A MYST family histone acetyltransferase, MoSAS3, is required for development and pathogenicity in the rice blast fungus

Abstract

Histone acetylation has been established as a principal epigenetic regulatory mechanism in eukaryotes. Sas3, a histone acetyltransferase belonging to the largest family of acetyltransferase, MYST, is the catalytic subunit of a conserved histone acetyltransferase complex. To date, the functions of Sas3 and its orthologues have been extensively studied in yeast, humans and flies in relation to global acetylation and transcriptional regulation. However, its precise impact on development and pathogenicity in fungal plant pathogens has yet to be elucidated. Considering the importance of Sas3 in H3K14 acetylation, here we investigate the roles of its orthologue in the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Unlike a previously reported Sas3 deletion in yeast, which led to no remarkable phenotypic changes, we found that MoSAS3 deletion alone had a profound effect on fungal growth and development, including asexual reproduction, germination and appressorium formation in M. oryzae. Such defects in pre-penetration development resulted in complete loss of pathogenicity in the deletion mutant. Furthermore, genetic analysis of MoSAS3 and MoGCN5 encoding a Gcn5-related N-acetyltransferase family histone acetyltransferase suggested that two conserved components of histone acetylation are integrated differently into epigenetic regulatory mechanisms in the yeast and a filamentous fungus. RNA-seq analysis of ΔMosas3 showed two general trends: many DNA repair and DNA damage response genes are up-regulated, while carbon and nitrogen metabolism genes are down-regulated in ΔMosas3. Our work demonstrates the importance of MYST family histone acetyltransferase as a developmental regulator and illuminates a degree of functional variation in conserved catalytic subunits among different fungal species.

Keywords: MoSAS3; development; histone acetyltransferase; pathogenicity; rice blast.

Figure 1

MoSAS3 as a histone acetyltransferase. (A) Domain architecture of MoSAS3 and its orthologues. (B) Localization of MoSAS3 in Magnaporthe oryzae spores. Green fluorescence in conidia expressing MoSAS3‐GFP (middle panel) and DAPI staining pattern (bottom panel). (C) Quantification of western blot signal for H3K14 acetylation, relative to H3. The western blot image was quantified using ImageJ software (https://imagej.nih.gov/ij/index.html).

Figure 2

Growth and asexual reproduction of wild‐type Magnaporthe oryzae (KJ201, WT), ΔMosas3 (KO) and complementation strain (Comp, MoSAS3c). (A) Colony morphology and pigmentation on oatmeal agar plates. (B) Growth rate measured in colony diameter at 9 days post‐inoculation. (C) Asexual sporulation measured in the number of spores produced from individual strains. (D) Conidiophore development. Mycelial mat was scraped off and then a small agar block (1 × 1 cm) was excised and incubated for 8 h under constant fluorescence light to induce sporulation before being observed under a microscope. Asterisks indicate statistically significant differences (P < 0.001, Tukey HSD test).

Figure 3

Defect in conidial germination and appressorium formation of Magnaporthe oryzae ΔMosas3. (A) Quantitative analysis of spore germination and appressorium formation in wild‐type (KJ201), ΔMosas3 and complementation strain (MoSAS3c) after 8 h of incubation on hydrophobic surface (glass coverslip) at 25 °C. Percentages of ungerminated, germinated and appressorium‐forming conidia were counted under a light microscope. (B) Representative image for germination and appressorium formation of different strains on hydrophobic surface (plastic coverslip). (C) Spore viability test using FUN‐1 staining. Dead spores were prepared by subjecting them to microwave treatment for 1 min. Live cells localize dye to vacuoles, while dead cells display diffuse signals throughout the cytoplasm.

Figure 4

Dissection of pathogenicity defect in MoSAS3 deletion mutant. (A) Lesion development in rice leaves 7 days post‐spray inoculation. (B) Lesion development in wound‐inoculated rice leaves 5 days post‐inoculation. Distilled water was used as a mock experiment. (C) Observation of invasive growth using rice sheath assay. Invasive growth in rice sheath cells was monitored under microscope at 48 h post‐inoculation.

Figure 5

Test for genetic interaction between MoSAS3 and MoGCN5. (A) Comparison of growth on oatmeal agar plates for ΔMosas3, ΔMogcn5 and the double deletion mutant (ΔMosas3/ΔMogcn5). (B) Growth of individual strains measured as colony diameter. (C) Asexual sporulation measured as the number of spores produced from individual strains. Different letters in bar plots indicate significantly different mean values (Tukey HSD test, P < 0.001).

Figure 6

Summary of GO enrichment analysis (biological process) for selected terms. The whole GO enrichment analysis is available in the Supporting Information. GO terms that are enriched with up‐regulated and down‐regulated genes are shown in (A) and (B), respectively (Fisher's exact test, P < 0.01). The area of the circle represents the number of genes assigned to the particular GO term. The colour of the circle indicates the proportion of genes assigned to the GO term in our dataset among the total number of genes having that GO term in the genome.