Regulation of subtilisin‐like protease prC expression by nematode cuticle in the nematophagous fungus Clonostachys rosea

Abstract

Nematophagous fungi have been used as biological control agents against nematodes parasitic to plants and animals. These fungi can secret subtilisin-like extracellular serine proteases during the infection of nematodes. The expression of these subtilisin-like serine proteases is regulated by nitrogen sources, including nematode cuticle. However, the mechanisms underlying the nitrogen sources-induced expression of these serine proteases is not well understood. In this study, we investigated the effect of nitrogen sources on the expression of a subtilisin-like extracellular protease, prC, in the nematophagous fungus Clonostachys rosea. Disruption of prC attenuated infection of the fungus to nematodes, indicating that this gene functions as a virulence factor. The inhibition of basal expression of prC by the preferred nitrogen sources (glutamine, ammonia) occurred at the transcriptional level. In contrast, nematode cuticle induced the expression of prC at the post-transcriptional level. The inducible expression of prC by nematode cuticle was significantly suppressed by glutamine, ammonia and phenylmethylsulfonyl fluoride (an inhibitor of serine protease). Thus, the existence of active PrC, albeit at a low level in the medium, is probably essential for further induction of this gene by nematode cuticle. Moreover, the low molecule weight (< 3 kD) degradation products of nematode cuticle could significantly induce the expression of prC. Ammonia suppresses the virulence of C. rosea against nematodes, probably by inhibiting prC expression. Thus, the nematophagous fungi probably could not function well as biocontrol agents in fields fertilized with a large amount of ammonium salt.

Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction

“…In this paper, we quantified expression of a serine protease gene, sep, to confirm the reliability of EF1 as a control gene. Serine protease is an important fungal cell wall degrading enzyme, which plays an essential role in infecting plant pathogens (Zou et al, 2010;Liu and Yang, 2013). We sequenced and analyzed transcriptome of C. rosea parasitizing on sclerotia of S. sclerotiorum and in vegetative growth on PDA medium in different stages (data not shown), and found that the expression levels of sep were consistent with the transcriptome analysis, indicating that EF1 can act as a suitable reference gene in C. rosea with different modes of nutrition.…”

The DAF-16/FOXO Transcription Factor Functions as a Regulator of Epidermal Innate Immunity

“…Recent studies have demonstrated that fungal infection and epidermal injury activate similar signaling pathways in C. elegans 19,24. Infection by nematophagous fungi causes nematode cuticle damage [18], [19], [20], [21], [22]. We have previously reported a unique fungal structure, called the spiny ball, on the vegetative hyphae of the fungus Coprinus comatus that damages the nematode cuticle [25].…”

“…Pathogenic bacteria, including P. aeruginosa , S. aureus , E. faecalis , S. enterica , and human pathogenic yeast Candida albicans infect the nematode intestine [2], [17], whereas natural nematophagous fungi, such as Drechmeria coniospora and Clonostachys rosea , infect the epidermis of nematode, leading to epidermal cell damage [18], [19], [20], [21], [22]. When comparing gene expression profiles of C. elegans infected with D. coniospora [23] and predicted DAF-16 transcriptional target genes [8], we found that there was a significant overlap between D. coniospora -upregulated genes and DAF-16 target genes.…”

The Pochonia chlamydosporia Serine Protease Gene vcp1 Is Subject to Regulation by Carbon, Nitrogen and pH: Implications for Nematode Biocontrol

“…Preferred nitrogen sources such as ammonium chloride have been shown to repress subtilisin-like serine proteases from other pathogens e.g. Candida albicans [37], M. anisopliae [29], Arthrobotrys oligospora [31] and Clonostachys rosea [38]. At 4 h after transfer to new medium, this was also the case for all P. chlamydosporia isolates tested, and at 8 h for Pc147 and Pc392.…”

“…In the nematophagous fungus Clonostachys rosea the PacC-mediated pH response has been shown to play an important role in pathogenesis [47]. Expression of the subtilisin-like extracellular protease PrC of C. rosea was upregulated in the presence of nematode cuticles but this only occurred under alkaline growth conditions [38]. Under acidic conditions, cuticles failed to stimulate the expression of PrC.…”