Combining ChIP-chip and expression profiling to model the MoCRZ1 mediated circuit for Ca/calcineurin signaling in the rice blast fungus

Abstract

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen.

Figure 1. Establishment of Chromatin immunoprecipitation.

(A) Nuclear localization of MoCRZ1 was visualized in the eGFP tagged strain under the native promoter. FK506 blocks nuclear localization of MoCRZ1::eGFP. Bar indicates 10 µm. (B) ChIP-chip experimental design to identify MoCRZ1 targets activated by calcium treatment. The Ca²⁺/FK506 treated sample served as the negative control treatment. (C) RT-PCR to verify that PMC1 was up-regulated in the Ca²⁺ treated but not in Ca²⁺/FK506 treated sample. (D) Quantitative PCR was conducted with DNA after ChIP with antiGFP antibody. 30% input DNA collected prior to pull down was used as control. 1 µl each of ChIPed and input DNA was used for real-time PCR. Fold changes were calculated by 2^ΔΔCt, where ΔΔCt = (Ct_{input DNA}-Ct_{ChIPed DNA}) _{Ca2+ treated sample} - (Ct_{input DNA}-Ct_{ChIPed DNA}) _{Ca2+/FK506 treated sample}.

Figure 2. Genome-wide distribution of putative MoCRZ1 targets.

Target genes mapped to M. oryzae supercontigs version 5.

Figure 3. Expression dynamics of MoCRZ1 targets.

(A) Expression microarray design. Wild type strain KJ201 and MoCRZ1 deletion mutant (Δmocrz1) were treated with Ca²⁺ and/or FK506 as depicted. Agilent M. oryzae whole genome microarray chip ver. 2 was hybridized in a single channel design with four biological replications per treatment. After global normalization of signal intensities to the average expression level of all probes among the 16 data sets, pairwise comparison between treatments was conducted. (B) Venn diagram showing number of genes identified from ChIP-chip and up-regulated in transcriptome profiling described in panel A. Number of genes with more than 2 fold differential expression with P<0.05 were noted as red for up-regulation and green for down-regulation in Ca²⁺ treated wild type samples in each comparison. (C) Hierachical clustering resulted in two large groups with differential expression.

Figure 4. Real-time RT-PCR to validate differential expression.

cDNA relevant to 25 ng total RNA was used to run real-time RT-PCR. Log₂ ^{(Fold changes)} calculated by 2^−ΔΔCt were displayed. ΔΔCt = (Ct_{gene of interest}-Ct_{control gene})_{test condition}-(Ct_{gene of interest}-Ct_{control gene})_{control condition}. Black bar represents signal ratio from microarray data, while grey bar shows fold changes from real-time RT-PCR. Fold changes were calculated by normalizing Ct values as in Figure 3A, where (a) compares expression level between Ca²⁺ treated vs. no treatment in wild type strain KJ201; (b), Ca²⁺ vs. Ca²⁺+FK506 in KJ201; (c), Ca²⁺ treatments in KJ201 vs. in Δmocrz1.

Figure 5. Putative MoCRZ1 binding motifs identified.

(A) Analysis schema to identify putative MoCRZ1 binding motifs. (B) WebLogo of Top 2 motifs and the best hits in the yeast motif database. The consensus sequences of the putative motif sequences (MEME) calculated using WebLogo server were displayed in order of predominance from top to bottom at each position with large letters being higher frequency. Best hit from yeast motif database was displayed for comparison (TOMTOM). (C) Electrophoretic mobility shift assay. Probe DNA was amplified by PCR from the promoter regions of MoCRZ1 (left panel, lanes 1 to 3) and CBP1 (right panel, lanes 4 to 6) with 5′-Biotin-labeled primer pairs, purified by gel isolation, and allowed to bind to purified MoCRZ1 protein. Lanes 1 and 4, Biotin-labeled probe DNA; lanes 2 and 5, probe DNA with purified MoCRZ1 protein; lanes 3 and 6, competition reaction with 200 fold molar excess of unlabeded DNA. FP, free probe; SP, shifted probe. Reaction mixture was run on 5% polyacrylamide gel in 0.5X TBE and transferred onto Hybond N⁺ (GE Healthcare) membrane. Signals were detected using chemiluminescence.

Figure 6. Proposed model of MoCRZ1 regulation of downstream genes.

Consensus model of calcineurin/CRZ-1 signaling with green arrows indicating feedforward regulation and red arrows feedback regulation. Regulation of calcineurin occurs in multiple layers. (a) direct feedback by induction of expression of calcineurin regulators CBP1 or CTS1. (b) indirect feedback by induction of PMC1 leading to sequestration of cytosolic Ca²⁺ ions to inactivate calcineurin.