이 작품은 베이징 농업 대학 (YQ201603)의 특별 한 과학 연구 프로젝트와 베이징 교육 위원회 (KM201610020005)의 과학 프로젝트에 의해 지원 되었다.

1. 스프레이 접종 M. grisea Conidia의 서 스 펜 션
<ol><li>
M.grisea 에 대 한 곰 팡이 문화
	<ol>
<li>곰 팡이 긴장에 대 한 오트밀 토마토 agar (OTA) 문화 매체를 준비 합니다.</li>
		<li>오트밀의 30-50 g를 무게, 소 주/이온된 수 (ddH2O)의 800 mL에 이것을 추가 하 고 전기 냄비에 30 분을 위한 혼합물을 비등.</li>
		<li>거 즈 조각을 통해 비 커에 끓인된 오트밀 주스를 필터링 합니다.</li>
		<li>토마토 주스와 한 천 20g 150 mL 비 커에 여과 액을 더하고 ddH2O 최대 1000 mL.</li>
	</ol>
</li>
	<li>
실험 자료의 준비
	<ol>
<li>3 d ddH2O에서에서 벼 (Oryza sativa) 품종 Lijiangxintuan-heigu (LTH)의 약 50 씨앗을 담가 또는 보 리의 약 50 씨앗을 담가 (Hordeum vulgare 이력서 골든 약속) ddH2오 약 1 d.</li>
		<li>촉촉한 거 즈에 쌀 이나 보 리의 씨앗을 포장 하 고 직경 10 cm x 10 cm에서 28 ° c.의 약 30 접시에 축축한 필터 종이에 그들을 발 아 쌀 종자 발 아에 약 2-3 d, 보 리 씨앗 발 아 시간이 약 2 d. 상대 습도 온실에서 대략 70% 이다.</li>
		<li>압력가 화분 용 흙 및 급수를 사용 하 여 냄비에 쌀 이나 보 리의 묘를 설치 하 고 질 석의 계층으로 그들을 커버.</li>
		<li>1 약 25 ° C에서 캐비닛 적합 한 유리 공장 또는 성장에 식물을 배치 ~ 2 주.</li>
		<li>원형에서 필터 종이의 3 층 (각 동그라미는 8 cm 직경 있어야 함) 무 균 수술가 위로 잘라 100mm 살 균 플라스틱 접시에 그들을 배치.</li>
		<li>필터 종이를 각 접시 ddH2O를 추가 합니다.</li>
		<li>필터 종이 완전히 젖은 것을 확인 하지만 여분의 물 추가.</li>
		<li>진공 펌프를 가진 어떤 과잉의 물을 제거 합니다.</li>
		<li>쌀/보 리를 지원 하기 위해 문화 접시에 장소 2 살 균 이쑤시개 잎; 이쑤시개는 공간 ~ 2-3 cm 떨어져.</li>
		<li>씨앗을 파 종 후 2 주 쌀의 나뭇잎을 수집 하거나 보 리 7 d의 잎 씨앗을 파 종 후.</li>
		<li>쌀/보 리의 묘 목 4-6 잎을 사용 하 여, 정상에서 ~ 5 센티미터에 줄기의 아래 부분을 잘라 하 고 나뭇잎을 수집 합니다.</li>
	</ol>
</li>
	<li>
스프레이 접종 프로토콜
	<ol>
<li>문화 ~ 4 d 서 모스 탯 인큐베이터 (25 ° C)에서 OTA 접시에 곰 팡이 부담.</li>
		<li>DdH2O 0.5-5 mL 피 펫 각 4-하루-오래 된 접시를 사용 하 여 ~ 2 mL를 추가 합니다.</li>
		<li>접종 루프와 사체 잔해에 M. grisea 야생-타입 긴장과 돌연변이 스트레인의 사체를 다쳤어요.</li>
		<li>사체 잔해를 수집 하 고 새로운 OTA 접시에 그것을 전송. 받아 사체 파편 타격 건조 깨끗 한 벤치에.</li>
		<li>24-48 시간 (10 h) 밤에 25 ° C (14 h) 하루에 23 ° C에서 온실에서 conidia의 성장에 필요한 습도 되도록 거 즈의 3 레이어 플레이트 커버.</li>
		<li>각 접시 ddH2O의 2 개 mL를 추가 하 고 뒤에 렌즈 종이의 2 층을 통해 여과 멸 균 면봉으로 부드럽게는 conidia를 다쳤어요. 문화 매체의 표면을 긁을 조심 해야.</li>
		<li>100-1000 µ L 피펫으로 새로운 50 mL 튜브에 conidia 현 탁 액을 전송 합니다.</li>
		<li>25 ° c.에 x g 5000의 최소한 5 분 동안 원심 분리기</li>
		<li>제거는 상쾌한 고 mL 당 2 × 104 conidia 0.025% (v/v) 트윈 20 솔루션에에서 주려고 펠 릿 resuspend. 트윈-20 솔루션은 일반적으로 약 10-20 mL.</li>
		<li>휴대용 분무기에 포자 현 탁 액을 붓는 다.</li>
		<li>Spay 2 주 된 쌀 묘 또는 보 리 쌀 잎에 conidial 서 스 펜 션의 약 10 mL 보 리 7-하루-오래 된 묘 목의 잎 하 고 0.025% (v/v) 트윈 20 솔루션 컨트롤 식물 25 ° c ~ 24 h. 스프레이 대 한 어두운, 습 한 약 실에서 그들을 품 어.</li>
		<li>12 h photoperiod 잎 25 ° C에서 형광등 불빛 아래에서 또 다른 습 챔버로 전송.</li>
		<li>접종 후 5 d에 질병 증상을 기록 합니다. 길이 ~ 6 cm의 병 쌀/보 리 잎을 검사 합니다. 표준 평가 국제 쌀 연구 연구소 (IRRI)의 폭발 저항에 대 한 채 점 시스템에 따라 이다. 폭발 저항에 대 한 채 점 시스템의 세부 정보는 표 1에 나와 있습니다.</li>
		<li>사진 테스트 긴장의 감염을 평가 하는 나뭇잎. 감염 병 변 3.6 c m2당 수로 평가 했다.</li>
	</ol>
</li>
</ol>2. 접종 균 큐브 또는 방울 Conidial 정지의 M. grisea 부상
<ol><li>분리 된 쌀/보 리 잎;의 주요 혈관에 3 2-3 cm 긴 상처를 긁어 해부학 바늘을 사용 하 여, 알아서 하지 잎에 침투.</li>
	<li>이쑤시개에 긁힌 것된 잎을 넣고 0.02% (v/v) 트윈 20 솔루션 방울의 층을 형성 하는 잎에 스프레이.</li>
	<li>OTA 접시에서 각 M. grisea 스트레인 (야생-타입, 돌연변이, 보완 긴장, 또는 다른 테스트 긴장)에 대 한 0.5 c m x 0.5 c m 바로 플러그를 잘라.</li>
	<li>바로 플러그를 넣어 또는 부상에 conidial 서 스 펜 션의 25 µ L 방울 단풍 잎 3-8 d의 습기 챔버에 25 ° c를 품 어.</li>
	<li>5-7 d 후 접종에 장애를 검사 합니다. 시험 방법은 같은 스프레이 접종 방법으로 (단계 1.3.13 참조).</li>
	<li>길이 ~ 6 cm의 병 쌀/보 리 잎을 검토 하 고 테스트 긴장의 감염을 평가 하기 위해 사진을. 감염 병 변 3.6 c m2당 수로 평가 했다.</li>
</ol>

<ol><li>Li, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+natural+allele+of+a+transcription+factor+in+rice+confers+broad-spectrum+blast+resistance%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A natural allele of a transcription factor in rice confers broad-spectrum blast resistance.</a> Cell. 170 (1), 114-126 (2017).</li>
