StRAB4 gene is required for filamentous growth, conidial development, and pathogenicity in Setosphaeria turcica

Abstract

Setosphaeria turcica, the fungal pathogen responsible for northern corn leaf blight in maize, forms specialized infectious structures called appressoria that are critical for fungal penetration of maize epidermal cells. The Rab family of proteins play a crucial role in the growth, development, and pathogenesis of many eukaryotic species. Rab4, in particular, is a key regulator of endocytosis and vesicle trafficking, essential for filamentous growth and successful infection by other fungal pathogens. In this study, we silenced StRAB4 in S. turcica to gain a better understanding the function of Rab4 in this plant pathogen. Phenotypically, the mutants exhibited a reduced growth rate, a significant decline in conidia production, and an abnormal conidial morphology. These phenotypes indicate that StRab4 plays an instrumental role in regulating mycelial growth and conidial development in S. turcica. Further investigations revealed that StRab4 is a positive regulator of cell wall integrity and melanin secretion. Functional enrichment analysis of differentially expressed genes highlighted primary enrichments in peroxisome pathways, oxidoreductase and catalytic activities, membrane components, and cell wall organization processes. Collectively, our findings emphasize the significant role of StRab4 in S. turcica infection and pathogenicity in maize and provide valuable insights into fungal behavior and disease mechanisms.

Keywords: RNA sequencing; Setosphaeria turcica; StRAB4 gene; conidia; gene silencing; infection ability.

Figure 1

StRab4 is required for S. turcica hyphal development and is essential for maize leaf infection. (A) Images of WT and StRab4i strains growing on PDA plates at 25°C. (B) Growth curve of WT and StRab4i strains growing on PDA plates over the course of 7 days. Error bars represent the standard error of three biological replicates. (C) Conidial production of WT and StRab4i strains. Bar chart shows the numbers of conidia produced by WT and StRab4i strains. ND indicates not detected. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent experimental replicates (^**p < 0.01 and ^***p < 0.001). Error bars represent the standard deviation of three replicates.

Figure 2

StRab4 regulates conidial and mycelial morphology. (A) Conidial morphology of WT and StRab4i strains as observed under a microscope (scale bar, 20 μm). (B) StRAB4 expression in the StRab4i-S strain. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test (^*p < 0.05). Error bars represent the standard deviation (SD) of three replicates. (C) Colony morphology of the WT and StRab4i-S strains on PDA plates after 7 days of growth at 25°C and microscopic views of conidia (scale bar, 20 μm). (D) Conidiophore morphology of WT and StRab4i-S strains (scale bar, 10 μm). (E) Bar chart showing the percentage of conidia with WT-like (normal) and abnormal morphology produced by the WT and StRab4i-S strains (left) and the length of conidia from the WT and StRab4i-S strains (right). Asterisks indicate statistical significance, as determined by two-tailed student’s t-test (^*p < 0.05). Error bars represent SD of three replicates. (F) Reverse transcription-quantitative PCR (RT-qPCR) analysis of the relative expression of septin genes in the WT and StRab4i-S. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test (^**p < 0.01). Error bars represent the SD of three replicates. (G) Early infection counts of maize leaves infected with conidia from the WT and StRab4i strains at 2 days post-inoculation. (H) Quantification of early infection counts caused by the WT and StRab4i strains. ND indicates not detected. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test (^*p < 0.05 and ^***p < 0.001). Error bars represent the SD of three replicates. (I) WT and StRab4i-S strains were subcultured on PDA plates for multiple generations: first (1G), second (2G), and third (3G) generations.

Figure 3

StRab4 regulates conidial germination and hyphal penetration. (A) Micrographs depicting germ tube, appressorium, and penetration peg formation from individual conidia of the WT and StRab4i strains growing on a cellophane layer over PDA. Images were taken at 6, 12, and 24 h post-inoculation (hpi) (scale bar, 10 μm). (B) Quantification of germ tubes, appressoria, and penetration pegs from conidia of WT and StRab4i strains growing on a cellophane layer over PDA. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent replicates (^*p < 0.05 and ^**p < 0.01). Error bars represent the standard deviation (SD) of three replicates. (C) Images depicting penetration by WT and StRab4i strains growing on a cellophane layer over PDA. Images were taken at 5 and 10 days post-inoculation.

