광범위 병저항성 유전자발굴 연구동향

안일평,박상렬,황덕주,장안철,배신철 한국육종학회 2012 한국육종학회지 Vol.44 No.4

Disease is one of the devastating obstacles in the stable crop production. Numerous agronomical and chemical controls have been developed to overcome this problem, but the former is not sufficient for the maintaining the disease under the economic threshold level and the latter is not free from the environmental regulation. One of the most ideal solutions is resistance breeding. Resistance breeding has been majorly dependent on the resistance (R) genes conferring race-specific vertical resistance effective for limited populations within the pathogen species harboring avirulence (Avr) genes encoding effectors exactly matching with R gene products. In spite of its outstanding efficiency, improper management of above cultivars frequently resulted in the resistance break down due to the appearance and domination of the new races in the field. During the last two decades, mechanisms of disease resistance have been characterized and analyzed in the respect of genetics, biochemistry, molecular biology, cell biology, and evolution. Especially, a growing body of investigations has been focused on the resistance effective for multiple races or even more species of pathogen’s infections and also durable and long-lasting. In this manuscript, we will introduce the investigations searching for durable and broad-spectrum resistance and the considerations for their applications in the crop production will be presented.

High Throughput Screening of Antifungal Metabolites Against Colletotrichumgloeosporioides

안일평,김순옥,이용환 한국식물병리학회 2008 Plant Pathology Journal Vol.24 No.1

Colletotrichum gloeosporioides forms an appressorium, a specialized infection structure, to infect its hosts. Among 400 and 600 culture filtrates from fungi and class Actinomycetes, six methanol extracts (A5005, A5314, A5387, A5560, A5597, and A5598) from the class Actinomycetes significantly inhibited appressorium formation in C. gloeosporioides infecting pepper fruits in a dose-dependent manner, while conidial germination was slightly enhanced. Two (A5005 and A5560) of them also exhibited distinctive inhibitory effect on the disease progress of pepper anthracnose. Water fractions of both culture filtrates also specifically inhibited appressorium formation in C. gloeosporioides and pepper anthracnose disease. Inhibition of appressorium formation by culture filtrate of A5005 was partially restored by the exogenous calcium. This results suggests that chemicals within A5005 extents its biological activity through disturbance of intracellular Ca2+ regulation during prepenetration morphogenesis by C. gloeosporioides. Together, cell-based and target-oriented screening system used in this study should be applicable for other plant pathogenic fungi prerequisite appressorium formation to infect their hosts.

총설 : 단백질 분해가 식물의 진균 병 진전에 미치는 영향

안일평 ( Il Pyung Ahn ),박상렬 ( Sang Ryeol Park ),배신철 ( Shin Chul Bae ) 한국균학회 2010 韓國菌學會誌 Vol.38 No.2

Plant pathogenic fungi are the most diverse and drastic causal agents of crop diseases threatening stable food production all over the world. Plant have evolved efficient innate immune system to scout and counterattack fungal invasion and pathogenic fungi also developed virulence system to nullify plant resistance machinery or signaling pathways and to propagate and dominate within their niche. A growing body of evidences suggests that post translational modifications (PTMs) and selective/nonselective degradations of proteins involved in virulence expression of plant pathogenic fungi and plant defense machinery should play pivotal roles during the compatible and incompatible interactions. This review elucidates recent investigations about the effects of PTMs and protein degradations on host defense and fungal pathogens` invasions.

