# Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game involving a chromatin remodeler and a specific transcription factor

Colin Clairet, Elise Gay, Antoine Porquier, Françoise Blaise, Claire-line Marais, Thierry Rouxel, Marie-hélène Balesdent, Jessica Soyer, Isabelle Fudal

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## ► To cite this version:

Colin Clairet, Elise Gay, Antoine Porquier, Françoise Blaise, Claire-line Marais, et al.. Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game involving a chromatin remodeler and a specific transcription factor. 17th European Conference on Fungal Genetics, Mar 2025, Dublin, Ireland., 242 (1), pp.247-261, 2024. [hal-05250850](https://hal.science/hal-05250850v1)

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https://hal.science/hal-05250850v1

Submitted on 11 Sep 2025

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# Regulation of effector gene expression as concerted waves in *Leptosphaeria maculans*: a two-player game involving a chromatin remodeler and a specific transcription factor

C. Clairet, E.J. Gay, A. Porquier, F. Blaise, C.L. Marais, T. Rouxel, M-H. Balesdent, J.L. Soyer, I. Fudal

UR Bioger, INRAE, Université Paris-Saclay, 91120 Palaiseau, France

## 01/ Context: effector-genes are enriched in heterochromatin domains and expressed as concerted waves during infection

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Successives waves of *L. maculans* gene expression during infection of oilseed rape Left panel: heatmap of the genes overexpressed *in planta* compared to *in vitro* conditions; right panel: up-regulated genes *in planta* are enriched in heterochromatin *in vitro*.

* Effector-genes of *Leptosphaeria maculans* are expressed as waves during infection of *Brassica napus* (oilseed rape)

* Genes induced during infection are associated with heterochromatin (H3K9me3- and / or H3K27me3-domains) *in vitro*

* Previous finding that **KMT1** (responsible for H3K9me3 deposition) represses effector-gene expression *in vitro*

* The transcription factor (TF) **Pf2** increasingly found as involved in pathogenicity and regulation of effector-gene expression in plant-pathogenic fungi

**Our working hypothesis: a two-layer regulatory network**

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TF

Effector-genes are repressed due to their localisation in H3K9me3- or H3K27me3-domains

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Effector-genes are expressed in fine-tuned successive waves due to removal of H3K9me3 and action of one / several TFs (transcription factors)

**Objective: investing role of KMT1 and LmPf2 in the regulation of (effector) gene expression in L. maculans**

## 02/ Methodology

* Analysis of expression of *LmPf2*, *KMT1* and cloned effector-genes

* CRISPR-Cas9 inactivation of *LmPf2* and *KMT1*

* Over-expression of *LmPf2* in a WT and a *ΔKMT1* background (i.e. WT_oPf2, *ΔKMT1_oPf2*)

* Phenotypic characterization of the generated transformants

* Analyses of gene expression in the transformants via RNA-seq

1. Inactivations of *LmPf2* or *KMT1*

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2. Over-expression of *LmPf2*

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Generation of CRISPR-Cas9 mutants for *KMT1* or *LmPf2* and of *LmPf2* over-expressed transformants in a WT or a *ΔKMT1* mutant

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: LmPf2

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: KMT1

## 03/ Expression of *KMT1*, *LmPf2* and avirulence genes of *L. maculans*

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* Avirulence effector genes are repressed *in vitro*, highly expressed during biotrophic infection of oilseed rape (i.e, cotyledon 5, 7, 9 dpi; petiole 7 dpi; stem 14, 28 dpi)

* **KMT1** has an **opposite expression pattern** to that of avirulence effector-genes

* *LmPf2* expression follows the same pattern as avirulence effector genes

## 04/LmPf2 and KMT1 are involved in pathogenicity and regulation of avirulence effector genes of *L. maculans*

Inoculation of *ΔLmPf2* and *ΔKMT1* on a susceptible cultivar of *B. napus*

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A, Pictures of symptoms 13 dpi; B, fluorescence images of cotyledons infected by GFP mutants and the WT 5 and 7 dpi (confocal microscope); C, fluorescence images of cotyledons infected by GFP mutants and the WT 8 and 13 dpi (fluorescence binocular). Scale bars, 1mm.

