근권 서식 미생물간의 상호작용 기작에 관한 분자수준의 이해 및 응용 : New molecular insights into microbial interaction mechanisms to understand microbial community in the rhizosphere원문보기
근권 환경에는 다양한 종류의 미생물이 공존하며 이들간의 상호작용 연구를 통해 식물 유용 세균들의 생태학적인 특성을 결정하는 중요한 정보를 얻을 수 있고 이를 바탕으로 식물병의 생물학적 방제나 식물생장 촉진 효과를 개선할 수 있을 것이다. 본 연구에서는 벼의 뿌리조직 내에서 서식하는 내생세균 16주를 분리 확보하였고 생물학적 특성에 기초하여 우수 내생균인 Burkholderia 균을 선별하였다. 선별된 내생세균인 Burkholderia sp. KJ006을 숙주균으로 이용하여 기주식물에 외래 형질을 도입할 수 있는 host-vector system을 구축하였다. 세균 세포간의 ...
근권 환경에는 다양한 종류의 미생물이 공존하며 이들간의 상호작용 연구를 통해 식물 유용 세균들의 생태학적인 특성을 결정하는 중요한 정보를 얻을 수 있고 이를 바탕으로 식물병의 생물학적 방제나 식물생장 촉진 효과를 개선할 수 있을 것이다. 본 연구에서는 벼의 뿌리조직 내에서 서식하는 내생세균 16주를 분리 확보하였고 생물학적 특성에 기초하여 우수 내생균인 Burkholderia 균을 선별하였다. 선별된 내생세균인 Burkholderia sp. KJ006을 숙주균으로 이용하여 기주식물에 외래 형질을 도입할 수 있는 host-vector system을 구축하였다. 세균 세포간의 신호전달 물질을 분해 하는 homoserine lactonase 유전자(aiiA)를 발현하도록 Burkholderia sp. KJ006 (pKPE-aiiA) 형질전환체를 제작하여 homoserine lactone의 분해능과 E. carotovora의 감자무름병 억제능을 in vitro상에서 확인하였다. Burkholderia sp. KJ006 (pKPE-aiiA) 재조합균을 벼의 뿌리조직에 도입하여 난방제성 벼 병원세균인 B. glumae의 병발생률을 현저히 감소시킨 결과를 얻었다. 세균성 병원균의 신호전달 체계를 방해하는 재조합 내생균을 식물에 미리 도입할 경우 식물병의 효율적인 억제가 가능하다는 새로운 관점을 제시하였다. 다른 한편 토양 내 서식하는 곰팡이 일종인 P. citrinum과 식물 유용 세균인 P. polymyxa E681의 대치배양 시 bacteria의 swarm motility가 증가하는 현상을 관찰하였다. LC/MS 분석을 통해 P. citrinum의 이차 대사산물 중 mycotoxin으로 잘 알려진 citrinin이 bacteria의 motility를 증가 시키는 물질임을 확인 하였다. Citrinin에 의해 P. polymyxa E681의 sigD와 hag유전자의 발현이 증가 됨을 RT PCR을 통해 확인하였고 flagella staining후 광학현미경 관찰을 통해 flagella 수가 증가 됨을 확인 하였다. Swim plate를 이용하여 citrinin이 chemo-attractant로 작용할 수 있다는 결과를 얻었으며 P. polymyxa ATCC 842, B. subtilis C9 과 더불어 P. fluorcence pf5의 swarm motility도 citrinin에 의해 증가됨을 확인하였다. 또한 다른 종류의 mycotoxin인 fusaric acid, patulin, ochratoxin A에 의해서도 P. polymyxa E681의 swarm motility가 증가됨을 확인하였다. 본 연구는 그동안 인축에 대한 독성물질로 잘 알려진 mycotoxin류의 곰팡이 유래 2차대사산물이 세균과 곰팡이의 상호작용에 있어 signal molecule로 작용 할 수 있다는 새로운 관점을 제시 하였다.