PCR방법을 이용한 국화흰녹병균 검출

전푸름,박상렬,김정구,안일평,황덕주,배신철 한국화훼산업육성협회 2014 화훼연구 Vol.22 No.4

우리나라의 절화국화는 국내 화훼작물 중 큰 비중을 차 지하고 있다. 수출 또한 2011년에는 2001년대비 약 1.9배 증가하였지만 생산단가 상승과 주변 경쟁국들과의 가격 경쟁력으로 수출농가가 어려움을 겪고 있다. 이를 극복하기 위해서는 소비자의 기회에 맞는 품종을 개발하 고 또한 고품질 국화를 생산하는 것이 필요하다. 국화 품 질을 저하시키는 요인 중 하나는 병해충 감염이며, 국화 흰녹병이 가장 심각한 피해를 주고 있다. 국화흰녹병 병 발생을 근절할 수 있는 근본적인 해결책은 저항성 품종 개발이지만 이는 많은 시간이 필요하기에 단기적 방법으 로 가시적으로 병징이 나타나기 이전에 국화흰녹병 감염 여부를 검정할 수 있는 기술을 개발한다면 병 발생 피 해를 최소화할 수 있을 것이다. 국화흰녹병 검정 기술 개 발을 위해 실험실 조건에서 이병주의 연중 생산이 필수 적이기에 이병주를 생산하는 시스템을 우선적으로 확립 하였다. 그리고 자체 생산한 이병주에서 증식된 병원균 의 담자포자의 형태적 특성 및 rDNA의 DNA 염기서열 분석 결과로 국화흰녹병균임을 입증하였다. 또한 국화흰 녹병균 유전체 정보를 이용하여 국화흰녹병균만을 검출 할 수 있는 유전자를 선발하였고 이 유전자를 대상으로 국화흰녹병균 게놈 DNA 0.25ng까지 검출할 수 있는 프 라이머 쌍을 개발하였다. 동일한 프라이머를 이용하여 Real-time PCR를 실시하면 게놈 DNA 6pg까지 검출이 가능하였다.향후 이러한 방법을 이용하여 병원균 접종후 육안으로 병징이 확인되기 이전에 국화흰녹병균의 검출 을 확인할 예정이다. In South Korea, chrysanthemum cut flowers have a large share in the flower industry and, compared to 2001, the export of chrysanthemum cut flower increased by 1.9 times in 2011. However, Korean growers have faced with difficulty in keeping competitiveness in world chrysanthemum cut flower market due to increase production cost and selling price competition. In order to overcome these problems, it is needed to develop cultivars that consumers demand and to produce cut flower chrysanthemum with high quality. Diseases and insects can cause severe quality problem of cut flower chrysanthemum. The most serious damage is caused by chrysanthemum white rust. The best option to minimize damage caused by white rust is to develop cultivars resistant to chrysanthemum white rust, but it will take so long time to develop a resistant cultivar. Therefore, an alternative way can be development of technique for early detection of infection by chrysanthemum white rust, which can minimize damage of cut flower chrysanthemum. We preferentially established the system for production of white rust susceptible chrysanthemum plants because it is required to supply susceptible plants in laboratories throughout the year to develop the detection technique for white rust. Pathogens sampled from susceptible plants were identified as chrysanthemum white rust through investigation of shape and size of basidiospore using microscope and analysis of rDNA sequence of the pathogen. In addition, the specific gene for chrysanthemum white rust was selected from genome database of Puccinia horiana and 0.25 ng of its genomic DNA could be obtained by PCR using primers designed to target the specific gene of chrysanthemum white rust. Six pg of its genomic DNA could be also amplified by Real-time PCR. In the near future, we will confirm if chrysanthemum white rust can be detected through the PCR method mentioned above before symptom caused by infection of white rust is become visible.

Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

황인선,안일평 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.3

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conven-tional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strat-egies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inef-ficient.Here, we introduce a cloning system that uti-lizes multi-fragment assembly by In-Fusion to gener-ate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1), which is associated with fumonisin B1 bio-synthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments us-ing confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms ex-hibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene dis-ruption in fungi.

국화 흰녹병균 생태 및 생명공학적 진단에 대한 연구동향

박상렬,안일평,황덕주,장안철,임기병,배신철 한국화훼산업육성협회 2013 화훼연구 Vol.21 No.3

Chrysanthemum is an economically important horticultural crop in chrysanthemum-growing areas including Korea. White rust is the most dangerous disease caused by an obligate pathogen Puccinia horiana. Control of white rust is dependent on chemical treatment and other cultural practices. However, chemical control of disease results in appearance of resistant race of white rust pathogen. Hence, development of chrysanthemum cultivar resistant to white rust is the most desirable. Nevertheless, not much research has been done about white rust pathogen (e.g infection process, pathogen diversity, etc). In order to investigate the way of chrysanthemum to overcome white rust we reviewed literatures reported about white rust pathogen, especially in terms of disease/life cycle and ecology of P. horiana and disease assessment based on polymerase chain reaction in this study. This review will be helpful for disease control and molecular breeding of chrysanthemum resistant to white rust. 국화는 국내외적으로 주요 화훼작물이지만 국화 흰녹병은 안정적 생산에 큰 걸림돌이 되고 있다. 현재까지 국화 흰녹병 방제는 농약살포를 통한 화학적 방제와 다습한 재배환경을 개선하는 재배적 방제에 의존하고 있지만, 살균제 저항성을 갖는 새로운 병원균 출현으로 농약 살포를 통한 병 방제에는 한계가 있어 병 저항성 품종 개발이 절실하다. 흰녹병은 순활물기생균인 Puccinia horiana에 의해 발생하는 곰팡이 병으로 이로 인한 경제적 손실이 크지만, 다른 주요 작물병과 비교하여 상대적으로 연구가 미흡하다. 특히 병원균의 전파 경로와 병원성의 다양성(pathogenic diversity), 병원균 침입기작 및 기주식물과의 상호작용에 대한 전반적인 지식 부족으로 효과적 농약 살포 기준 설정과 병저항성 품종개발에 어려움이 있다. 따라서 본 논문에서는 이러한 과제를 해결하는데 기초자료를 제공하고자 각 연구분야별로 현재까지 발표된 연구결과를 정리하고 앞으로 요구되는 연구과제에 대하여 모색하여 보았다. 특히 효과적 약제처리 기준 설정과 병저항성 품종 개발의 출발점이 되는 흰녹병균의 생활사와 생태, 병원균 DNA 정보를 이용한 조기진단, 병원균의 병원형 등에 관해 현재까지의 연구결과들을 정리하였다. 앞으로 흰녹병균의 식물체 침입 기작에 관한 종합적 이해의 폭을 넓히고, 병원균 침입 시 식물체내에서 발병과 병저항성 유도 요인에 대한 분자 수준에서 연구가 진행된다면 흰녹병균 저항성 품종 육성 시기를 앞당길 수 있으리라 사료된다.

