded using a scale ranging from 0 to 5, with 0 for healthy plants and 5 for dead plants.

Figure 5

The deletion of FomYjeF affected the ROS accumulation and sensibility to oxidative stress of Fusarium oxysporum f. sp. momordicae. (A) The wild-type strain SD-1, FomYjeF-KO-2, and FomYjeF-CO-2 were inoculated to bitter gourd leaves of similar size and physiological state in the same part. MOCK leaf was inoculated with PDA. The red circle indicates the site of the disease and observation part. Bars: 1 cm. (B) Glutathione peroxidase activity in SD-1 and FomYjeF-KO-2. * over the histogram indicates the standard error of the mean (SEM) at p < 0.05 (t-test). Ns indicates no significant difference. Error bars represent the SEM of three biological replicates with three technical replicates.

Figure 6

The transcription levels of Type II myosin, Wor1-like, NoxR, and NoxA in the wild-type SD-1 and FomYjeF-KO-2 according to q-PCR using the Type II myosin-F/R, Wor1-like -F/R, NoxR-F/R, and NoxA-F/R primer sets. Error bars represent the SEM of three biological replicates with three technical replicates.