근권 환경에는 다양한 종류의 미생물이 공존하며 이들간의 상호작용 연구를 통해 식물 유용 세균들의 생태학적인 특성을 결정하는 중요한 정보를 얻을 수 있고 이를 바탕으로 식물병의 생물학적 방제나 식물생장 촉진 효과를 개선할 수 있을 것이다. 본 연구에서는 벼의 뿌리조직 내에서 서식하는 내생세균 16주를 분리 확보하였고 생물학적 특성에 기초하여 우수 내생균인 Burkholderia 균을 선별하였다. 선별된 내생세균인 Burkholderia sp. KJ006을 숙주균으로 이용하여 기주식물에 외래 형질을 도입할 수 있는 host-vector system을 구축하였다. 세균 세포간의 신호전달 물질을 분해 하는 homoserine lactonase 유전자(aiiA)를 발현하도록 Burkholderia sp. KJ006 (pKPE-aiiA) 형질전환체를 제작하여 homoserine lactone의 분해능과 E. carotovora의 감자무름병 억제능을 in vitro상에서 확인하였다. Burkholderia sp. KJ006 (pKPE-aiiA) 재조합균을 벼의 뿌리조직에 도입하여 난방제성 벼 병원세균인 B. glumae의 병발생률을 현저히 감소시킨 결과를 얻었다. 세균성 병원균의 신호전달 체계를 방해하는 재조합 내생균을 식물에 미리 도입할 경우 식물병의 효율적인 억제가 가능하다는 새로운 관점을 제시하였다. 다른 한편 토양 내 서식하는 곰팡이 일종인 P. citrinum과 식물 유용 세균인 P. polymyxa E681의 대치배양 시 bacteria의 swarm motility가 증가하는 현상을 관찰하였다. LC/MS 분석을 통해 P. citrinum의 이차 대사산물 중 mycotoxin으로 잘 알려진 citrinin이 bacteria의 motility를 증가 시키는 물질임을 확인 하였다. Citrinin에 의해 P. polymyxa E681의 sigD와 hag유전자의 발현이 증가 됨을 RT PCR을 통해 확인하였고 flagella staining후 광학현미경 관찰을 통해 flagella 수가 증가 됨을 확인 하였다. Swim plate를 이용하여 citrinin이 chemo-attractant로 작용할 수 있다는 결과를 얻었으며 P. polymyxa ATCC 842, B. subtilis C9 과 더불어 P. fluorcence pf5의 swarm motility도 citrinin에 의해 증가됨을 확인하였다. 또한 다른 종류의 mycotoxin인 fusaric acid, patulin, ochratoxin A에 의해서도 P. polymyxa E681의 swarm motility가 증가됨을 확인하였다. 본 연구는 그동안 인축에 대한 독성물질로 잘 알려진 mycotoxin류의 곰팡이 유래 2차대사산물이 세균과 곰팡이의 상호작용에 있어 signal molecule로 작용 할 수 있다는 새로운 관점을 제시 하였다.
From studies of microbial interactions in rhizosphere various information on modes of action and relevant molecular mechanisms can be obtained and it will be an important base for the improvement of biological control and/or plant growth promotion efficacy of biological agents. Among sixteen endophy...
From studies of microbial interactions in rhizosphere various information on modes of action and relevant molecular mechanisms can be obtained and it will be an important base for the improvement of biological control and/or plant growth promotion efficacy of biological agents. Among sixteen endophytic bacteria isolated from tissue of rice root Burkholderia sp. KJ006 strain was selected as a superior strain based on biological activity. By using the KJ006 strain, a host-vector system was constructed to introduce and express a foreign gene into the endophytic bacterium, and resultantly the expressed product can be delivered to host plant harboring the endophyte. Recombinant Burkholderia sp. KJ006 (pKPE-aiiA) containing a foreign homoserine lactonase gene was constructed, and this strain was shown to degrade bacterial quorum signal molecule effectively that in vitro analysis, hexanoyl homoserine lactone (HHL) as a substrate was completely degraded in 2 h incubation and the disease incidence of potato rot caused by E. carotovora was significantly reduced by pretreating of the recombinant strain. In an in vivo experiment the recombinant strain KJ006 (pKPE-aiiA) could reduce the pathogenecity of B. glumae remarkably so that the rice seedling harboring the AiiA-recombinant strain showed significantly reduced seedling rot symptoms than control. The results obtained in the present study showed that plant diseases caused by bacterial pathogens could be controlled efficiently by using an engineered endophyte which has interfering activity on quorum-sensing system of bacterial pathogens. In the second part of this study the interaction between fungi and bacteria was investigated. Interestingly, it was observed that the swarming motility of P. polymyxa strain E681, isolated from the rhizosphere of winter barley, was greatly increased by adjacent growth of a fungal strain, P. citrinum KCTC6549. From a culture supernatant of the fungal strain which showed the same effect on E681, an active compound was purified and identified as citrinin which is one of mycotoxins by LC/MS analysis. It was confirmed through reverse transcriptase PCR that transcriptional expressions of sigD and hag were increased in the presence of citrinin compared with non-treats. Microscopic observation revealed that the number of flagella of the P. polymyxa E681 cells was significantly increased in the presence of citrinin. It was also observed that P. polymyxa E681 could sense the citrinin as a chemo-attractant