Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii

Abstract

Metarhizium robertsii is a versatile fungus with saprophytic, plant symbiotic and insect pathogenic lifestyle options. Here we show that M. robertsii mediates the saprophyte-to-insect pathogen transition through modulation of the expression of a membrane protein, Mr-OPY2. Abundant Mr-OPY2 protein initiates appressorium formation, a prerequisite for infection, whereas reduced production of Mr-OPY2 elicits saprophytic growth and conidiation. The precise regulation of Mr-OPY2 protein production is achieved via alternative transcription start sites. During saprophytic growth, a single long transcript is produced with small upstream open reading frames in its 5' untranslated region. Increased production of Mr-OPY2 protein on host cuticle is achieved by expression of a transcript variant lacking a small upstream open reading frame that would otherwise inhibit translation of Mr-OPY2. RNA-seq and qRT-PCR analyses show that Mr-OPY2 is a negative regulator of a transcription factor that we demonstrate is necessary for appressorial formation. These findings provide insights into the mechanisms regulating fungal lifestyle transitions.

Fig. 1

Pathogenicity of WT, the mutants ΔMr-OPY2 and ΔMr-STE50, and their respective complementation strains. a LT₅₀ (time taken to kill 50% of insects) values when the insects were inoculated by topical application. The bioassays were repeated three times with 40 insects per repeat. Data are expressed as the mean ± SE. Values with different letters are significantly different (n = 3, P < 0.05, Tukey’s test in one-way ANOVA). b Upper panel: mycelial growth and conidiation on the surface of cadavers (scale bar represents 10 mm). Lower panel: mycelia in cross sections of cadavers (scale bar represents 5 mm). Each image is representative of ~ 120 insect cadavers (three replicates with 40 insects per replicate). c Formation of appressoria (stained with Calcofluor Brightener White 2B) against a hydrophobic plastic surface. AP: appressorium; CO: conidium; SP: septum. Left: bright field microscopy. Note: the hyphal tips of ΔMr-OPY2 and ΔMr-STE50 do not swell to form appressoria. Right: fluorescence microscopy. Note: the septum between the appressorium and its appressorial mother cell. Images are representative of at least three independent experiments for each condition. Scale bar represents 10 μm

Fig. 2

Transcription and translation analyses of the Mr-OPY2 gene. a Representative Northern blot image of RNA isolated from undifferentiated mycelia grown in SDY or hemolymph (HE), and from germlings differentiating appressoria on locust wings (AP). The uncropped Northern blot is shown in Supplementary Fig. 11a. b 5′RACE with the RLM RT-PCR kit. The uncropped image of the agarose gel is shown in Supplementary Fig. 11b. c A diagrammatic representation of differences between the two mRNA variants of Mr-OPY2. Note: the two transcripts share the same major ORF but they have different 5′UTR. d Representative western blot image and analysis to quantify Mr-OPY2 levels in ΔMr-OPY2 and WT grown in SDY or hemolymph (HE) (Upper panel). The OPY2 levels of hemolymph-grown WT mycelium (1.4) is calculated relative to growth in SDY which is set at 1. ΔMr-OPY2 is included as a negative control. Lower panel: a portion of the loading control gel (for the Western blot analysis in upper panel) stained with Coomassie Brilliant Blue. M: Protein ladder (Thermo scientific, USA). The uncropped images of the western blot and the SDS-PAGE gel are shown in Supplementary Fig. 11c, d. e Immunohistochemical staining of the Mr-OPY2 protein in germlings differentiating appressoria against a plastic surface, and in non-differentiating germlings from 1/2 SDY shake cultures. Scale bar represents 10 μm. FITC: Fluorescein isothiocyanate; Merge: FITC and bright field pictures are merged; AP: appressorium. Note: no fluorescent signal in ΔMr-OPY and a weak signal in WT grown in 1/2 SDY. Images are representative of at least three independent experiments for each condition

