The bZIP Transcription Factor LtAP1 Modulates Oxidative Stress Tolerance and Virulence in the Peach Gummosis Fungus Lasiodiplodia theobromae

Abstract

Lasiodiplodia theobromae is one of the primary causal agents in peach gummosis disease, leading to enormous losses in peach production. In our previous study, a redox-related gene, LtAP1, from the fungus was significantly upregulated in peach shoots throughout infection. Here, we characterized LtAP1, a basic leucine zipper transcription factor, during peach gummosis progression using the CRISPR-Cas9 system and homologous recombination. The results showed that LtAP1-deletion mutant had slower vegetative growth and increased sensitivity to several oxidative and nitrosative stress agents. LtAP1 was highly induced by exogenous oxidants treatment in the L. theobromae wild-type strain. In a pathogenicity test, the deletion mutant showed decreased virulence (reduced size of necrotic lesions, less gum release, and decreased pathogen biomass) on infected peach shoots compared to the wild-type strain. The mutant showed severely reduced transcription levels of genes related to glutaredoxin and thioredoxin in L. theobroame under oxidative stress or during infection, indicating an attenuated capacity for reactive oxygen species (ROS) detoxification. When shoots were treated with an NADPH oxidase inhibitor, the pathogenicity of the mutant was partially restored. Moreover, ROS production and plant defense response were strongly activated in peach shoots infected by the mutant. These results highlight the crucial role of LtAP1 in the oxidative stress response, and further that it acts as an important virulence factor through modulating the fungal ROS-detoxification system and the plant defense response.

Keywords: AP1 transcription factor; Lasiodiplodia theobromae; fungal virulence; oxidative stress response; peach gummosis disease; plant defense response.

Figure 1

Effect of LtAP1 deletion on the mycelial growth of Lasiodiplodia theobromae. Morphological visualization of fungal colony (A), aerial hyphae growth (B), and diameter quantification (C) of the colony growth of WT (wild type), two ΔLtap1 deletion mutants (ΔLtap1-8 and -10), and the ΔLtap1/AP1 complementary strain after 36h at 28°C in darkness. Different letters on top of bars represent a statistically significant differences at p<0.05. Bars show mean growth averaged across three biological replicates, and error bars represent standard deviation.

Figure 2

Mycelial growth of the L. theobromae WT and mutants in response to stress treatments. (A) Cultures of the WT, two ΔLtap1 deletion mutants and the ΔLtap1/AP1 strain, grown on PDA media supplemented with 2.5mgml⁻¹ Congo red, 1M KCl, 0.02% SDS, 1M sorbitol, 1M glucose, and 0.05mgml⁻¹ calcofluor white (CFW) or water (mock) at indicated concentrations after 36h. (B) Percent growth inhibition of WT and mutants on PDA with the inhibitors. Different letters on top of bars represent a statistically significant differences at p<0.05. Bars show mean inhibition of growth rate averaged across three biological replicates, and error bars represent standard deviation.

Figure 3

Defects of LtAP1 on the response of L. theobromae to oxidative stress. (A) Mycelial growth of WT, two deletion strains and the complementary strain ΔLtap1/AP1, cultured on PDA media amended with oxidants 2.5mm H₂O₂, 0.68mm cumene H₂O₂, 0.5mm tert-butyl-hydroperoxide (TBHP), and 0.1mm menadione or water (mock) at the indicated concentrations after 36h. (B) Inhibition rate of fungal growth on PDA with oxidants compared with PDA without stress exposure. Different letters on top of bars represent a statistically significant differences at p<0.05. Bars show mean inhibition of growth rate averaged across three biological replicates, and error bars represent standard deviation. (C,D) Time-course response of LtAP1 transcripts to H₂O₂ or TBHP exposure. Transcript levels were normalized with reference gene LtTUB and are displayed relative to the transcript level in samples at time zero (which was therefore set to one). Asterisks indicate the significant difference relative to the initial point (0min) at p<0.01. Values are means ± SD of three biological and three technical replicates.

Figure 4

Involvement of LtAP1 of L. theobromae in response to nitro-oxidative stress. (A) Fungal growth of WT and the deletion mutant strain ΔLtap1 cultured on PDA stressed with 1mm H₂O₂ and/or 5mm Sodium nitroferricyanide dihydrate (SNP), or water (mock) at the indicated concentration after 36h. (B) Inhibition rate of fungal growth on PDA with stress treatment in relation to the mock. Values are means ± SD of three biological replicates. Different letters on top of paired bars represent a statistically significant difference at p<0.05.

Figure 5

Virulence test of the LtAP1 mutants into peach shoots. (A) Peach gummosis progression in the detached shoots inoculated with different genotypes of L. theobromae (WT, two deletion mutants, and the complementary strain ΔLtap1/AP1) at 5 dpi. Bar represents 5mm. (B) Quantification of lesion size on inoculated peach shoots. (C) Quantitative real-time PCR (qRT-PCR) analysis of L. theobromae amounts in the infected peach shoots. In panels, different letters on top of bars indicate statistically significant differences at p<0.05.

Figure 6

Effect of LtAP1 deficiency on ROS generation and its related genes transcripts in infected peach shoots. (A,B): Accumulation of superoxide anion and hydrogen peroxide in peach shoots inoculated with L. theobromae WT or ΔLtap1 mutant at 5 dpi. (C, D): Transcript abundance of ROS production-related genes PpRBOHD and PpRBOHF in infected peach shoots at 5 dpi. Relative transcript levels of genes compared with that of the control using reference gene PpTEF2 for normalization. All data are means ± SD of three biological replicates. Asterisks indicate the significant difference between two genotypes for the same parameter comparison at p<0.01.

Figure 7

Impact of diphenylene iodonium (DPI) on pathogenicity of LtAP1 mutants. (A): Symptom of different genotypes of L. theobroame-inoculated peach shoots was treated with 0.4μm DPI [dissolved in dimethyl sulfoxide (DMSO) and then diluted with water; an NADPH oxidase inhibitor] or not (mock, 0.04% DMSO) at 5 dpi. Bar represents 5mm. (B) Quantification of lesion size in infected shoots. All data are means ± SD of three biological replicates. Different letters indicate the significant difference at p<0.05.

Figure 8

qRT-PCR analysis of the glutaredoxin and thioredoxin systems genes in the WT, LtAP1 deletion mutant under H₂O₂ treatment and in infected peach shoots. Mycelial samples of WT and the deletion mutant ΔLtap1 treated with water (A) and 2.5mm H₂O₂ (B) were collected after 1h culture at 28°C in darkness. (C) RNA samples were collected from the border of L. theobromae-colonized peach shoots at 5 dpi. The transcript levels were normalized with LtTUB and are displayed in relation to the transcript levels in the corresponding WT samples (which was therefore set to one). The values are means ± SD of three biological replicates. Asterisks indicate significant differences for genes between two genotypes, with ^*p<0.01 and ^**p<0.05.

Figure 9

Impact of LtAP1 deficiency on the transcripts of defense-responsive genes in infected peach shoots. RNA samples were collected from peach shoots inoculated with L. theobromae WT or ΔLtap1 mutant at 5 dpi. (A–F): The relative expression of pathogenesis-related (PR) genes, including PpPR1a, PpPR8, PpPR10-1, PpPR10-4, PpDFN1, and PpLTP1. (G–I): Expression pattern of SA biosynthetic (PpPAL1 and PpICS1) and signaling (PpNPR1) genes. Relative transcript levels of genes compared with that of the control using reference gene PpTEF2 for normalization. Values are means ± SD of three biological replicates. Asterisks indicate a significant difference between two genotypes for genes at p<0.01.