by chemotaxis experiment and could colonize around fungal hypha during co-culture. The swarm motilities of other bacterial species such as P. polymyxa ATCC842 (paenibacillus type strain), B. subtilis C9 and P. fluorcence Pf-5 were also increased with the addition of citrinin to the culture medium. Also, other mycotoxins such as fusaric acid, ochratoxin A and patulin could increase the swarm motility of P. polymyxa E681. The results presented here will provide an insight for understanding ecological role of fungal mycotoxins besides their well-known toxicity to man and/or animals. That is, we found that mycotoxins such as citrinin, fusaric acid and ochratoxin A have a newly identified function of inducing bacterial motility, and this finding will contribute to understanding interaction between bacteria and fungi in nature.
From studies of microbial interactions in rhizosphere various information on modes of action and relevant molecular mechanisms can be obtained and it will be an important base for the improvement of biological control and/or plant growth promotion efficacy of biological agents. Among sixteen endophytic bacteria isolated from tissue of rice root Burkholderia sp. KJ006 strain was selected as a superior strain based on biological activity. By using the KJ006 strain, a host-vector system was constructed to introduce and express a foreign gene into the endophytic bacterium, and resultantly the expressed product can be delivered to host plant harboring the endophyte. Recombinant Burkholderia sp. KJ006 (pKPE-aiiA) containing a foreign homoserine lactonase gene was constructed, and this strain was shown to degrade bacterial quorum signal molecule effectively that in vitro analysis, hexanoyl homoserine lactone (HHL) as a substrate was completely degraded in 2 h incubation and the disease incidence of potato rot caused by E. carotovora was significantly reduced by pretreating of the recombinant strain. In an in vivo experiment the recombinant strain KJ006 (pKPE-aiiA) could reduce the pathogenecity of B. glumae remarkably so that the rice seedling harboring the AiiA-recombinant strain showed significantly reduced seedling rot symptoms than control. The results obtained in the present study showed that plant diseases caused by bacterial pathogens could be controlled efficiently by using an engineered endophyte which has interfering activity on quorum-sensing system of bacterial pathogens. In the second part of this study the interaction between fungi and bacteria was investigated. Interestingly, it was observed that the swarming motility of P. polymyxa strain E681, isolated from the rhizosphere of winter barley, was greatly increased by adjacent growth of a fungal strain, P. citrinum KCTC6549. From a culture supernatant of the fungal strain which showed the same effect on E681, an active compound was purified and identified as citrinin which is one of mycotoxins by LC/MS analysis. It was confirmed through reverse transcriptase PCR that transcriptional expressions of sigD and hag were increased in the presence of citrinin compared with non-treats. Microscopic observation revealed that the number of flagella of the P. polymyxa E681 cells was significantly increased in the presence of citrinin. It was also observed that P. polymyxa E681 could sense the citrinin as a chemo-attractant by chemotaxis experiment and could colonize around fungal hypha during co-culture. The swarm motilities of other bacterial species such as P. polymyxa ATCC842 (paenibacillus type strain), B. subtilis C9 and P. fluorcence Pf-5 were also increased with the addition of citrinin to the culture medium. Also, other mycotoxins such as fusaric acid, ochratoxin A and patulin could increase the swarm motility of P. polymyxa E681. The results presented here will provide an insight for understanding ecological role of fungal mycotoxins besides their well-known toxicity to man and/or animals. That is, we found that mycotoxins such as citrinin, fusaric acid and ochratoxin A have a newly identified function of inducing bacterial motility, and this finding will contribute to understanding interaction between bacteria and fungi in nature.
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