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. 2022 Sep 15:13:908682.
doi: 10.3389/fpls.2022.908682. eCollection 2022.

The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens

Guillermo Reboledo  1 , Astrid Agorio  1 , Lucía Vignale  1 , Alfonso Alvarez  2 , Inés Ponce De León  1
Affiliations

The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens

Guillermo Reboledo et al. Front Plant Sci. .

Abstract

APETALA2/ethylene response factors (AP2/ERFs) transcription factors (TFs) have greatly expanded in land plants compared to algae. In angiosperms, AP2/ERFs play important regulatory functions in plant defenses against pathogens and abiotic stress by controlling the expression of target genes. In the moss Physcomitrium patens, a high number of members of the ERF family are induced during pathogen infection, suggesting that they are important regulators in bryophyte immunity. In the current study, we analyzed a P. patens pathogen-inducible ERF family member designated as PpERF24. Orthologs of PpERF24 were only found in other mosses, while they were absent in the bryophytes Marchantia polymorpha and Anthoceros agrestis, the vascular plant Selaginella moellendorffii, and angiosperms. We show that PpERF24 belongs to a moss-specific clade with distinctive amino acids features in the AP2 domain that binds to the DNA. Interestingly, all P. patens members of the PpERF24 subclade are induced by fungal pathogens. The function of PpERF24 during plant immunity was assessed by an overexpression approach and transcriptomic analysis. Overexpressing lines showed increased defenses to infection by the fungal pathogens Botrytis cinerea and Colletotrichum gloeosporioides evidenced by reduced cellular damage and fungal biomass compared to wild-type plants. Transcriptomic and RT-qPCR analysis revealed that PpERF24 positively regulates the expression levels of defense genes involved in transcriptional regulation, phenylpropanoid and jasmonate pathways, oxidative burst and pathogenesis-related (PR) genes. These findings give novel insights into potential mechanism by which PpERF24 increases plant defenses against several pathogens by regulating important players in plant immunity.

Keywords: AP2/ERF; Physcomitrium patens; defense; pathogens; transcription factor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Unrooted phylogenetic tree of AP2 domains of moss, liverwort, hornwort, and angiosperms. Unrooted maximum likelihood phylogenetic tree was visualized with iTOL. Subfamilies are indicated with different colors: ERFs (blue), DREBs (red), and moss-specific proteins (green). The moss-specific clade, the PpERF24 subclade, ERFs, and DREBs clades are supported by bootstrap values ≥ 95 (black asterisk). A bootstrap of 92 separate ERFs and DREBs (red asterisk). Pp3c11_14690 (PpERF24) is highlighted in black. See Supplementary Table 4 for complete information.
FIGURE 2
FIGURE 2
Conserved motifs in the AP2 domain of mosses ERF family clades (A). Sequence logos of AP2 domain from P. patens DREB clade, P. patens ERF clade, and moss-specific clades. (B) Alignment of PpERF24 and several AP2 domains of the moss-specific clade and corresponding sequence logos. Conserved YRG and RAYD elements are indicated with a black line, and conserved amino acids 14 and 19 for members of the DREB and ERF clades and 15 for PpERF24 and moss-specific AP2 domains are marked with red arrows. The β-sheets and α-helix predicted for PpERF24 are shown.
FIGURE 3
FIGURE 3
Cell death measurement in wild-type and PpERF24 overexpressing plants. Measurement of cell death by Evans blue staining 24 h after inoculation of wild type, PpERF24-OX-2 (OX2), and PpERF24-OX-4 (OX4) moss colonies with B. cinerea, P. irregulare, and treatment with elicitors of P.c. carotovorum. Cell death measurement for plant tissues inoculated with C. gloeosporioides was performed at 72 hpi. Data were expressed as the absorbance (Abs) at 600 nm per milligram of dry weight (DW). Values are means with standard deviations of eight independent replicate moss samples. Experiments were repeated thrice with similar results. Asterisks indicate a statistically significant difference between the wild-type and overexpressing PpERF24 plants [two-way ANOVA test, with Tukey’s honest significant difference test as post hoc test using P ≤ 0.001 (***)].
FIGURE 4
FIGURE 4
Differentially expressed genes of wild type and PpERF24-OX-4 overexpressing line during control treatment and B. cinerea infection. (A) Number of differentially expressed genes (DEGs), up- and downregulated, in wild type vs. PpERF24-OX-4 (OX4) tissues treated with water (Ctrl), and wild type (Wt) and PpERF24-OX-4 tissues inoculated with B. cinerea (Bcin) vs. water-treated tissues at 24 hpi. (B) Venn diagram of B. cinerea-responsive P. patens genes in wild-type and PpERF24-OX-4 plants and PpERF24-OX-4 vs. wild-type control tissues. Genes were considered as DEGs when | log2 FC| ≥ 1 and FDR ≤ 0.05.
FIGURE 5
FIGURE 5
Heatmap of hierarchical clustering of wild type and PpERF24-OX-4 overexpressing line during control treatment and B. cinerea infection. DEGs correspond to the 391 DEGs of PpERF24-OX-4 control vs. wild-type control plants. Selected DEGs had | log2 FC| ≥ 1 and FDR ≤ 0.05. See Supplementary Table 7 for complete information.
FIGURE 6
FIGURE 6
Expression levels of defense genes in control wild-type and PpERF24 overexpressing plants. Transcript levels in control PpERF24 overexpressing lines were expressed relative to the corresponding levels in control wild-type samples, using Ubi2 as reference gene. Results are reported as means ± standard deviation (SD) of four samples for each treatment. Asterisks indicate a statistically significant difference between wild-type and overexpressing plants (Student’s t-test, *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001; ****P < 0.0001).
FIGURE 7
FIGURE 7
Expression levels of defense genes in fungal-inoculated wild-type and PpERF24 overexpressing plants. Transcript levels in PpERF24 overexpressing lines inoculated with B. cinerea or C. gloeosporioides at 24 hpi were expressed relative to the corresponding levels in pathogen-inoculated wild-type plants, using Ubi2 as reference gene. Results are reported as means ± standard deviation (SD) of four samples for each treatment. Asterisks indicate a statistically significant difference between wild-type and overexpressing plants (Student’s t-test, *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001; ****P < 0.0001).
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