Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, Yunnan 650091, China.
Environ Microbiol. 2010 Dec;12(12):3243-52. doi: 10.1111/j.1462-2920.2010.02296.x.
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.
食线虫真菌已被用作防治寄生在动植物上的线虫的生物防治剂。这些真菌在感染线虫时会分泌类似于枯草杆菌蛋白酶的细胞外丝氨酸蛋白酶。这些类似枯草杆菌蛋白酶的丝氨酸蛋白酶的表达受氮源调控，包括线虫的角质层。然而，氮源诱导这些丝氨酸蛋白酶表达的机制尚不清楚。在这项研究中，我们研究了氮源对食线虫真菌玫瑰色拟青霉中类似于枯草杆菌蛋白酶的细胞外蛋白酶 prC 表达的影响。prC 的敲除削弱了真菌对线虫的感染，表明该基因是一种毒力因子。优选氮源（谷氨酰胺、氨）在转录水平上抑制 prC 的基础表达。相比之下，线虫角质层在转录后水平诱导 prC 的表达。线虫角质层诱导 prC 的可诱导表达显著被谷氨酰胺、氨和苯甲基磺酰氟（丝氨酸蛋白酶抑制剂）抑制。因此，尽管在培养基中 PrC 的活性水平较低，但它的存在对于进一步诱导该基因被线虫角质层诱导可能是必需的。此外，线虫角质层的低分子量（<3 kD）降解产物可显著诱导 prC 的表达。氨通过抑制 prC 的表达来抑制 C. rosea 对线虫的毒力。因此，在大量施用铵盐的田间，食线虫真菌可能无法很好地作为生物防治剂发挥作用。
