The fatty acid 2-hydroxylase CsSCS7 is a key hyphal growth factor and potential control target in Colletotrichum siamense

Abstract

SCS7 is a fatty acid 2-hydroxylase required for the synthesis of inositol phosphorylceramide but is not essential for normal growth in Saccharomyces cerevisiae. Here, we demonstrate that the Colletotrichum siamense SCS7 homolog CsSCS7 plays a key role in hyphal growth. The CsSCS7 deletion mutant showed strong hyphal growth inhibition, small conidia, and marginally reduced sporulation and also resulted in a sharp reduction in the full virulence and increasing the fungicide sensitivity. The three protein domains (a cytochrome b5 domain, a transmembrane domain, and a hydroxylase domain) are important to CsSCS7 protein function in hyphal growth. The fatty acid assay results revealed that the CsSCS7 gene is important for balancing the contents of multiple mid-long- and short-chain fatty acids. Additionally, the retarded growth and virulence of C. siamense ΔCsSCS7 can be recovered partly by the reintroduction of homologous sequences from Magnaporthe oryzae and Fusarium graminearum but not SCS7 of S. cerevisiae. In addition, the spraying of C. siamense with naked CsSCS7-double-stranded RNA (dsRNAs), which leads to RNAi, increases the inhibition of hyphal growth and slightly decreases disease lesions. Then, we used nano material Mg-Al-layered double hydroxide as carriers to deliver dsRNA, which significantly enhanced the control effect of dsRNA, and the lesion area was obviously reduced. These data indicated that CsSCS7 is an important factor for hyphal growth and affects virulence and may be a potential control target in C. siamense and even in filamentous plant pathogenic fungi.IMPORTANCECsSCS7, which is homologous to yeast fatty acid 2-hydroxylase SCS7, was confirmed to play a key role in the hyphal growth of Colletotrichum siamense and affect its virulence. The CsSCS7 gene is involved in the synthesis and metabolism of fatty acids. Homologs from the filamentous fungi Magnaporthe oryzae and Fusarium graminearum can recover the retarded growth and virulence of C. siamense ΔCsSCS7. The spraying of double-stranded RNAs targeting CsSCS7 can inhibit hyphal growth and reduce the disease lesion area to some extent. After using nano material Mg-Al layered double hydroxide as carrier, the inhibition rates were significantly increased. We demonstrated that CsSCS7 is an important factor for hyphal growth and affects virulence and may be a potential control target in C. siamense and even in filamentous plant pathogenic fungi.

Keywords: Colletotrichum siamense; control target; fatty acid hydroxylase CsSCS7; hyphal growth factor.

Fig 1

Phylogenetic analysis and protein domains of CsSCS7. (A) Phylogenetic analysis of CsSCS7 and its orthologs in other fungal species, plants, and mammals. A phylogenetic tree was constructed with MEGA 7.0 using the maximum-likelihood method. The CsSCS7 protein amino acid sequence in this study is indicated with a red star. (B) SMART analysis of the CsSCS7 orthologs from fungi, mammals, and plants.

Fig 2

Comparison of colony and conidial characterization of the tested strains. (A) Colony morphology and (B) colony diameter of the tested strains grown on four types of plates for 7 days. (C) Morphology and (D) conidial size of the tested strains. (E) Spore germination rates of the tested strains. Different letters indicate an extremely significant difference (P < 0.01) (one-way analysis of variance [ANOVA] and Duncan’s test), and the error bars represent the standard deviations.

Fig 3

Pathogenicity assays of the strains tested in this study. (A) Disease lesions and dot plot of the lesion area of the wild-type strain HN08, ΔCsSCS7 strain, and complementary strain ΔCsSCS7/CsSCS7 on rubber tree leaves 3 days after inoculation with wounding. (B) Disease lesions and dot plot of the lesion area of the three tested strains on light-green rubber tree leaves 3 days after inoculation without wounding. (C) Disease lesions and dot plot of six tested strains on bronzing leaves 3 days after inoculation with wounding. (D) Disease lesions and dot plot of six tested strains on bronzing leaves 3 days after inoculation without wounding. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and Duncan’s test; the error bars show the standard deviations.

Fig 4

Fungicide sensitivity, the plasma membrane permeability, and gene expression of HN08, ∆CsSCS7, and ∆CsSCS7/CsSCS7. (A) Mycelial growth of tested strains on CM containing prochloraz. (B) Mycelial growth of tested strains on CM containing fludioxonil. (C) Mycelial growth of tested strains on CM containing tebuconazole. (D) Evan’s blue content in HN08, ∆CsSCS7, and ∆CsSCS7/CsSCS7. The experiment was repeated three times. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and Duncan’s test; the error bars show the standard deviations. (E) The expression of drug efflux pump-related genes on three tested strains. ZEB2 (XP_036488279.1), ABCA3 (XM_036646271.1), and PMd1 (XM_036645902.1) from ATP-binding cassette transporters, hxnp-3 (XP_036497635.1) and mfsd1 (XP_036489548.1) from the major facilitator superfamily (MFS). ATP-binding cassette transporters and major facilitator superfamily are the main gene families of drug efflux pumps.

Fig 5

Schematic diagram, colony morphology, and colony diameter of transformants expressing different protein domains. (A) Schematic diagram of the structures of the sequences containing different domains of the CsSCS7 protein. (B) Colony morphology and (C) diameter of transformants expressing the different protein domains grown on four types of plates for 7 days. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and Duncan’s test; the error bars show the standard deviations.

Fig 6

Content of fatty acids in the wild-type strain and ∆CsSCS7. (A) Total content of four types of fatty acids in the wild-type and ∆CsSCS7 strains. SCFAs, short-chain fatty acids; SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids. (B) Content of seven short-chain fatty acids in the wild-type strain HN08 and ∆CsSCS7. (C) Content of 32 medium- and long-chain fatty acids in the wild-type strain HN08 and ∆CsSCS7. The asterisk indicates significant differences within each measurement group (*P < 0.1 and **P < 0.01 according to one-way ANOVA and Duncan’s test). The error bars show the standard deviations.

Fig 7

Effects of dsRNAs targeting CsSCS7 gene on C. siamense growth and virulence. (A) Schematic diagram of dsRNAs targeting different regions of the CsSCS7 gene. (B) Colony morphology and (C) colony diameter of HN08 coinoculated or not coinoculated with Cytb5-dsRNA, Mid-dsRNA, and FA-dsRNA and grown on CM plates for 4 days. (D) Expression levels of CsSCS7 in C. siamense HN08 grown on CM plates 4 days after spraying with dsRNAs. (E) Germinating spores and (F) spore germination rate of HN08 coinoculated or not coinoculated with three dsRNAs for 4 and 6 h. (G) Disease lesions and (H) lesion area of rubber tree leaves caused by C. siamense conidia coinoculated or not coinoculated with three dsRNAs. (I) Disease lesions and (J) lesion area of rubber tree leaves caused by C. siamense conidia coinoculated or not coinoculated with three BioClay. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and Duncan’s test; the error bars show the standard deviations.