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. 2018 Oct 3;8(1):14718.
doi: 10.1038/s41598-018-32825-0.

A Rust Fungal Effector Binds Plant DNA and Modulates Transcription

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Free PMC article

A Rust Fungal Effector Binds Plant DNA and Modulates Transcription

Md Bulbul Ahmed et al. Sci Rep. .
Free PMC article

Abstract

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Sequence alignment and phylogenetic tree of the M. larici-populina CPG2811 SSP family. (A) Schematic representation of Mlp124478 protein topology, signal peptide (SP), nuclear localization sequence (NLS) and DNA-binding domain are shown. (B) Multiple sequence alignment of the nine members of the M. larici-populina CPG2811 SSP family. Predicted Signal peptides (SP) are boxed. Identical/highly conserved residues (*); semi conserved residues (:) and conserved residues (.) are marked. Predicted nuclear localization signal (NLS) is indicated by solid black underline. (C) Phylogenetic tree of the nine members of the CPG2811 gene family obtained with COBALT using Kimura distance value and neighbor joining tree method. Amino acid length is indicated in parenthesis.
Figure 2
Figure 2
Overview of functional approaches applied to Mlp124478. (A) Mlp124478 was mined from CPG2811 family and has a signal peptide (SP), a putative nuclear localization signal (NLS) and a putative DNA-binding domain (DBD). (B) The mature coding sequences of Mlp124478 was cloned in frame with the green fluorescent protein (GFP). (C) Mlp124478 was recombined into pVSPPsSpdes vector for Pst infection assay (effector delivery) and pB7FWG2.0 was then inserted into A. tumefaciens strain C58C1. (D) Pst expressing Mlp124478 was syringe infiltrated into the abaxial side of the leaves of Arabidopsis thaliana. (E) A. tumefaciens strain C58C1 expressing Mlp124478 was used to develop stable transgenic A. thaliana plants expressing Mlp124478 and perform transient expression, both were viewed by confocal microscopy (F) Transcriptomic study was performed with cDNA library preparation from the RNA extracted from the transgenic A. thaliana expressing Mlp124478 and control.
Figure 3
Figure 3
Mlp124478 carries a putative nuclear localization signal and a putative DNA-binding domain. (A) Morphology of 4-week-old soil grown A. thaliana Col-0 and stable transgenic plant expressing Mlp124478 grown at 22 °C under 14 h/10 h photoperiod in growth chamber. (B) Immunodetection of GFP protein in Col-0 and stable transgenic seedlings from 12 days old plantlets. (C) Live cell imaging using confocal microscope of epidermal cells of 4-days-old A. thaliana stable transgenic Mlp124478-GFP plantlets. GFP in the Col-0 background was used as control. Left panel shows GFP, middle panel shows DIC and right panel shows merge. Nucleoli are pointed with black or white arrowheads.
Figure 4
Figure 4
Mlp124478 Nuclear Localization Signal (NLS) is required for nucleolar accumulation. (A) Schematic representation of the constructs (Mlp124478 and Mlp124478∆29–38) used for transient expression. (B) Subcellular accumulation of Mlp124478-GFP and Mlp124478∆29–38-GFP in N. benthamiana epidermal cells at 4-days post-infiltration, the nucleus was stained by DAPI and epidermal cells were observed under the blue channel (left panel), green channel (middle panel) and merge of all channels (right panel). Arrowheads point the nucleolus. (C) Nuclear-nucleolar distribution of the fluorescent fusion proteins according to the fluorescence intensity ratios: nucleolar intensity (INo) divided by nuclear intensity (IN).
Figure 5
Figure 5
Mlp124478-GFP and Mlp124478∆29–38-GFP increase H. arabidopsidis growth on A. thaliana. (A) Col-0 plant showing normal leaves (i); wavy leaves phenotype observed in Mlp124478-GFP (ii); strongly enhanced leave waviness and early bolting in Mlp124478∆29–38-GFP (iii) morphology of eds1-1 (iv). (B) Live cell imaging using confocal microscopy of epidermal cells of 4-days-old stable transgenic Mlp124478∆29-38-GFP and Mlp124478-GFP plantlets. Left panel displays GFP, middle panel shows DIC, right panel shows the merge. Nucleoli are pointed with arrows. Scale bar: 10 µm. (C) Four weeks old soil grown Col-0, stable transgenic Mlp124478, Mlp124478∆2938-GFP and eds1-1 plants were spray inoculated with Hyaloperonospora arabidopsidis Noco2 (50,000 conidiospores/mL) and the number of conidiospores were quantified at 7 days after inoculation. Statistical significance was evaluated using student’s t test. Asterisk denotes significant difference to Col-0, p < 0.0001 for Mlp124478 and p < 0.002 for Mlp124478∆29-38. Experiments were repeated three times with similar results.
Figure 6
Figure 6
The expression of Mlp124478 in plant cells alters A. thaliana transcriptome. (A) Schematic illustration of transcriptomic work flow. RNA was isolated from 4-days old A. thaliana Mlp124478 stable transgenic and Col-0 plants and sequenced using Ion torrent. Transcripts were analyzed using iPlantCollaborative DNA subway and deregulated genes were considered for further analysis. (B) Go term enrichment analysis was performed with deregulated genes filtered with Q-value ≤ 0.05 and fold-change ≥ 2 using the Cytoscape software (version 3.1.1). Cytoscape was performed with the plug-in ClueGO and CluePedia to visualize functions enriched in the deregulated genes. The GO terms presented are significantly enriched in up-regulated and down-regulated genes with FDR ≤ 0.05 (Benjamini-Hochberg p-value correction) and revealed 15 GO terms belonging to 7 functional groups.
Figure 7
Figure 7
Regulation of gene expression level. Heat map of biotrophic pathogens response of genes in two groups: (A) upregulated genes and (B) down regulated genes. Genevestigator was used for differential expression analysis.
Figure 8
Figure 8
Mlp124478 binds DNA. Two-weeks-old plants tissues of Col-0 expressing GFP or stable transgenic Mlp124478 were used for chromatin preparation using ChIP assay with antibody against GFP as described in the material and methods section and A. thaliana genomic DNA was used as a positive control. TGA1a associated site was PCR amplified with TGA1a specific primer pair. Expected bands (211 bp) was obtained from transgenic and Arabidopsis genomic DNA for TGA1a at the promoter region of AT2G34450 gene. Mlp124478-GFP indicates chromatin IP from that line. Col-0 indicates that chromatin was immunoprecipitated from Col-0 expressing GFP (negative control). Genomic Col-0 DNA, not immunoprecipitated served as a positive control. The other genes shown (At4g08870, At2g39250, At2g47750, At4g39800) are examples of non specific reaction. Col-0 expressing GFP DNA: negative control; A. thaliana genomic DNA: positive control.

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