Slt2-MAPK/RNS1 Controls Conidiation via Direct Regulation of the Central Regulatory Pathway in the Fungus Metarhizium robertsii

Abstract

Ascomycete fungi usually produce small hydrophobic asexual conidia that are easily dispersed and essential for long-term survival under a variety of environmental conditions. Several upstream signaling regulators have been documented to control conidiation via regulation of the central regulatory pathway that contains the transcription factors BrlA, AbaA and WetA. Here, we showed that the Slt2-MAPK signaling pathway and the transcription factor RNS1 constitute a novel upstream signaling cascade that activates the central regulatory pathway for conidiation in the Ascomycetes fungus M. robertsii. The BrlA gene has two overlapping transcripts BrlAα and BrlAβ; they have the same major ORF, but the 5' UTR of BrlAβ is 835 bp longer than the one of BrlAα. During conidiation, Slt2 phosphorylates the serine residue at the position 306 in RNS1, which self-induces. RNS1 binds to the BM2 motif in the promoter of the BrlA gene and induces the expression of the transcript BlrAα, which in turn activates the expression of the genes AbaA and WetA. In conclusion, the Slt2/RNS1 cascade represents a novel upstream signaling pathway that initiates conidiation via direct activation of the central regulatory pathway. This work provides significant mechanistic insights into the production of asexual conidia in an Ascomycete fungus.

Keywords: BrlA; MAPK; Metarhizium; RNS1; conidiation; fungi; phosphorylation.

Figure 1

RNS1 regulates conidiation. (A) Colony growth curve on PDA plates. WT: the wild-type strain; ∆Rns1: the deletion mutant of Rns1; C-∆Rns1: the complemented strain of ∆Rns1. Data are shown as the mean ± SE. (B) Colony morphology (upper panel; scale bar: 3 cm) and conidiophores (lower panel; scale bar: 10 μm) on the PDA plates. In this study, all shown images are representative of at least three independent experiments. (C) Conidial yields. Conidial yield assay was repeated three times with three replicates. Data are shown as the mean ± standard error (SE). Values with different letters are significantly different (p < 0.05, Tukey’s test in one-way ANOVA). (D) qRT-PCR analysis of the expression of Rns1 at the four conidiation stages of the WT strain on PDA plates. The values represent the fold-change of expression of Rns1 at a conidiation stage compared with the expression at 2 days post-inoculation, which is set to 1.

Figure 2

EMSA and ChIP-qPCR analysis of the binding of RNS1 to the putative the BM2 motifs in the promoters of the gene BrlA and AbaA. (A) EMSA analysis of the binding of the recombinant protein RNS1-DBD (the DNA binding domain in RNS1) to the Biotin-labeled BM2 motif in the BrlA promoter, and (B) in the AbaA promoter. The binding activity was demonstrated by the shift of the labeled DNA band prior to the addition of the specific competitor (the unlabeled DNA probe) in a 200-fold excess. (C) ChIP-qPCR analysis of the binding of the fusion proteins RNS1::FLAG to the BrlA promoter, and (D) to the AbaA promoter. WT-FLAG: a strain expressing the tag FLAG in the WT strain; WT-RNS1-FLAG: a strain expressing the fusion protein RNS1::FLAG protein in the WT.

Figure 3

Identification and characterization of the transcripts BrlAα and BrlAβ of the BrlA gene. (A) 5′RACE (upper panel) and 3′RACE (lower panel) amplification of the BrlA transcripts using the RLM RT-PCR kit. Note: two PCR products were obtained with 5′RACE. (B) A diagrammatic representation of the genome sequence of the gene BrlA (upper panel) and its two transcripts (BrlAα and BrlAβ) (down panel). The position of the BM2 motif is shown. Note: the two transcripts have the same major ORF and different 5′UTR. TSS: transcription start site. (C) qRT-PCR analysis of the expression of BrlAβ with Primer set 1, and of BrlAα and BrlAβ using the Primer set 2 during conidiation. The relative positions of the primers are shown in (B). (D) qRT-PCR analysis of the expression level of BrlA (the Primer set 2), AbaA and WetA in WT, the deletion mutants ∆Rns1 and ∆Slt2. The values in each figure represent the fold-changes of expression of a gene in a mutant compared with WT, which is set to 1.

