Role of Sugars in Early Stage of Spore Germination in Filamentous Fungi, Aspergillus nidulans

사상균인 Aspergillus nidulans의 무성포자 발아와 당의 역할

Abstract

Initiation of spore germination in filamentous fungi such as Aspergillus nidulans and Botrytis cinerea requires the presence of nutrients. In this study, involvement of sugar sensing machinery was suggested in the germination of A. nidulans spores. Germination did not occur when the spores of A. nidulans were incubated in distilled water, whereas they were successfully germinated in the presence of 5% glucose with a germination rate of over 98% after 6hr incubation. Similar results were obtained when the spores were incubated in the presence of various sugars such as fructose, sucrose, and starch. Interestingly, spore germination was not observed in the presence of D-arabinose, whereas L-arabinose could induce germination as determined by the formation of germ tubes, indicating the presence of sugar sensing machinery that distinguish between the enantiomers of sugars. This inference was further supported by a decrease in germination rate (less than 25%) upon treatment of spores with trypsin. Subsequent MALDI-TOF mass spectrometry analysis of the surface proteins of spores identified ten proteins among which eight were involved in sugar metabolism. Taken together, our results suggest that spore germination in A. nidulans is initiated by the interaction of sugars with sugar binding proteins on the surface of spores.

Aspergillus nidulans와 Botrytis cinerea와 같은 사상균들은 당과 같은 영양분이 존재하지 않은 조건에서는 발아하지 않는다. 본 연구에서는 A. nidulans의 포자에 당을 인식하는 기구가 존재할 것이라는 증거를 제시하였다. A. nidulans의 포자에 증류수를 가하였을 때에는 발아가 관찰되지 않는 반면에 5%의 글루코오스를 가해 주었을 때에는 98%이상의 포자가 발아하였다. 뿐만 아니라 프록토오스, 설탕, 녹말과 같은 단당류, 이당류, 다당류를 가해 주어도 유사한 결과를 관찰할 수 있었다. 특이한 것은L-형의 아라비노오스를 가해 준 포자는 발아관을 형성하였으나D-형의 아라비노오스를 가해주면 증류수를 가해주었을 때와 같이 발아관을 형성하지 못 하였다. 포자를 트립신으로 처리한 후에 글루코오스를 가해주면 발아율이 25%롤 감소하였다. 포자 표면에 존재하는 단백질을 용출하여 분리한 후 MALDI-TOF 질량분석기로 단백질을 동정한 결과 동정된 10종의 단백질 중 8종의 단백질은 당의 대사와 관련된 효소들 임을 확인하였다.

Fig. 1. Germination rate of Aspergillus nidulans spores in the presence of sugar. Asexual spores of A. nidulans were incubated in the solution of water (○), 5% of glucose (●), D-arabinose (△), and L-arabinose (▲) at 37℃, respectively. Germination rate was calculated by counting the spores formed germ tubes.

Fig. 2. Germination rate of Aspergillus nidulans spores in the presence of mixture of D- and Larabinose. Spores were incubated in the solution containing various molar ratio of D- and L- arabinose. Germination rate was calculated by counting the germinated spores by time interval.

Fig. 3. Germination rate of Aspergillus nidulans spores treated with glucose and L-arabinose for short period. Asexual spores were treated with 5% of glucose and L-arabinose for 5 min, respectively. After washing out 3 times with water, spores were incubated in water without sugar for 6 hr. The germ tubes were observed by inverted microscopy after incubation (ⅹ400).

Fig. 4. Germination rate of Aspergillus nidulans spores treated with trypsin. Spores were incubated with trypsin for 2 hr. After washing out added trypsin, spores were further incubated in the presence of 5% glucose at 37℃ (□). The spores treated with BSA instead of trypsin were used as control (■).

Fig. 5. Analysis of proteins extracted from spore surface of Aspergillus nidulans by SDS-polyacrylamide gel electrophoresis. Proteins extracted from asexual spores using 1 M NaCl were treated with/without trypsin. The arrows and numbers indicated protein band to be cut out for protein identification by MALDI-TOF mass spectrometry.

Table 1. The germination rate of Aspergillus nidulans spores incubated in the presence of various sugars

Table 2. List of identifed proteins extracted from spore surface of Aspergillus nidulans