Fig. 3

The uORFs suppress translation efficiency of the major ORF in Mr-OPY2-L. a Top panel: graphic representation of WT transcripts Mr-OPY2-L, Mr-OPY2-S and the mutated Mr-OPY2-L (Mr-OPY2-L ^ΔAUGs) that has GUGs in place of AUGs (highlighted in red). The blue and green labelled sequences in the 5′UTR represent the positions of the two different uORFs (these color conventions are also used in the top panel in b. Middle panel: Mr-OPY2 protein levels in SDY grown cultures of: (1) ΔMr-OPY2; (2) WT; (3) T-Mr-OPY2-L ^AUG (the native Mr-OPY2 gene was replaced by a native genomic Mr-OPY2 clone); and (4) T-Mr-OPY2 ^ΔAUGs (the native Mr-OPY2 gene was replaced by a genomic Mr-OPY2 clone with mutated uORFs). Bottom panel: a portion of the loading control gel (for the Western blot analysis in the middle panel) stained with Coomassie Brilliant Blue. M: Protein ladder. b The short Mr-OPY2-S transcript is more efficiently translated than the longer Mr-OPY2-L. Top panel: graphic representation of the constructs containing the transcript Mr-OPY2-L, Mr-OPY2-S, and Mr-OPY2-L ^ΔAUGs driven by the Pgpd-NUTR. The 'No 5′UTR' indicates that the 5′UTR of the M. acridum gpd gene is excluded. These constructs were transformed into the Mr-OPY2 deletion mutant ΔMr-OPY2. Middle panel: representative Western blot image and analysis to quantify Mr-OPY2 levels in SDY grown cultures of: (1) ΔMr-OPY2; (2) WT; (3) ΔMr-OPY2::Mr-OPY2-L; (4) ΔMr-OPY2::Mr-OPY2-S, and (5) ΔMr-OPY2::Mr-OPY2-L ^ΔAUGs. Bottom panel: a portion of the loading control gel (for the western blot analysis in the middle panel) stained with Coomassie Brilliant Blue. M: Protein ladder. For western blot analysis (a, b middle panels), the Mr-OPY2 levels are calculated relative to the WT which is set at 1 (ΔMr-OPY2 is a negative control). The uncropped images of the western blots and the SDS-PAGE gels are shown in Supplementary Fig. 12. Images are representative of at least three independent experiments

Fig. 4

Precise regulation of Mr-OPY2 levels is important for saprophytic growth and infection. a Constructing strains with manipulated Mr-OPY2 levels. Left panel: diagram illustrating constructs where Mr-OPY2’s ORF is driven by the two Pgpd promoters, P683 and P404, with high and low activity, respectively. These constructs were transformed into ΔMr-OPY2 to produce strains where the Mr-OPY2 level was exclusively controlled by the promoters. The 'With 5′UTR' indicates the 5′UTR of the M. acridum gpd gene is included. Middle panel: western blot analysis of Mr-OPY2 levels in the transformants: (1) ΔMr-OPY2; (2) WT; (3) P404-Mr-OPY2; and (4) P683-Mr-OPY2. Mr-OPY2 levels are calculated relative to the WT which is set at 1. ΔMr-OPY2 is a negative control. Right panel: a portion of the loading control gel (for the Western blot analysis in the middle panel) stained with Coomassie Brilliant Blue. M: Protein ladder. The uncropped images of the western blot and the SDS-PAGE gel are shown in Supplementary Fig. 13. b Colony phenotype (left upper panel), conidiophores (left lower panel) and conidial yields (right panel) of strains with different Mr-OPY2 levels. Note: strain P683-Mr-OPY2 with elevated Mr-OPY2 level relative to WT has a fluffy colony with impaired conidiophores and reduced conidial yield. Colony pictures were taken 18 days after inoculation. Scale bar in the left upper panel represents 10 mm, left lower panel 10 μm. In the right panel, values with different letters are significantly different (n = 9, P < 0.05, Tukey’s test in one-way ANOVA). c Pathogenicity of strains with different Mr-OPY2 levels. Left panel: appressorial development on a hydrophobic surface. At each time point, values with different letters are significantly different (n = 3, P < 0.05, Tukey’s test in one-way ANOVA). Right panel: survival curves of G. mellonella larvae infected by the WT, P404-Mr-OPY2 and P683-Mr-OPY2. Control: insects treated with 0.01% Triton X-100 solution. Bioassays were repeated three times with 40 insects per repeat. Conidial yield and appressorium formation assays were repeated three times with three replicates per repeat. Data are expressed as the mean ± SE. Images are representative of at least three independent experiments for each condition

