Molecular Mechanisms Of Nitric Oxide Regulated Conidiation In Coniothyrium Minitans

Coniothyrium minitans is a sclerotial mycoparasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and an effective biocontrol agent against Sclerotinia disease. Understanding signaling pathways that modulate conidiation of mitosporic fungi is of both practical and theoretical importance. Previous studies have demonstrated that L-arginine-derived nitric oxide (NO) regulate conidia formation of C. minitans. Despite the importance of NO signaling in fungi, the mechanisms responsible for the transduction and turnover of NO bioactivity are not well understood. The enzymatic origin of NO, the NO signal transmission and gene up-regulated during conidiation of C. minitans were investigated.The second messenger cGMP have just been reported in several model fungus, but the mechanism of synthesis and biological function was rarely understand. This research is the first evidence in fungi that cGMP was downstream of NO signal and involve in conidiation. The activity of a nitric oxide synthase-like (NOS-like) enzymew as detected in C. minitans as evidenced by the conversion of L-arginine to L-citrulline. Guanylate cyclase (GC) activity was also detected indirectly in C. minitans with the GC-specific inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), which significantly reduced production of cyclic GMP (cGMP). The dynamics of NOS activity were closely mirrored by the cGMP levels during pycnidial development, with the highest levels of both occurring at the pycnidial initiation stage of C. minitans. Furthermore, the NO donor, sodium nitroprusside (SNP), stimulated the accumulation of cGMP almost instantly in mycelium during the hyphal growth stage. When the activity of NOS or GC was inhibited with Noo-nitro-L-arginine or ODQ, conidial production of C. minitans was suppressed or completely eliminated; however, the suppression of conidiation by ODQ could be reversed by exogenous cGMP. The results also showed that conidiation of an L-arginine auxotroph could be restored by the NO donor SNP, but not by cGMP. Thus, NO-mediated conidiation has more than one signal pathway, including the cGMP signal pathway and another yet-unknown pathway, and both are essential for conidiation in C. minitans.Protein S-nitrosylation modification is an important pathway of NO function. To understand whether this chemical modification plays roles in conidiation, C. minitans S-nitrosoglutathione reductase (CmGSNOR) was identified and the biofunction was studied. The protein of GSNOR was demonstrated in previous research to metabolize the NO-related species S-nitrosoglutathione (GSNO) in vivo, was identified in the filamentous fungus C. minitants. The CmGSNOR deletion mutants and gene complement strains were obtained and their phenotypes were characterized. Loss of CmGSNOR function blocked the ability to metabolize GSNO completely and lead to increase the intracellular levels of S-nitrosothiol (SNO) and NO. The⊿Cmgsnor strains have many phenotype changes, including abnormal colony and hyphal morphology, reduced conidial production, atypical conidial shape and enhanced germination. These abnormal phenotypes could be restored in complement strains.Moreover, in the absence of CmGSNOR, the nitrosative stress resistance to GSNO was seriously impaired, but the antioxidant resistance to hydrogen peroxide (H2O2) was enhanced unexpectedly. The expression of CmGSNOR was positively regulated by the extracellular GSNO, however, it was not altered by the H2O2 treatment. Furthermore, the ACmgsnor mutant was more resistant to high osmotic environments induced by sodium chloride than the wild type strain. These data imply SNO formation and turnover regulate multiple processes in filamentous fungal normal growth and responses to environmental stress. In total, our results demonstrate the expanded importance of GSNOR-regulated SNO homeostasis to filamentous fungal development and abiotic stresses.Previous studies have shown that L-arginine can increase the level of NO in mutant ZS-1T2029 and restore its sporulation phenotype. NO is an essential molecule in many fungal physiological processes, but there has not been an extensive survey of the transcriptional changes induced by endogenous NO in fungi. Here, a L-arginine auxotroph ZS-1T2029, which has low levels of NO during pycinidial development growing on potato dextrose agar (PDA) was used to probe genes up-regulated by endogenous NO with a suppression subtractive hybridization (SSH) technique. The mycelial mass of ZS-1T2029 during pycnidial development harvested from PDA medium amended or not amended with L-arginine was and subjected to SSH analyses A cDNA library with approximately 2000 clones was constructed from these two mRNA samples. Among these,421 clones were confirmed to have abundant mRNA during conidiation. After sequencing and analysis,226 unique up-regulated genes were identified.The functions of these genes involve general categories such as metabolism (17.9%), energy (8.6%), protein synthesis (13.2%), protein fate (9.9%), and transcellular transport (7.9%) among others. The expression levels of 13 genes were confirmed to be up-regulated by NO with time-course semi-quantitative RT-PCR analyses. Furthermore, two up-regulated genes, CmFDS1 and CmCAP1, were confirmed to be involved in conidiation with the RNAi technique. This study provides the insight into C. minitans genes up-regulated during conidiation, and is important clues for studying NO-responsive genes involved in conidiation of fungi.Together, the possible signal transduction pathways of NO-mediaed conidiation in C. minitans was initial clarified:the second messenger cGMP and protein S-nitrosylation modification play an important regulatory role in conidia development. Furthermore, we identified of a variety of genes and metabolic pathways which up-regulated during conidiation by constructing SSH library. These findings can expand our understanding the molecular mechanism of NO signal and conidiation in C. minitans.