Regulatory effects of nitric oxide on reproduction and melanin biosynthesis in onion pathogenic fungus Stemphylium eturmiunum

Highlights

• Nitric oxide is required for the reproduction in S. eturmiunum.

• Nitric oxide dictates melanin biosynthesis in S. eturmiunum.

• Melanin biosynthesis maters the reproduction in S. eturmiunum.

Abstract

The formation of propagules is the critical stage for transmission of the pathogenic fungus Stemphylium eturmiunum. However, how the development of these propagules is regulated remains to be fully understood. Here, we show that nitric oxide (NO) is necessary for reproduction in S. eturmiunum.Application of NO scavenger carboxy-CPTIO (cPTIO) or soluble guanylate cyclase (sGC) inhibitor NS-2028 abolishes propagules formation, which was increased by a supplement of sodium nitroprusside (SNP). SNP supplement also triggered increased biosynthesis of melanin, which can be inhibited upon the addition of arbutin or tricyclazole, the specific inhibitors for DOPA and DHN synthetic pathway, respectively. Intriguingly, enhanced melanin biosynthesis corelates with an increased propagules formation; The SNP-induced increment propagules formation can be also compromised upon the supplement of cPTIO or NS-2028. RT-PCR analysis showed that SNP promoted transcription of brlA, abA and wetA at 0.2 mmol/L, but inhibited at 2 mmol/L. In contrast, SNP increased transcription of mat1, and mat2, and the synthetic genes for DHN and DOPA melanins at 2 mmol/L. However, the increased transcription of these genes is down-regulated upon the supplement of cPTIO or NS-2028. Thus, NO regulates reproduction and melanin synthesis in S. eturmiunum possibly through the NO-sGC-GMP signaling pathway.

Introduction

The foliar fungal pathogen Stemphylium eturmiunum is a homothallic filamentous ascomycete causing severe leaf blight of onions (Fernandez and Rivera-vargas, 2008). In addition, S. eturmiunum is also a postharvest spoiler of fresh tomatoes (Andersen and Frisvad, 2004; Trinetta et al., 2013). The infection at the lesions of fresh tomatoes by the fungus results in the production of toxic metabolites infectopyrone and macrosporin (Andersen and Frisvad, 2004). In natural habitats, S. eturmiunum develops asexual conidiophores for conidiation, and sexual fruiting bodies pseudothecia for producing ascospore (Simmons, 2001). The conidia that easily reach the host alveoli are the major infective propagules (Muñiz-Paredes et al., 2017). Notably, the conidial cell wall, directly in contact with host cells, consists of melanins in addition to the presence of a-(1,3)-glucan and proteinaceous rodlets. The fungal melanins, synthesized via DOPA or DNH pathway (Chang et al., 2019) both possess the potential to scavenge free radicals (Pacelli et al., 2020). In filamentous fungi, the presence of melanins in the conidial cell wall confers resistance to the attack of free radicals from host organisms, which, in turn, increases their pathogenicity for effective infection and transmission (Chamilos and Carvalho, 2020; Amin et al., 2014; Cunha et al., 2010). The sexual fruiting body of fungi was constructed by tightly interwoven melanin-contained hyphae (Engh et al., 2007). Similar to conidia, the unique structure of pseudothecia also confers fungi the capacity to survive in a harsh environment (Lambou et al., 2008; Zhao et al., 2017). Thus, the involvements of melanin in the construction of conidia and pseudothecia endow the resistance of fungi against environmental insults and the effective infection and transmission (Pal et al., 2014).

Asexual sporulation is the most common mode for reproduction and transmission in filamentous fungi (Park et al., 2012) and is timed and genetically programmed (Adams et al., 1998; Lee et al., 2010). In Aspergillus, the conidial sporulation is determined by a central regulatory pathway, where the transcriptional factors brlA, abaA, wetA coordinate conidiation-specific gene expression and determines the order of gene activation during the formation of conidiophores and maturation of conidia (Boylan et al., 1987; Mirabito et al., 1989).

Similar to asexual sporulation, the formation of pseudothecia is also regulated by multiple genes. Previously, we showed that the development of pseudothecia in S. eturmiunum is regulated not only by the genes encoding MAT1, MAT2, and the protein in G-protein signaling pathway but also by magl, the gene responsible for the synthesis of arachidonic acid (Zhao et al., 2019). Treatment of S. eturmiunum with 5-azacytidine (5-AC) resulted in the complete silence of magl followed by the disruption in the development of pseudothecia and melanin synthesis. Interestingly, the impaired development of pseudothecia can be restored by an external supplement of arachidonic acid (Zhao et al., 2019), which implicates the significance of arachidonic acid, the common source for the genesis of oxylipins (Aukema et al., 2016), in sexual development of S. eturmiunum.

The successful survival of fungi in nature depends on efficient communication with their surroundings (Hassan et al., 2019). In filamentous fungi, the communication with habitats is achieved by complex signaling systems that determine the fungal proliferation, development, and in some cases virulence (Kozubowski et al., 2009). The well-known signaling pathways in fungi include the protein kinase A/cyclic AMP (cAMP), protein kinase G/cyclic GMP (cGMP), protein kinase C (PKC)/mitogen-activated protein kinase (MAPK), lipid signaling cascades, and the calcium-calcineurin signaling pathway (Kozubowski et al., 2009). PKG/cGMP pathway, which seems to exist in filamentous fungi, is activated by free radical molecule NO (Zhao et al., 2020). The NO, highly diffusible within the cell or through the cell membrane (Lancaster, 1997), can work as a transient, local, intra- or intercellular signaling molecule in miscellaneous biological systems including fungi (Culotta and Koshland, 1992). In fungi, NO signaling is involved in conidiation, the formation of sexual fruiting bodies, and the regulation of secondary metabolism (Zhao et al., 2020). Established pieces of evidence have shown that NO can function as the activator of sGC able to bind selectively with the hemoprotein in sGC even in the presence of oxygen (Boon and Marletta, 2005). The binding of NO with Heme iron triggers the change sGC conformation and subsequently activates its catalytic domain, where GTP can be catalyzed to cyclic GMP (cGMP) (Friebe and Koesling, 2003). The produced cGMP then combines with PKG, which constitutes a central downstream mediator of the NO-cGMP-PKG signaling pathway, and orchestrates pathway-specific cellular response through the phosphorylation of phosphorylation-dependent transcriptional factors (Contestabile, 2008).

Studies have shown that NO-mediated signaling networks are involved in conidiation (Wang and Higgins, 2005; Gong et al., 2007), the formation of the parasitic structure of appressoria (Prats et al., 2008), asexual structures of sporangiophores (Maier et al., 2001), and sexual structures of cleistothecia in Aspergillus species (Baidya et al., 2011). In our previous attempts to unravel the regulatory mechanisms for sexual development of S. eturmiunum, we found that treatment of the fungus with 5-AC resulted in the formation of albinism phenotype and the silence of the genes responsible for melanins synthesis in addition to magl. Application of SNP partially restored biosynthesis of melanins in 5-AC treated S. eturmiunum. Intriguingly, SNP supplement in non-treated S. eturmiunum increased the number of conidia and pseudothecia. However, how NO signaling affects asexual and sexual development remains to be established. In this study, we used SNP as the external NO source to probe the regulatory effects of NO on conidiation, sexual fruiting body formation, and production of melanin. Our data showed that NO-induced increment in conidiation, the formation of pseudothecia, and melanin accumulation is possibly mediated by the NO-cGMP signaling pathway, where NO works as an sGC activator triggering the signal transduction cascade.