Figure 4

StRab4 regulates cell wall integrity and surface hydrophobicity in S. turcica. (A) WT and StRab4i strains were inoculated on PDA plates containing 100 μg/mL Congo Red, 20 μg/mL calcofluor white (CFW), 0.01% sodium dodecyl sulfate (SDS), and control (CK) plates containing no additional chemicals. (B) Inhibition rate of WT and StRab4i strains growing on PDA plates containing 100 μg/mL Congo Red, 20 μg/mL CFW, and 0.01% SDS was quantified by normalizing to growth on the CK negative control plate. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent replicates (^*p < 0.05 and ^**p < 0.01). Error bars represent the standard deviation of three replicates. (C) Determination of surface hydrophobicity in WT and StRab4i strains. Colonies were treated with a 30 μL H₂O, 0.2% gelatin, 250 mg/mL Tween-20, or 0.2% SDS with 5 mM EDTA. Images were taken at 0 and 12 h post-inoculation (hpi). A: H₂O; B: 0.2% gelatin; C: 250 mg/mL Tween 20; D: 0.2% SDS with 5 mM EDTA.

Figure 5

StRab4 regulates melanin synthesis in S. turcica. Intracellular melanin was extracted from WT and StRab4i strains in liquid culture with shaking. Extracellular melanin was extracted from fungal strains growing in liquid culture in the presence (shaking) and absence (static) of shaking. (A) Intracellular melanin in fungal extracts was black-to-light brown in color depending on the concentration and intracellular melanin content from WT and StRab4i cultures was quantified. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent replicates (^**p < 0.01). Error bars represent the standard deviation of three replicates. (B,C) Extracellular melanin in fungal extracts was black-to-light brown in color depending on the concentration and extracellular melanincontent from WT and StRab4i cultures was quantified in the presence (shaking) and absence (static) of shaking. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent replicates (^**p < 0.01). Error bars represent the standard deviation of three replicates.

Figure 6

Differential gene expression and functional enrichment analysis between the WT and StRab4i strains. (A) Volcano plot depicting the differentially expressed genes (DEGs) between the WT and StRab4i-2. Red and green dots signify significantly up-and down-regulated genes, respectively (adjusted p-value ≤0.01 and log2FC >2). Blue dots signify genes that did not have significant changes in gene expression. Fold changes were calculated using fragments per kilobase million (FPKM) values. (B) Clusters of Orthologous Groups (COG) classification of all assembled genes. (C) Representation of the top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among the DEGs. (D) Visualization of Gene Ontology (GO) terms enriched among the DEGs. (E) Heatmap expression of selected genes that are differentially expressed during conidia development. The color gradient from blue to red corresponds to an ascending order of expression.

Figure 7

The validation of differentially expressed genes in WT and StRab4i-2. (A) Expression levels of the related genes of peroxisome. Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent replicates (^*p < 0.05 and ^**p < 0.01). Error bars represent the standard deviation of three replicates. (B) Validation levels of the differentially expressed genes (DEGs), including HSP12 (gene ID: St005597), CPP (gene ID: St011758), 3HNR (gene ID: St002127), GPI-CFEM (gene ID: St003016), SCD1 (gene ID: St006222). Asterisks indicate statistical significance, as determined by two-tailed student’s t-test with three independent replicates (^*p < 0.05 and ^**p < 0.01). Error bars represent the standard deviation of three replicates.

Figure 8

A functional model of the StRab4i of S. turcica. StRAB4 gene plays an important role in S. turcica. In this study, the StRAB4 gene functions in filamentous growth, conidial development, and pathogenicity of S. turcica. Further investigations revealed StRab4 is a positive regulator of cell wall integrity and melanin secretion. In the StRab4i mutant, functional enrichment analysis of differentially expressed genes (DEGs) highlighted primary enrichments in peroxisome pathways, cell wall organization processes, membrane components, and melanin metabolic processes.