Expression of avirulence effector genes *in vitro*, in the transformants generated, compared to the WT (RNA-seq data)

<table><thead><tr><th>name</th><th>TE<sup>a</sup></th><th>H3K4me2<sup>b</sup></th><th>H3K9me3<sup>b</sup></th><th>H3K27me3<sup>b</sup></th><th>Δkmt1<sup>c</sup></th><th>ΔLmPf2<sup>c</sup></th><th>JN2_oPf2<sup>c</sup></th><th>Δkmt1_oPf2<sup>b</sup></th></tr></thead><tbody><tr><td>AvrLm3</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>up</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm4-7</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>-</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm14</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>-</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm6</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>-</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm2</td><td>yes</td><td>-</td><td>5_included</td><td>-</td><td>down</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm10_A</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>down</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm10_B</td><td>yes</td><td>-</td><td>all_included</td><td>all_included</td><td>-</td><td>down</td><td>-</td><td>up</td></tr><tr><td>AvrLmS-Lep2</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>-</td><td>-</td><td>-</td><td>up</td></tr><tr><td>AvrLm5-9</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>AvrLm11</td><td>yes</td><td>-</td><td>all_included</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

<sup>a</sup>Genes located in a TE-rich genomic context;

<sup>b</sup>Genes associated with H3K4me2, H3K9me3 or H3K27me3 during axenic culture in the wild type strain;

<sup>c</sup>Genes deregulated in a given transformant compared to the WT strain during axenic culture.

* ΔLmPf2 mutants: unable to colonize *B. napus*; ΔKMT1 mutants: less symptoms than the WT

* Over-expression of *LmPf2* in a WT background: no influence on avirulence gene expression

* Over-expression of *LmPf2* in a *ΔKMT1* background: up-regulation of avirulence effector genes located in heterochromatin-domains

## 05/ LmPf2 and KMT1 regulate expression of effector genes

<table><thead><tr><th></th><th>Total genes</th><th>Effector genes</th><th>H3K4me2 genes<sup>a</sup></th><th>H3K9me3 genes<sup>a</sup></th><th>H3K27me3 genes<sup>a</sup></th><th>H3K9me3/H3K27me3 genes<sup>a</sup></th></tr></thead><tbody><tr><td rowspan="4">Up-regulated</td><td>Δkmt1</td><td>129</td><td>22*</td><td>22*</td><td>3</td><td>64*</td><td>4*</td></tr><tr><td>ΔLmPf2</td><td>202</td><td>20</td><td>68*</td><td>4</td><td>57*</td><td>5*</td></tr><tr><td>WT_oPf2</td><td>247</td><td>46*</td><td>46*</td><td>2</td><td>101*</td><td>6*</td></tr><tr><td>Δkmt1_oPf2_B</td><td>931</td><td>154*</td><td>233*</td><td>25*</td><td>329*</td><td>24*</td></tr><tr><td rowspan="4">Down-regulated</td><td>Δkmt1</td><td>143</td><td>27*</td><td>35*</td><td>10*</td><td>42*</td><td>9*</td></tr><tr><td>ΔLmPf2</td><td>225</td><td>40*</td><td>58*</td><td>4</td><td>88*</td><td>10*</td></tr><tr><td>WT_oPf2</td><td>277</td><td>19</td><td>110*</td><td>1</td><td>60*</td><td>3</td></tr><tr><td>Δkmt1_oPf2_B</td><td>861</td><td>83</td><td>364*</td><td>11</td><td>182*</td><td>14*</td></tr></tbody></table>

<sup>a</sup>Genes associated with H3K4me2, H3K9me3, H3K27me3 or H3K9me3 and H3K27me3 during axenic growth of the WT isolate;

<sup>b</sup>Genes statistically under-represented in a given category; *Genes statistically enriched in a given category; Statistical analyses were performed using Chl<sup>2</sup>, P value < 0.05.

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Carbohydrate uptake

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Oxydo-reduction processes

Over-expression of *LmPf2* in a *ΔKMT1* background (*ΔKMT1_oPf2*):

* Enhanced effect on gene expression, compared to effect observed by removal of H3K9me3 or over-expression of *LmPf2*, individually

* 20 out of 36 effector-genes associated with H3K9me3 are up-regulated

* Among the 256 effector-genes over-expressed naturally *in planta* in the WT, 45% are up-regulated in at least one of the transformants, 34% in the *ΔKMT1_oPf2* transformants

## 06/ Take-home message & future work

* KMT1 inhibits expression of genes naturally over-expressed *in planta*, *LmPf2* positively regulates these genes

* Regulation of the expression of effector-genes is a two layer mechanism

* What are the direct targets of *LmPf2*?

* What is the signal allowing *in planta* effector gene expression?

### Contacts

jessica.soyer@inrae.fr

Isabelle.fudal@inrae.fr

### Fundings

INRAE SPE Chromadyn

ANR CHOPIN

PhD Grant Univ. Paris-Saclay (C. Clairet)

### References

Clairet et al., New Phytol. 2024. DOI: 10.1111/nph.19581.

Gay et al., BMC Biol. 2021 DOI: 10.1186/s12915-021-00989-3

Soyer et al., Chrom. Res. 2021. DOI: 10.1007/s10577-021-09658-1

Soyer et al., Curr. Op. Plant Biol. 2015. DOI: 10.1016/j.pbi.2015.05.025

Soyer et al., PLOS Genet. 2014. DOI: 10.1371/journal.pgen.1004227