배양기간 , 온도 , pH가 인삼 근부병균 Cylindrocarpon destructans ( Zinssm. ) Scholten의 균사생육에 미치는 영향 ( Effect of Incubation Period , Temperature and pH on Mycelial Growth of Cylindrocarpon destructans ( Zinssm. ) Scholten Causing Root-rot of Ginseng )

조대휘,안일평,유연현,오승환,이호자 고려인삼학회 1995 Journal of Ginseng Research Vol.19 No.2

Generation of Chinese cabbage resistant to bacterial soft rot by heterologous expression of Arabidopsis WRKY75

최창현,박상렬,안일평,배신철,황덕주 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.5

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is a serious disease in Chinese cabbage (Brassica rapa L. subsp. pekinensis). To reduce the severity of soft rot symptoms in Chinese cabbage, Arabidopsis AtWRKY75 was introduced into Chinese cabbage by Agrobacterium-mediated transformation, which was previously reported to reduce susceptibility to Pcc infection in Arabidopsis. Three independent Chinese cabbage transgenic lines carrying AtWRKY75 were obtained. The growth phenotypes of AtWRKY75 overexpression (OE) lines were normal. Bacterial soft rot symptoms and Pcc growth were reduced in AtWRKY75-OE Chinese cabbage lines compared with WT plants. In contrast, overexpression of AtWRKY75 had no effect on infection with a hemibiotrophic pathogen, Xanthomonas campestris pv. campestris (Xcc) causing black rot disease. These results are consistent with those observed in the transgenic Arabidopsis. We found that AtWRKY75 activated a subset of Chinese cabbage genes related to defense against Pcc infection, such as Meri15B, BrPR4, and BrPDF1.2 (but not BrPGIP2). Moreover, overexpression of AtWRKY75 caused H2O2 production and activation of H2O2 scavenge enzyme genes, suggesting that H2O2 played a role in AtWRKY75- mediated resistance to Pcc. Together, these results demonstrated that AtWRKY75 decreased the severity of Pcc-caused bacterial soft rot and activated a subset of Pcc infection defense-related genes in Chinese cabbage similar to in Arabidopsis. It is suggested that AtWRKY75 is a candidate gene for use in crop improvement, because it results in reduced severity of disease symptoms without concurrent growth abnormalities.

무름병에 감수성인 애기장대 돌연변이체 Atstp1 선발

최창현,황덕주,김민갑,안일평,박상렬,배신철 한국식물병리학회 2010 식물병연구 Vol.16 No.3

Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft rot disease in various plants. Although many studies about Pcc have been going on, little is known yet about the defense genes from plants. To identify defense associated genes in response to Pcc, we screened about 20 thousand Arabidopsis T-DNA knock out lines by inoculation with Pcc. We obtained a line (Atspt1) showing more susceptible symptom compared to WT (Col-0) on 1 day after the inoculation of Pcc on leaves of Arabidopsis with toothpicks. In this study, we optimized the system to select resistant and susceptible lines to Pcc from T-DNA inserted pool of Arabidopsis and expect the system and Atspt1 might be used for molecular breeding to produce resistant vegetables against Pcc. 본 연구는 애기장대에서 무름병에 대한 저항성 유전자를 탐색하고자 2만여개의 T-DNA 삽입 돌연변이군을 이용하여 Pcc에 대한 스크리닝을 수행하고 이 방법을 소개한 연구다. 1차 선발을 통하여 15개의 저항성 line과 20개의 감수성 line을 선발하였으며, 이로부터 2차 선발하여 3개의 저항성 line과 4개의 감수성 line을 선발하였고, 최종적으로 3차 선발을 통하여 1개의 감수성 line (Atstp1)을 선발할 수 있었다. 현재 Atstp1을 이용해 flanking sequencing 하여 유전자를 탐색하고 있으며, 앞으로 클로닝을 통하여 다양한 무름병 저항성 식물 개발에 유용하게 이용될 것으로 기대한다.

액체배양을 이용한 단기 벼 형질전환 방법

양대화,서석철,장안철,안일평,김해정,김동헌,이효연 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.1

Rice is one of the most important cereal crops as a model plant for functional genomics of monocotyledons and usually transformed using Agrobacterium tumefaciens. However, the transformation’s process using previous method is still time consuming and uneconomical, low efficiency. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the infection and co-cultivation, Agrobacterium elimination,infected calli’s selection steps using liquid media. We directly inoculated Agrobacterium containing a ZjLsL gene under the control of constitutive promoter into the 1- to 3-week-old rice calli derived from mature seeds. After 3days of co-cultivation, the infected calli were transferred onto liquid media of Agrobacterium elimination and calli’s selection for 3 days. The calli were transferred to calli’s growth solid media for 14 days and then the calli transferred to shoot induction and root induction media. Putative transformants were initially selected on the medium containing phosphinothricin, and the PAT protein verified by PAT strip test. This method in this study would lead to reduction of substantial labor and time to generate transgenic plants.