Table_3_The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens.XLSX

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.</p

Table_6_The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens.XLSX

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.</p

Table_4_The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens.XLSX

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.</p

Table_7_The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens.xlsx

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.</p

Table_2_The moss-specific transcription factor PpERF24 positively modulates immunity against fungal pathogens in Physcomitrium patens.XLSX

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.</p

Expression profiles of <i>TAD3-1</i> alleles in Col-0 x Nok-1 hybrids determined by pyrosequencing.

<p>Expression profiles of <i>TAD3-1</i> alleles in Col-0 x Nok-1 hybrids determined by pyrosequencing.</p

Sequence length distribution of the small RNAs over the <i>TAD3-1</i> locus.

<p>sRNA reads were counted in three different genomic regions: the first one (Chr5: 8,446,240–8,447,463) comprises two transposons upstream of <i>TAD3-1</i>, the second includes the promoter of <i>TAD3-1</i> (Chr5: 8,447,463–8,447,954) and the last one corresponds to the <i>TAD3-1</i> gene (Chr5: 8,447,954–8,451,218). Transposable elements are depicted in orange and <i>TAD3-1</i> in grey. Counts are given in reads per million of mapped reads. The precise distribution of sRNA reads is described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s011" target="_blank">S11 Fig</a>.</p

The methylation of the <i>tad3-1 epiallele</i> is reversible.

<p>(A) Genomic DNAs (300 ng) from the F9 plants indicated were digested with <i>McrBC (+McrBC</i>) and then amplified using primers specific for the regions indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a> to localize the amplicons within <i>TAD3</i>). We verified the presence of the polymorphisms between Nok-1 and Col-0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>) by sequencing the PCR fragments obtained without <i>McrBC</i> treatment (-<i>McrBC</i>) for PCR#2, #3, #5 and #6, excluding a genetic recombination between Col-0 and Nok-1 in this region. All plants are from the progeny of an F8 revertant that was fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5. <i>K1ColK5Nok</i> are F9 plants fixed Col-0 at chromosome 1 and Nok-1 at chromosome 5. <i>K1ColK5Col</i> are F9 plants fixed Col-0 at both chromosomes. For the Nok-1 plants, only PCR#2 and #3 are specific for chromosome 5. (B) The methylation rates within the promoter and the gene body of <i>TAD3</i> were determined in plants described in (A). Data were obtained by amplifying the regions indicated, namely ‘<i>Prom</i>.’ for the promoter and ‘<i>Gene’</i> for the gene body, after bisulfite conversion (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s007" target="_blank">S7 Fig</a>). (C) Expression of <i>TAD3</i> analyzed by qRT-PCR in plants described in (A) using primers corresponding to PCR#3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>), specific for chromosome 5. (D) Segregation of the Nok-1 and Col-0 alleles at chromosome 5 in the progeny of the revertant. We genotyped the progeny of two F9 plants (#3 and #2), descending from the revertant, and fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5. The control is an F9 plant fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5, coming from a lineage independent of the revertant. The numbers of plants genotyped are indicated in parentheses.</p

<i>TAD3-1</i> is methylated in Nok-1 and Est-1, preventing its expression.

<p>(A) DNA methylation of <i>TAD3-1</i> 5’-UTR analysed by digesting the indicated genomic DNA with <i>McrBC</i> followed by PCR amplification. Regions amplified correspond to <i>PCR#2</i> and <i>PCR#3</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>) and are specific for <i>TAD3-1</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s004" target="_blank">S4 Fig</a>). (B) <i>TAD3-1</i> is silenced in Nok-1 and Est-1. Plants were grown on medium containing 0 (-) or 10 (+) μg/ml of 5-aza-2’deoxycytidine (<i>Aza</i>) for seven days. RNAs of plants were extracted and cDNAs were amplified using primers corresponding to PCR#3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>), specific for chromosome 5. <i>ATEF</i> amplifications served as controls. (C) <i>TAD3-1</i> qRT-PCR analyses using PCR#3 primers that are specific for chromosome 5 and plants described in (B).</p

Methylation patterns of <i>TAD3-1</i> in incompatible plants carrying a <i>cmt3</i> mutation.

<p>(A) Methylation patterns of <i>TAD3-1</i> in incompatible plants carrying a <i>cmt3</i> mutation. Genomic DNAs (300 ng) were digested with <i>McrBC (+McrBC</i>) and then amplified using primers specific for the PCR fragments indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a> to localise the amplicons within the <i>TAD3-1</i> gene). Plants noted “<i>CN</i>” or “<i>CC</i>” are all in the <i>cmt3-11</i> background. “<i>C</i>” indicates that the Col-0 allele is fixed, “<i>N</i>” indicates that the Nok-1 allele is fixed. For the Nok-1 plants, only PCR#2 and #3 are specific for chromosome 5. (B) Methylation rates within the promoter regions in incompatible plants carrying a <i>cmt3-11</i> mutation. Data (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s013" target="_blank">S13 Fig</a>) were obtained by amplifying from leaf genomic DNA the promoter region corresponding to PCR#7 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s007" target="_blank">S7A Fig</a>). The numbers indicate the plants analyzed, as shown in (A). In a <i>cmt3</i> background, the allele inherited from Nok-1 at chromosome 5 becomes specifically hypomethylated in the CHG context compared to the Nok-1 parent (plants #6 and #46). (C) Segregation of the Nok-1 and Col-0 alleles at chromosome 5 in the progeny (n = 128) of a <i>cmt3</i> mutant. The progeny of a plant fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5 and sibling of the plants presented on (A) and (B) were genotyped.</p