l-Arginine is essential for conidiation in the filamentous fungus Coniothyrium minitans

Abstract

Conidiation is important in the life cycles of mitosporic fungi for survival and transmission. A full-length cDNA of one gene named CMCPS1 encoding l-arginine-specific carbamoyl-phosphate synthase was obtained from Coniothyrium minitans, a sclerotial parasite of the plant pathogenic fungus Sclerotinia sclerotiorum. T-DNA insertional disruption of CMCPS1 resulted in conidiation deficiency of mutant ZS-1T2029, and this was confirmed with the RNAi technique. The phenotype was restored by complementation with l-arginine, and the effect of l-arginine on conidiation may be mediated by nitric oxide, which is catalyzed by nitric oxide synthase (NOS). Conidiation of ZS-1T2029 was restored by sodium nitroprussiate, a NO donor; and conidiation of wild type strain ZS-1 could be suppressed by l-NAME, an inhibitor of NOS. The highest amount of NO in mycelia was detected at an early stage of conidiation (72 hpi) in liquid shake culture medium. Staining with the NO-sensitive fluorescent probe, DAF-FM DA, gave strong fluorescent signals in primordia and young pycnidia. This work presents the first report that l-arginine is involved in conidiation of C. minitans, and the possibility of l-arginine-derived nitric oxide-mediated conidiation among fungi and possible modes of action are discussed.

Introduction

The production of asexual spores known as conidia is important in the life cycle of many fungi. Environmental factors, such as light, nutrition, air, temperature and secondary metabolism products, could influence conidiation (Calvo et al., 2002, Roncal et al., 2002, Sánchez-Murillo et al., 2004, Flaherty and Dunkle, 2005). Upon receipt of conidiation signals, fungi switch to the asexual reproductive phase from pure hyphal growth.

In hyphomycete fungi, such as Aspergillus nidulans, the asexual reproductive phase includes initiation of the conidiation pathway, formation of spore-bearing structures (conidiophores, metulae, vesicle and phialides), budding and maturation of conidia (Adams et al., 1998). In coelomycete fungi, asexual reproduction involves more complicated structures, and conidiophores are differentiated, developing within specialized structures such as sporodochia, acervuli or pycnidia. Although the molecular biology of conidiation of filamentous fungi has been characterized in detail for several hyphomycete fungi, including A. nidulans, Neurospora crassa, Magnaporthe grisea and Cryphonectria parasitica (Lengeler et al., 2000), limited work on the molecular biology of conidiation in coelomycete fungi has been done.

Coniothyrium minitans is a coelomycetous fungus, and significant as a sclerotial parasite of a worldwide plant pathogenic fungus, Sclerotinia sclerotiorum. Coniothyrium minitans was first reported by Campbell (1947), and its potential for biological control of plant diseases caused by Sclerotinia species was evaluated by scientists working in different countries (Whipps and Gerlagh, 1992). Conidia may be delivered into soil or aerial parts to destroy the sclerotia and hyphae of S. sclerotiorum, and subsequently control diseases induced by S. sclerotiorum (Bennett et al., 2006, Jones et al., 2004, Li et al., 2006).

Efficient production of conidia of C. minitans is a key step for commercial use; and much research has focused on the environmental factors (such as nutrition, oxygen and pH of the growth medium) which may influence conidiation of C. minitans (De Vrije et al., 2001). Our previous work found that several strains of C. minitans could sporulate well in shaken liquid medium while others did not (Cheng et al., 2003). This phenomenon suggests that there exists some endogenous difference in conidiation of C. minitans among strains. To better understand the molecular biology of conidiation of C. minitans, a T-DNA insertional mutagenesis library was constructed with strain ZS-1, which produces conidia abundantly in liquid shake culture (Li et al., 2005).

From this T-DNA insertional library, several conidiation-deficiency mutants were selected (Li et al., 2005), including ZS-1T2029, a conidiation deficiency mutant, which could grow hyphae on potato dextrose agar (PDA), and parasitize sclerotia of S. sclerotiorum when inoculated as hyphae fragments, but did not produce conidia in culture. We found that ZS-1T2029 could produce pycnidia and conidia on sclerotia, but single-spore-isolation from cultures of ZS-1T2029 still gave a ZS-1T2029 phenotype, lacking conidial production in culture. We hypothesized that there are some active components in sclerotia that are necessary for conidiation of mutant strains of C. minitans such as ZS-IT2029, while wild type strains can synthesize these compounds since they could produce conidia in culture without the stimulation of the host. The T-DNA insertional disrupted gene in ZS-1T2029 was previously cloned with TAIL-PCR, and then cDNA was obtained with RT-PCR. Here we report a novel signal pathway involved in conidiation of fungi based on the study of conidiation deficiency mutant ZS-1T2029 of C. minitans.