Figure 4

qRT-PCR analysis of the expression of the gene Rns1 during conidiation in the WT strain, the deletion mutant ∆Fus3 (Fus3-MAPK), ∆Hog1 (Hog1-MAPK) and the three deletion mutants in the Slt2-MAPK cascade: ∆Bck1 (MAPKKK), ∆Mkk^1/2 (MAPKK) and ∆Slt2 (MAPK). The values represent the fold-changes of Rns1 expression in a mutant compared with WT, which is set to 1.

Figure 5

Phosphorylation of RNS1 by Slt2 during conidiation. (A) Phosphorylated peptide spectra identified by LC–MS/MS. The serine residue at position 306 was phosphorylated. The WT-RNS1-FLAG strain was used to conduct mass spectrometry assay. (B) Yeast two-hybrid analysis showing the physical interaction between the Slt2 and RNS1. Upper panel: colonies grown in SD-His-Ade-Leu-Trp + X-α-gal + AbA. Slt2/RNS1: cells expressing Slt2 and RNS1. Lower panel: colonies grown in SD-His-Trp-Ade plus X-a-Gal. BD-Slt2:: Y2HGold cells expressing Slt2; NC: negative control; PC: positive control. (C) Co-IP confirmation of the interaction of Slt2 and RNS1. Immunoprecipitation was conducted with anti-Myc antibody. Proteins were detected by immunoblot analysis with anti-Myc and anti-FLAG antibodies. (D) Phos-tag analysis of RNS1 phosphorylation by Slt2. Proteins were detected with the anti-FLAG antibody. (1) WT-RNS1-FLAG (RNS1::FLAG expressed in WT strain); (2) WT-RNS1^S306A-FLAG (RNS1^S306A::FLAG (Ser-306 substituted to Ala in RNS1::FLAG) expressed in the WT strain); (3) ∆Slt2-RNS1-FLAG (RNS1::FLAG expressed in ∆Slt2); (4) ∆Slt2-RNS1^S306A-FLAG (RNS1^S306A::FLAG expressed in ∆Slt2. (E) Western blot analysis of the protein RNS1::FLAG in the strains WT-RNS1-FLAG, WT-RNS1^S306A-FLAG, ∆Slt2-RNS1-FLAG and ∆Slt2-RNS1^S306A-FLAG.

Figure 6

Phosphorylation of the Sec-306 of RNS1 by Slt2 facilitates the binding of RNS1 to its own promoter to self-induce expression during conidiation. (A) ChIP-qPCR analysis of the binding to the BM2 motif in the Rns1 promoter of the proteins RNS1::FLAG or RNS1^S306A::FLAG. WT-FLAG: a strain expressing the tag FLAG in the WT strain; WT-RNS1-FLAG: a strain expressing the fusion protein RNS1::FLAG protein in the WT. WT-RNS1^S306A-FLAG: a strain expressing RNS1^S306A::FLAG protein in WT. ∆Slt2-RNS1-FLAG: a strain expressing RNS1::FLAG protein in ∆Slt2. Microscopic observation (B), qRT-PCR analysis (C) and Western blot analysis (D) of GFP expression during conidiation. WT-PRns1-gfp: a strain with gfp driven by the native Rns1 promoter (PRns1) in WT; WT-PRns1^BM2-gfp: gfp driven by PRns1^∆BM2 (the BM2 motif was mutated in Rns1’s promoter PRns1) in WT; ∆Rns1-PRns1-gfp: gfp driven by the promoter PRns1 in ∆Rns1; ∆Slt2-PRns1-gfp: gfp driven by the promoter PRns1 in ∆Slt2. Bars, 10 μm. (E) qRT-PCR analysis of the expression of Rns1. ∆Rns1-gRns1^S306A: a strain constructed by complementation of the mutant ∆Rns1 with a mutated genomic clone of Rns1 (gRns1) with the serine residue at position 306 changed to alanine; ∆Rns1-gRns1^∆BM2: a strain constructed by complementation of the mutant ∆Rns1 with a gRns1 mutant with the BM2 motif mutated.