The formation of conidia and pseudothecia of the onion pathogenic S. eturmiunum is the critical stage for its transmission. However, how the development of the two propagules is regulated remains to be fully understood. In the study, we showed that NO is necessary for conidiation and the formation of pseudothecia in S. eturmiunum. Application of NO scavenger cPTIO abolishes the formation of conidia and pseudothecia, as well as melanin production. In control cultures supplement of SNP results in an increased formation of conidia at 0.2 mmol/L, and pseudothecia at 2 mmol/L. SNP supplement also triggered increased biosynthesis of melanin, which can be inhibited upon addition of either arbutin or tricyclazole, the specific inhibitors for DOPA and DHN melanin synthetic pathway, respectively. Moreover, the enhanced biosynthesis of melanin coincides with the increased formation of the two propagules that are impaired following the inhibition of melanin biosynthesis (Fig. 10).
The small free radical NO is a short-lived but highly reactive diatomic gas (Brüne 2010). Because it is highly diffusible within the cell and through cell membranes (Lancaster 1997), NO functions as a transient, local, intracellular or intercellular signaling molecule in diverse biological systems (Culotta & Koshland 1992). One of the important mechanisms that NO regulates physiological processes of living system is the binding of NO with transition metals of metalloproteins such as sGC, a hemoprotein that has evolved to bind selectively with NO (Boon & Marletta 2005). The activation of sGC by NO results in the production of the secondary messenger cyclic GMP (cGMP) (Friebe & Koesling 2003) that binds to protein kinase G (PKG), and forms the central downstream mediator of NO-cGMP signaling pathway, and mediates pathway-specific cellular responses via the phosphorylation of phosphorylation-dependent transcriptional factors, such as the cAMP-response-element-binding protein (Contestabile 2008), and activates its downstream targets (Tomankova et al. 2017).
In filamentous fungi, endogenous production of NO correlates with mycelial growth and conidia formation (Marcos et al. 2020). In A. nidulans, increased production of NO was detected in the transition from vegetative growth to conidiation (Marcos et al. 2016). Similar to A. nidulans, our results showed that the presence of NO-producing capacity followed by intracellular production of NO correlates with the regular conidiation, which is compromised following the addition of NO scavenger cPTIO. This indicates that maintaining a certain level of cellular NO underpins the vegetative growth and asexual development in S. eturmiunum. Interestingly, addition of NO-releasing compound SNP resulted in an evident reduction in NO-producing capacity (Fig. 7A), which suggests that NO-producing capacity can be self-regulated in response to alteration of the cellular NO levels. In addition, SNP supplement at concentrations lower than 0.2 mmol/L also promotes conidiation, which implicates that external application of NO at lower concentrations favors the transcription of brlA, abaA, and wetA, the three transcriptional factors involved in conidiation (Chen et al. 2020).
Cellular NO levels also affect sexual development in filamentous fungi (reviewed by Zhao et al. 2020). In Aspergillus nidulans, the increase in cellular NO levels by disrupting the genes encoding flavohemoglobin or supplementing NO-releasing compound promotes the formation of cleistothecia (Baidya et a., 2011). Similar to A. nidulans, supplement of SNP at 2 mmol/L increased transcription of mat1 and mat2 followed by increased formation of pseudothecia, which indicates that the higher NO levels also tend to induce sexual development in S. eturmiunum.
Melanins production in fungi is believed to protect hyphae and propagules from environmental stresses (Bell and Wheeler 1986; Butler and Day 1998), and to serve as a virulence factor (Langelder et al. 2003; Nsanchuk and Casadevll, 2003). Disruption of melanin biosynthesis compromised fungal ability for survival and longevity (Engh et al. 2007). In Pestalotiopsis microspore, the production of polyketide-derived DNH melanin is required for the formation of integrated conidia and viability in addition to morphogenesis, germination and viability (Yu et al. 2015). Moreover, production of DNH melanin is also linked to the development of sexual fruiting body, and is controlled by specific regulatory genes involved in sexual differentiation in Sordaria macrospora (Engh et al. 2007). In our study, external addition of NO also promotes the transcription of the two genes encoding two polyketide synthases, and the gene coding for tyrosinase followed by increased melanin accumulation and formation of conidia and pseudothecia. Notably, inhibiting biosynthesis of DOPA and DNH melanin all lead to the impairment in the formation of conidia and pseudothecia. This indicates that biosynthesis of both DOPA and DNH melanin is required for asexual and sexual development in S. eturmiunum.
The melanins present in fungal cells are involved in their protection from UV radiation desiccation, salinity and oxidation (Pacelli et al. 2020). Melanins in phytopathogenic or human pathogenic fungi are also involved in the protection against ROS attack from host defense (Papon et al. 2020). In filamentous fungi, most ascomycetes produce DHN-melanin (Gonçalves et al. 2012). While in the ubiquitous human-pathogenic fungus Aspergillus fumigatus, the pathogen that causes fatal lung infection in immunocompromised individuals, produces both DOPA and DHN melanins (Langfelder et al. 2003). The presence of DHN melanin in conidia protects from phagocytic uptake and ROS-induced intracellular killing by frugivorous amoeba Protostelium aurantium and disrupts its autonomous defense (Ferling et al. 2020). In Sporothrix schenckii, production of DOPA melanin in the presence of tyrosine in fungal cell confers more resistance to nitrogen-derived oxidants and UV irradiation (Almeida-Paes et al. 2012). In our study, heterologous expression of pks1 and pks2 all resulted in the production of pigmented conidia with resistance to UV irradiation. In this context, the production of DOPA and DHN melanins found in phytopathogenic S. eturmiunum and their tight connections with the formation of conidia and pseudothecia implicate that this fungus possesses more resistance to the attacks from environmental stressors than those producing DNH melanins alone.
The primary sensor of NO soluble guanylate cyclase (sGC) orchestrates NO-cGMP-PKG signaling pathway, and has been implicated in many essential physiological processes and disease conditions in mammals (Kang et al. 2019). Several studies have reported that sporulation in filamentous fungi is promoted by application of external NO but inhibited by sGC inhibitor L-NAME or ODQ. However, sporulation can be restored by exogenous cGMP (Zhao et al. 2020). In our study, NO promoted formation of conidia and pseudothecia, and melanin production can also be compromised by sGC specific inhibitor NS-2080 even in the presence of SNP. This implicates the existence of NO-cGMP-PKG signaling pathway in S. eturmiunum that mediates the increased transcription of the genes encoding the downstream regulators for conidiation, formation of pseudothecia and melanin biosynthesis.
It should be noted that, in our study, in contrast to the increased formation of conidia and pseudothecia by lower concentrations of SNP (≤ 2 mmol/L), external supplement of SNP at higher concentrations (≥ 4 mmol/L) inhibited or compromised the formation of the two propagules and melanin biosynthesis. This indicates that higher concentrations of NO can covalently binds with thiols in active cysteine residues (Amal et al. 2019; Heinrich et al. 2013), leading to S-nitrosylation of active proteins and subsequent negative feedback of NO signaling (Zhao et al. 2016).
This study revealed the NO-mediated mechanisms in regulating asexual and sexual development of onion blight pathogen S. eturmiunum. Further studies should be directed to the identification of downstream transcription factors of NO-cGMP-PKG, which is undertaking in our lab. However, our data reveal, for the first time, that the cellular levels of NO determines the fate of asexual and sexual development of S. eturmiunum. Moreover, the specific requirement for NO by S. eturmiunum in the development of conidia and pseudothecia implicates a possible strategy to curb the transmission of this onion pathogen by applying higher concentration of NO-releasing compound to impede its asexual and sexual development.