Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora  litchii

Abstract

Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.

Keywords: ATG6; Peronophythora litchii; autophagy-related gene; mycelial growth; oxidative stress; pathogenicity; sporangium production.

Figure 1

PlATG6a is conserved in oomycetes and up-regulated in the asexual stage of Peronophythora litchii. (A) Amino acid sequence alignment of PlATG6a and its orthologs from Phytophthora sojae (Ps), P. capsici (Pc), Hyaloperonospora parasitica (Hp), Pythium ultimum (Pu), and Saccharomyces cerevisiae (Sc). (B) Expression pattern of PlATG6a during the asexual life cycle and infection stages was analyzed by quantitative reverse transcription PCR (qRT-PCR). MY: mycelia; SP: sporangia; ZO: zoospore; CY: cyst; GC: germination of cyst; hpi: hours post inoculation. Relative expression levels were calculated using the 2^−ΔΔCT method [23] with PlActin gene as the internal control. The MY value was set as “1”. Asterisks indicate significant difference compared with MY (** p < 0.01, t-test). These experiments were repeated three times.

Figure 2

CRISPR/Cas9-mediated deletion of PlATG6a in Peronophythora litchii. (A) Schematic representation of the strategy of CRISPR/Cas9-mediated mutagenesis of PlATG6a. Two single-guide RNAs targeted the PlATG6a gene sequence (indicated by black arrows). The Δplatg6a mutants were identified by genomic PCR (B) and sequencing (C) with primers F1 and R1 (Supplementary Table S1). (D) Transcription of PlATG6a was analyzed by qRT-PCR in wild-type (WT) WT, CK and Δplatg6a mutants (T32 and T47). “***” indicates significant difference compared with WT (p < 0.001, t-test). These experiments were repeated three times. (E) Visualization of autophagosome by MDC staining. The WT and Δplatg6a mutants were incubated in carrot juice agar (CJA) medium for 3 days. After 3 washes and incubation with sterile distilled water for 4 h, hyphae samples were stained with MDC and analyzed by microscopy. Bars = 10 μm. WT: wild-type strain; CK: the transformant failed to acquire PlATG6a mutation (control).

Figure 3

Growth rates of the wild-type (WT), CK (control; the transformant failed to acquire PlATG6a mutation), and the Δplatg6a mutants (T32 and T47). (A) The photographs were taken at 5 dpi. (B) The growth rate was measured and calculated. Bar chart depicts the mean ± SD. Asterisks represent significant difference compared with WT (** p < 0.01, n = 9, t-test).

Figure 4

Knockout of PlATG6a impaired the sporangium production of Peronophythora litchii and promoted zoospore release. Δplatg6a mutants (T32 and T47) and WT were cultured on CJA medium for 5 days and sporangia were collected and used for releasing zoospores. (A) Photographs were taken after collecting sporangia from CJA medium. Scale bar = 400 μm. (B) The sporangia number was calculated. (C,D) The sporangia length and width were measured. (E,F) The zoospore release rates of Δplatg6a mutants, CK and WT were calculated at 1 and 2 h post incubation of the sporangia in water. (G) Cyst germination rate. The bar charts depict the means ± SDs. Asterisks indicate significant difference vs. WT (* p < 0.05, ** p < 0.01, t-test). These experiments were repeated three times.

Figure 5

PlATG6a regulates sporangiophore morphology. (A–C) Microscopic images showing branches of sporangiophore. T32 and T47 are Δplatg6a mutants. (D) Number of branches per sporangiophore were calculated. Mean ± SD, derived from three independent biological repeats, for each strain. Asterisks represent a significant difference vs. WT (* p < 0.05) based on t-test.

Figure 6

Virulence assessment. The virulence of Δplatg6a mutants was tested on litchi leaves, with WT and CK as controls. Zoospores (A) or mycelial plugs (C) were inoculated on the litchi leaves. Photographs were taken at 48 hpi. (B,D) The lesion length was measured at 48 hpi, corresponding to (A,C), respectively. CK: control; the transformant failed to acquire the PIATG6a mutation. Asterisks indicate significant difference vs. WT (* p < 0.05, t-test). These experiments were repeated three times, each containing 3 leaves for each strain.

Figure 7

PlATG6a is involved in salt stress tolerance. (A) Mycelial of WT, CK and the Δplatg6a mutants (T32 and T47) grown on Plich medium with or without salt (0.05 M NaCl or 0.1 M CaCl₂) supplement. Images were taken at 7 dpi. (B) Colony diameter was measured at 7 dpi. Growth inhibition rate (%) was calculated. Mean ± SD (n = 9 for each strain). Asterisks denote significant differences vs. WT (* p < 0.05; ** p < 0.01; t-test).

Figure 8

PlATG6a regulates response to oxidative stress in Peronophythora litchii. (A) WT, CK and the Δplatg6a mutants were allowed to grow on Plich medium with or without 2 mM H₂O₂ at 25 °C. Images were taken at 7 dpi. (B) The colony diameters were measured at 7 dpi. Growth inhibition rate (%) was calculated. WT and CK strains were used as controls. (C) Transcriptional analysis of the PlATG6a gene under oxidative stress (5 mM H₂O₂, for 0, 5, 15, 55 min). Expression levels were normalized using the values at 0 min as ‘1′. (D) qRT-PCR analysis of P. litchii putative peroxidase-encoding genes in Δplatg6a mutants and WT strain under oxidative stress conditions (5 mM H₂O₂, for 5 min). Data are mean ± SD (n = 9). Asterisks represent significant differences vs. WT (** p < 0.01) based on t-test.

Figure 9

Assessment of sexual reproduction. (A) Photographs were taken at 10 dpi. The blue arrows indicate the representative oospores. (B) Oospore number was calculated. Data are mean ± SD (n = 9). There was no significant difference in oospore number between WT and the Δplatg6a mutant, T32 or T47 (p > 0.5; t-test).