The Basic Leucine Zipper Transcription Factor PlBZP32 Associated with the Oxidative Stress Response Is Critical for Pathogenicity of the Lychee Downy Blight Oomycete Peronophythora litchii

Abstract

Basic leucine zipper (bZIP) transcription factors are widespread in eukaryotes, including plants, animals, fungi, and oomycetes. However, the functions of bZIPs in oomycetes are rarely known. In this study, we identified a bZIP protein possessing a special bZIP-PAS structure in Peronophythora litchii, named PlBZP32 We found that PlBZP32 is upregulated in zoospores, in cysts, and during invasive hyphal growth. We studied the functions of PlBZP32 using the RNAi technique to suppress the expression of this gene. PlBZP32-silenced mutants were more sensitive to oxidative stress, showed a lower cyst germination rate, and produced more sporangia than the wild-type strain SHS3. The PlBZP32-silenced mutants were also less invasive on the host plant. Furthermore, we analyzed the activities of extracellular peroxidases and laccases and found that silencing PlBZP32 decreased the activities of P. litchii peroxidase and laccase. To our knowledge, this is the first report that the functions of a bZIP-PAS protein are associated with oxidative stress, asexual development, and pathogenicity in oomycetes.IMPORTANCE In this study, we utilized the RNAi technique to investigate the functions of PlBZP32, which possesses a basic leucine zipper (bZIP)-PAS structure, and provided insights into the contributions of bZIP transcription factors to oxidative stress, the production of sporangia, the germination of cysts, and the pathogenicity of Peronophythora litchii This study also revealed the role of PlBZP32 in regulating the enzymatic activities of extracellular peroxidases and laccases in the plant-pathogenic oomycete.

Keywords: Peronophythora litchii; bZIP transcription factor; laccase; pathogenicity; peroxidase.

FIG 1

Phylogenetic analysis of PlBZP32 protein and its orthologs. The evolutionary history was inferred using the neighbor-joining method (47). Evolutionary analyses were conducted in MEGA7 (48).

FIG 2

Transcriptional profile of the PlBZP32 gene. Relative transcriptional levels of PlBZP32 were determined by qRT-PCR with total RNA extracted from specific stages of the life cycle. MY, mycelia; SP, sporangia; ZO, zoospores; CY, cysts; GC, germinating cysts; Oo, oospores; IF1.5 to IF48, infection stages of 1.5, 3, 6, 12, 24, and 48 h postinoculation. Transcriptional levels were normalized using the MY values as “1,” and the P. litchii ACTIN gene served as an internal control. The experiment was repeated three times with independent samples.

FIG 3

PlBZP32 was not required for the mycelial growth of P. litchii. (A, B) Relative transcriptional levels of PlBZP32 in wild type (WT), CK (transformed with empty vector), and transformants. PlBZP32 transcription was normalized to that of the WT value (set as “1.0”). The P. litchii ACTIN gene served as an internal control. RNA used in qRT-PCR was extracted from mycelium (A) and zoospore (B). (C) Transcriptional level of Pl105397 in WT, CK, and PlBZP32-silenced transformants. (D) Colony diameter of WT, CK, and the three PlBZP32-silenced transformants. Bar charts A to D depict means ± SD derived from 3 independent repeats, each of which contained 3 replicates. Asterisks on the bars denote significant difference (P < 0.05) based on Duncan’s multiple range test method. (E) WT, CK, and the PlBZP32-silenced transformants were inoculated on CJA medium for 3 days at 28°C in darkness and then photographed. These experiments were repeated three times.

FIG 4

The PlBZP32-silenced mutants were hypersensitive to H₂O₂. (A) The wild type (WT), CK, and PlBZP32-silenced mutants were inoculated on Plich medium with or without 2 or 5 mM H₂O₂ and cultured at 25°C for 7 days. (B) The colony diameters of the tested strains were measured. Wild-type and CK strains were used as controls. The growth inhibition rate was calculated using the following formula: (the diameter of control − the diameter of treated strain)/(the diameter of control) ×100%. The bar chart depicts means ± SD derived from 3 independent repeats, and different letters represent a significant difference (P < 0.05) based on Duncan’s multiple range test method.

FIG 5

Transcription of PlBZP32 during H₂O₂ treatment. P. litchii mycelia were treated with 5 mM H₂O₂ for 5 min, 15 min, 30 min, 1.5 h, and 3 h, before collection for total RNA extraction and qRT-PCR analysis. Expression of the PlBZP32 gene in the untreated mycelia collected at 5 min was used as a reference and normalized to 1.0. The bar chart depicts means ± SD derived from 3 independent repeats. The same letter on the top of bars represents no significant difference (P > 0.05), based on Duncan’s multiple range test method.

FIG 6

PlBZP32 was required for full virulence of P. litchii. (A) Wild type (WT), CK, and three PlBZP32-silenced mutants (T15, T21, and T35) were inoculated on lychee leaves and kept at 25°C in the dark. Photographs were taken 36 h postinoculation (hpi). Representative images for each instance were displayed. (B) Lesion length was measured at 12, 24, and 36 hpi. The values are means ± SD derived from three independent biological repeats (n = 12 leaves for each strain). Asterisks denote a significant difference (P < 0.05) based on Duncan’s multiple range test method.

FIG 7

PlBZP32-silenced mutants produced more sporangia. (A, B) Sporangia were quantified at 5 days after inoculation. Bar chart represents mean ± SD (C, D). Branch tips of each sporangiophore were counted. These results are derived from three independent biological repeats, each of which contained 5 technical replicates. Different letters represent a significant difference (P < 0.05) based on Duncan’s multiple range test method.

FIG 8

Silencing of PlBZP32 impaired cyst germination of P. litchii. Cyst germination was quantified at 2 h after cyst formation. In the bar graph, mean values present the cyst germination rates of PlBZP32-silenced mutants or control strains. Different letters represent a significant difference (P < 0.05) based on Duncan’s multiple range test method. This experiment was repeated three times. Scale bars, 200 μm.

FIG 9

PlBZP32 regulated activities of extracellular peroxidases and laccases. (A) Peroxidase activity assay. Mycelial mats of WT, CK, and three PlBZP32-silenced transformants were inoculated onto solid Plich medium containing Congo red at a final concentration of 500 μg/ml, as an indicator. The discoloration of Congo red was observed after incubation for 1 day. (B) Laccase activity assay. Mycelial mats of the aforementioned strains were inoculated on lima bean agar (LBA) media containing 0.2 mM ABTS for 10 days. (C) The discoloration halo diameters were measured at 1 day postinoculation. (D) The diameters of oxidized ABTS (dark purple) were measured at 10 days postinoculation. Different letters in (C) and (D) bar charts represent a significant difference (P < 0.05) based on Duncan’s multiple range test method. These two experiments contains three independent biological repeats, each of which contained three replicas.