Fig. 5

Colony phenotypes and osmotic stress tolerance of ΔMr-OPY2, ΔMr-STE50 and WT. a Upper panel: colonies of the strains on PDA (Scale bar represents 10 mm). Middle panel: colonies on PDA plus 0.75 M KCl (scale bar represents 1 mm). Lower panel: bright field images of individual germlings in YE (0.01% yeast extract) plus 0.75 M KCl (Scale bar represents 10 μm). Colony pictures were taken 18 days after inoculation. Germling pictures were taken 16 h after inoculation. b Growth curves of the strains on PDA plates plus 0.75 M KCl. Growth assays were repeated three times with three replicates per repeat. Data are expressed as the mean ± SE. Note: growth of ΔMr-OPY2 and ΔMr-STE50 was greatly reduced. c Upper panel: Western blot analysis of Mr-OPY2 protein levels in ΔMr-OPY2 grown in SDY (1), WT grown in SDY (2) and in SDY plus 0.75 M KCl (3). The band intensity of WT in SDY is set at 1, and WT in SDY with KCl is relative to it; ΔMr-OPY2 is a negative control. Lower panel: a portion of the loading control gel (for the western blot analysis in the upper panel) stained with Coomassie Brilliant Blue. M: Protein ladder. The uncropped images of the Western blot and the SDS-PAGE gel are shown in Supplementary Fig. 13. d Upper panel: yeast two-hybrid confirmation of the physical interaction of Mr-SET50 with Mr-OPY2 and three MAPKKK proteins (SSK2, STE11 and BCK1). Colonies were grown in SD-His-Ade-Leu-Trp + X-α-gal + AbA (Takara, Dalian China). NC: negative control (yeast cells containing the plasmid pGADT7-T and pGBKT7-Lam). PC: positive control (yeast cells containing the plasmid pGADT7-T and pGBKT7-53). Lower panel: Mr-STE50 lacks autoactivation activity. Y2HGold cells with pGBKT7-Mr-STE50 cannot grow in SD-His-Trp-Ade + X-α-gal (Takara, Dalian China). NC and PC are the same as those in the upper panel. Images are representative of at least three independent experiments for each condition

Fig. 6

Regulation of MAPK signaling pathways by Mr-OPY2 and Mr-STE50 during saprophytic growth, pathogenesis and under high osmotic stress. β-Tubulin was used as a loading control. Numbers indicate band intensity for Phos-Fus3p (Phos-Hog1p) relative to total Fus3p (Hog1p). WT values were set to 1. Images are representative of at least three independent experiments for each condition. The uncropped images of the western blots are shown in Supplementary Fig. 14

Fig. 7

Identification and characterization of the appressorial formation transcription factor AFTF1 and its negative regulation by Mr-OPY2. a Venn diagram of RNA-seq analysis showing the distribution of shared DEGs in transcriptomes of WT and ΔMr-OPY2 during growth in SDY (SDY), the insect cuticle (Cuticle) and hemolymph (Hemolymph). Two biological repeats were established for each treatment. b qRT-PCR analysis of Aftf1 expression by the WT grown in hemolymph and on locust cuticle relative to expression during saprophytic growth in SDY (which is set to 1 in the figure). c qRT-PCR analysis of Aftf1 expression by the WT, ΔMr-OPY2, ΔMr-STE50, ΔMero-Hog1, ΔMero-Fus3 and ΔMero-Slt2 during appressorial formation. The expression level in the WT is set to 1. The qRT-PCR analyses were repeated three times with three replicates per repeat. Data are expressed as the mean ± SE. Values with different letters are significantly different (n = 3, P < 0.05, Tukey’s test in one-way ANOVA). d The % germlings differentiating appressoria on a hydrophobic plastic surface in the WT, ΔAftf1 (the Aftf1 disruption mutant), C-ΔAftf1 (the complemented strain of ΔAftf1) and Aftf1 ^OE (a strain overexpressing Aftf1). Appressorium formation assays were repeated three times with three replicates per repeat. Data are expressed as the mean ± SE. At each time point, values with different letters are significantly different (n = 3, P < 0.05, Tukey’s test in one-way ANOVA). e Survival curves of G. mellonella larvae infected by WT, ΔAftf1, C-ΔAftf1 and Aftf1 ^OE. Data are expressed as the mean ± SE. Control: insects treated with 0.01% Triton X-100 solution. The bioassays were repeated three times with 40 insects per repeat. f A schematic model of Mr-OPY2-mediated regulation of appressorial formation and osmotic stress tolerance. Mr-OPY2 regulates the phosphorylation level of Fus3 and Hog1-MAPK under high osmotic stress. During appressorial formation, Fus3-MAPK regulates the expression level of Aftf1, but regulation of Aftf1 by Mr-OPY2 is mediated by unidentified components. Slt2-MAPK also regulates Aftf1, but it remains to be determined whether Mr-OPY2 is involved in such regulation