Network and role analysis of autophagy in Phytophthora sojae

Abstract

Autophagy is an evolutionarily conserved mechanism in eukaryotes with roles in development and the virulence of plant fungal pathogens. However, few reports on autophagy in oomycete species have been published. Here, we identified 26 autophagy-related genes (ATGs) belonging to 20 different groups in Phytophthora sojae using a genome-wide survey, and core ATGs in oomycetes were used to construct a preliminary autophagy pathway model. Expression profile analysis revealed that these ATGs are broadly expressed and that the majority of them significantly increase during infection stages, suggesting a central role for autophagy in virulence. Autophagy in P. sojae was detected using a GFP-PsAtg8 fusion protein and the fluorescent dye MDC during rapamycin and starvation treatment. In addition, autophagy was significantly induced during sporangium formation and cyst germination. Silencing PsAtg6a in P. sojae significantly reduced sporulation and pathogenicity. Furthermore, a PsAtg6a-silenced strain showed haustorial formation defects. These results suggested that autophagy might play essential roles in both the development and infection mechanism of P. sojae.

Figure 1

Distribution of autophagy-related (ATG) proteins in representative species. ATG proteins identified from P. sojae (Ps), P. capsici (Pc), H. parasitica (Hp), Py. ultimum (Pu), T. pseudonana (Tp), A. thaliana (At), C. elegans (Ce), H. sapiens (Hs), S. cerevisiae (Sc), and M. grisea (Mg) were compared. The distribution of putative ATGs appeared to be mostly restricted to oomycete species. Evolutionarily, the autophagy pathway of oomycetes appears more closely related to that of plants and animals than to that of fungi.

Figure 2

Schematic representation of autophagy in P. sojae. According to the distribution of ATGs in P. sojae, autophagy divided into 4 steps, as it is in H. sapiens: initiation of autophagy, vesicle nucleation at the PAS, vesicle expansion and recycling.

Figure 3

Phylogenetic relationship and domain structures of selected ATGs. (A) Sequence features of Atg8 proteins. Atg8 proteins from H. sapiens (GABARAP and MAP1LC3B), A. thaliana (AtAtg8) and P. sojae (PsAtg8) are shown. Atg8 proteins of different species are highly conserved across their entire length, and very similar to each other in most positions. (B) and (C) Phylogenetic relationship and domain structures of Atg1 and Atg11. The phylogenetic trees of Atg1 (B) and Atg11 (C) proteins identified from P. sojae (Ps), P. capsici (Pc), H. parasitica (Hp), Py. ultimum (Pu), T. pseudonana (Tp), A. thaliana (At), C. elegans (Ce), H. sapiens (Hs), S. cerevisiae (Sc), and M. grisea (Mg). Pkinase: kinase domain. Atg11: Atg11 domain. DUF3543: DUF3543 domain. Atg17: Atg17 domain.

Figure 4

Heat map of expression profiles for P. sojae ATGs. Color bar represents the log2 expression values, ranging from green (0) to red (8). MY, mycelia; SP, zoosporangia; ZO, zoospores; CY, cysts; GC, germinated cysts; IF1.5 to IF24, indicates samples from 1.5, 3, 6, 12 and 24 h after infection of soybean leaves.

Figure 5

Induction of autophagy in response to rapamycin treatment in P. sojae. (A) Autophagy indicated by GFP-PsAtg8. GFP-PsAtg8-expressing P. sojae hyphae were incubated in 10% V8 juice. After treated with 100 nM rapamycin or DMSO (control) for 4 h, hyphae were analyzed by confocal microscopy. Bars = 10 μm. (B) Autophagy indicated by MDC dye. P. sojae wild-type hyphae were incubated in 10% V8 juice. After treated with 100 nM rapamycin or DMSO (control) for 4 h, hyphae samples concurrently stained with MDC were analyzed by confocal microscopy. Bars = 10 μm.

Figure 6

P. sojae sporangial production is regulated by autophagy. (A) Visualization of autophagic activation with MDC in P. soaje mycelia (MY) and zoosporangia (SP). Wild-type hyphae were incubated in 10% V8 juice for 30 h. After 3 washes and incubation with sterile distilled water for 4 h, hyphae were concurrently stained for MDC and analyzed by confocal microscopy. Bars = 10 μm. (B) Numbers of sporangia produced by PsAtg6a transgenic lines. Sporangia of the indicated samples were counted 12 h after the induction of sporangial production. Representative data are shown from three separate experiments. The bars indicate standard errors, and stars above bars indicate that the difference from the wild-type value was significant. **P < 0.01 (t-test). (C) Sporangial phenotypes forPsAtg6a transgenic lines. Micrographs were taken 12 h after sporangial induction. Black arrows indicate sporangia. Bars = 500 μm.

Figure 7

Autophagosome formation in the germinating cyst stage of P. sojae. Visualization of autophagy activation with MDC in cysts (CY) and germinated cysts (GC) of P. sojae. Cyst and germinated cyst samples were collected, concurrently stained with MDC, and analyzed by confocal microscopy. Bars = 10 μm.

Figure 8

Construction of PsAtg6a-silenced lines. (A) Verification of incorporation into genomic DNA by PCR using oligonucleotides from the Ham34 promoter and terminator regions as primers. The transgene should yield an amplified fragment 1520 bp in length. +: plasmid DNA; WT: wild-type strain P6497; M: molecular markers. (B) qRT-PCR measurement of the relative transcript levels of PsAtg6a and PsAtg6b in control (P6497), control (T9) and silenced (ST17, ST21 and ST39) transformants. The relative expression levels were calculated using TEF1 as the reference gene. The bars indicate standard errors, and stars above the bars indicate that the difference from the wild-type value was significant. (C) Visualization of autophagic activation with MDC in wild type and ST39. P. sojae wild-type or ST39 hyphae were incubated in 10% V8 juice for 30 h. After 3 washes and incubation with sterile distilled water for 4 h, hyphae samples were concurrently stained with MDC and analyzed by confocal microscopy. Bars = 10 μm.

Figure 9

PsAtg6a silencing reduced P. sojae virulence. (A) Phenotypes of soybean etiolated seedlings inoculated with wild-type P. sojae and recombinant strains. Approximately 250 zoospores were used for each inoculation. Photographs were taken 24 and 36 hpi. The edges of the lesions are indicated by arrows. (B) Lesion lengths measured at 24 hpi and 36 hpi on five seedlings in each of four independent experiments. The bars indicate the standard errors. **P < 0.01, *P < 0.05 (t-test). (C) Trypan blue staining of hyphae infecting soybean epidermal cells. Photographs were taken at 30 hpi. Black arrows: haustoria.