Pleiotropic Roles of ChSat4 in Asexual Development, Cell Wall Integrity Maintenance, and Pathogenicity in Colletotrichum higginsianum

Abstract

Potassium has an important role to play in multiple cellular processes. In Saccharomyces cerevisiae, the serine/threonine (S/T) kinase Sat4/Hal4 is required for potassium accumulation, and thus, regulates the resistance to sodium salts and helps in the stabilization of other plasma membrane transporters. However, the functions of Sat4 in filamentous phytopathogenic fungi are largely unknown. In this study, ChSat4, the yeast Sat4p homolog in Colletotrichum higginsianum, has been identified. Target deletion of ChSAT4 resulted in defects in mycelial growth and sporulation. Intracellular K+ accumulation was significantly decreased in the ChSAT4 deletion mutant. Additionally, the ΔChsat4 mutant showed defects in cell wall integrity, hyperoxide stress response, and pathogenicity. Localization pattern analysis indicated ChSat4 was localized in the cytoplasm. Furthermore, ChSat4 showed high functional conservation with the homolog FgSat4 in Fusarium graminearum. Taken together, our data indicated that ChSat4 was important for intracellular K+ accumulation and infection morphogenesis in C. higginsianum.

The MAPKKK CgMck1 Is Required for Cell Wall Integrity, Appressorium Development, and Pathogenicity in Colletotrichum gloeosporioides

“…Mycelial plugs of the wild type, the gene deletion mutants and the complemented strains were inoculated onto CM, PDA, Mathur’s agar, and minimal medium (MM), respectively. Plates were kept in an incubator at 25 °C and colony diameter was measured at 5 days post inoculation [ 22 ]. Fruiting bodies were induced to develop on V8 medium plates [ 19 ] after inoculation for 10 days at 25 °C under 16 h/8 h light and dark cycle.…”

“…Mycelial plugs were inoculated onto the CM agar plates with sodium dodecylsulfate (SDS) (0.005%), Calcofluor white (CFW) (50 and 100 µg mL −1 ), Congo red (CR) (200 and 600 μg mL −1 ), NaCl (0.7 M), sorbitol (1 M) and cultured in the dark for 5 days at 25 °C. Hyphal growth inhibition rate was calculated using the method described previously [ 22 ].…”

Calcineurin-Responsive Transcription Factor CgCrzA Is Required for Cell Wall Integrity and Infection-Related Morphogenesis in Colletotrichum gloeosporioides

“…The wild type (WT) C. gloeosporioides s.s. strain SMCG1#C was used for transformation. The WT, Δ CgcrzA mutant, and complemented strains were maintained on potato dextrose agar (PDA) medium ( Yang et al, 2018 ). Fungal growth assays were performed on PDA and complete medium (CM) at 25oC for 5 days in the dark.…”

“…The upstream and downstream flanking regions (~1.5 kb) of CgCRZA were amplified and fused on either side of the hygromycin B phosphotransferase gene ( HPH ) cassette to generate the gene replacement constructs. Then, the constructs were transformed into protoplasts of the WT as previously described ( Yang et al, 2018 ). Subsequently, the candidate transformants were screened by PCR analysis of open reading frame of CgCRZA and further confirmed by Southern blotting.…”

“…One microgram of the RNA was reverse transcribed into the first-strand cDNA according to the manual of HiScript II Reverse Transcriptase (Vazyme Biotech Co., Nanjing, China), and the cDNA was used as the templates of qRT-PCR. qRT-PCR reactions were conducted following the method described by Yang et al (2018) , and 18S rRNA was used as a reference gene. The experiment was performed three times, and each treatment had three replicates.…”