The Farnesyltransferase β-Subunit Ram1 Regulates Sporisorium scitamineum Mating, Pathogenicity and Cell Wall Integrity

Abstract

The basidiomycetous fungus Sporisorium scitamineum causes a serious sugarcane smut disease in major sugarcane growing areas. Sexual mating is essential for infection to the host; however, its underlying molecular mechanism has not been fully studied. In this study, we identified a conserved farnesyltransferase (FTase) β subunit Ram1 in S. scitamineum . The ram1 Δ mutant displayed significantly reduced mating/filamentation, thus of weak pathogenicity to the host cane. The ram1 Δ mutant sporidia showed more tolerant toward cell wall stressor Congo red compared to that of the wild-type. Transcriptional profiling showed that Congo red treatment resulted in notable up-regulation of the core genes involving in cell wall integrity pathway in ram1 Δ sporidia compared with that of WT, indicating that Ram1 may be involved in cell wall integrity regulation. In yeast the heterodimeric FTase is responsible for post-translational modification of Ras (small G protein) and a-factor (pheromone). We also identified and characterized two conserved Ras proteins, Ras1 and Ras2, respectively, and a MAT-1 pheromone precursor Mfa1. The ras1 Δ, ras2 Δ and mfa1 Δ mutants all displayed reduced mating/filamentation similar as the ram1 Δ mutant. However, both ras1 Δ and ras2 Δ mutants were hypersensitive to Congo red while the mfa1 Δ mutant was the same as wild-type. Overall our study displayed that RAM1 plays an essential role in S. scitamineum mating/filamentation, pathogenicity, and cell wall stability.

Identification and Functional Analysis of the Pheromone Response Factor Gene of Sporisorium scitamineum

“…This confirms that S. scitamineum a locus gene SsMFA1 and SsPRA1 are functional in regulating fungal dimorphic switch, and likely acting at downstream of SsPrf1. This result is also consistent with a recent report on SsMFA1 characterization (Sun et al, 2019).…”

“…As reported in this study, we identified a Prf1 ortholog in S. scitamineum and found that it was responsible for both basal and pheromone-induced expression of mating type genes of the a1 locus. We further showed that deletion of the a locus gene SsMFA1 or SsPRA1 resulted in similar phenotype (Figure 6A) as the T-DNA insertion mutation or deletion mutant of the SsPRF1 gene (Figure 3A), as well as the reported MFA1 deletion phenotype (Sun et al, 2019). Our results provide new insight into the mechanism of pathogenicity of this important sugarcane pathogen, by confirming the biological function of the a 1 locus gene in S. scitamineum mating/filamentation, and indicating that SsPrf1 plays a key role in pheromone signaling and filamentous growth in S. scitamineum through transcriptional induction of the mating locus.…”

“…The conserved b locus genes have also been functionally investigated, with the gene sequence encoding the pheromone receptors, PRA1 and PRA2 , annotated (Yan et al, 2016a, b). The MFA1 and MFA2 gene, respectively encoding the pheromone precursors in MAT-1 and MAT-2 mating-type strain, have both been annotated and characterized by reverse genetics (Lu et al, 2017; Sun et al, 2019). However, the pheromone response factor Prf1 that governs pheromone-induced transcription of a and b loci in U. maydis , has not yet been identified or characterized in S. scitamineum .…”

“…For fungal biomass assessment in the infected sugarcane seedlings, the sugarcane stem tissue infected by S. scitamineum sporidia (compatible mating types mixing) was collected at 72 hpi. Quantification of relative fungal biomass in infected sugarcane stem tissue was performed using the fungal ACTIN gene as reference, and sugarcane glyceraldehyde dehydrogenase ( GAPDH ) gene as reference for normalization, following the established protocol (Sun et al, 2019).…”

Ovarian Tumor Domain-Containing Proteases-Deubiquitylation Enzyme Gene SsCI33130 Involved in the Regulation of Mating/Filamentation and Pathogenicity in Sporisorium scitamineum

“…After the gene was knocked out, the sexual mating ability of WT17 and WT18 was completely lost, and they were unable to infect sugarcane (Yan et al, 2016b). Sun et al (2019) found that the gene Ram1 encoding the farnesyltransferase (FTase) β-subunit in S. scitamineum through gene knockout and gene complement techniques was related to sexual mating, cell wall stability, and pathogenicity in S. scitamineum. Zhu et al (2020) reported that the pheromone response factor SsPRF1 was involved in the regulation of mycelial growth, sexual mating, and pathogenicity in S. scitamineum.…”