<li>Chi, M. H., Park, S. Y., Kim, S., Lee, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+pathogenicity+gene+is+required+in+the+rice+blast+fungus+to+suppress+the+basal+defenses+of+the+host%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host.</a> PLoS Pathogens. 5 (4), 1000401(2009).</li>
<li>Jia, Y., Valent, B., Lee, F. N. <a target="_blank" href="https://apsjournals.apsnet.org/doi/10.1094/PDIS.2003.87.2.129">Determination of host responses to Magnaporthe grisea.on detached rice leaves using a spot inoculation method.</a> Plant Disease. 87 (2), 129-133 (2003).</li>
<li>Ebbole, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnaporthe+as+a+model+for+understanding+host-pathogen+interactions%5BTitle%5D)+AND+%22Annual+Review+of+Phytopathology%22%5BJournal%5D)">Magnaporthe as a model for understanding host-pathogen interactions.</a> Annual Review of Phytopathology. 45, 437-456 (2007).</li>
<li>Hamer, J. E., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infection-related+development+in+the+rice+blast+fungus+Magnaporthe+grisea%5BTitle%5D)+AND+%22Current+Opinion+in+Microbiology%22%5BJournal%5D)">Infection-related development in the rice blast fungus Magnaporthe grisea.</a> Current Opinion in Microbiology. 1 (6), 693-697 (1998).</li>
<li>Howard, R. J., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breaking+and+entering%3A+host+penetration+by+the+fungal+rice+blast+pathogen+Magnaporthe+grisea%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea.</a> Annual Review of Microbiology. 50, 491-512 (1996).</li>
<li>Chen, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-Glycosylation+of+Effector+Proteins+by+an+%CE%B1-1%2C3-+Mannosyltransferase+Is+Required+for+the+Rice+Blast+Fungus+to+Evade+Host+Innate+Immunity%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">N-Glycosylation of Effector Proteins by an α-1,3- Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity.</a> The Plant Cell. 26 (3), 1360-1376 (2014).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M.ARG1%2C+MoARG5%2C6+and+MoARG7+involved+in+arginine+biosynthesis+are+essential+for+growth%2C+conidiogenesis%2C+sexual+reproduction%2C+and+pathogenicity+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Microbiological+Research%22%5BJournal%5D)">M.ARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae.</a> Microbiological Research. 180, 11-22 (2015).</li>
<li>Du, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+serine%2Fthreonine-protein+phosphatase+PP2A+catalytic+subunit+is+essential+for+asexual+development+and+plant+infection+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Current+Genetics%22%5BJournal%5D)">A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae.</a> Current Genetics. 59 (1-2), 33-41 (2013).</li>
<li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+protein+com1+is+required+for+normal+conidium+morphology+and+full+virulence+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Molecular+Plant-Microbe+Interactions%22%5BJournal%5D)">A novel protein com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.</a> Molecular Plant-Microbe Interactions. 23 (1), 112-123 (2010).</li>
<li>Cao, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+ash1-like+protein+MoKMT2H+null+mutant+is+delayed+for+conidium+germination+and+pathogenesis+in+Magnaporthe+oryzae%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">An ash1-like protein MoKMT2H null mutant is delayed for conidium germination and pathogenesis in Magnaporthe oryzae.</a> BioMed Research International. 2016, 1575430(2016).</li>
<li>Bryan, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+amino+acid+difference+distinguishes+resistant+and+susceptible+alleles+of+the+rice+blast+resistance+gene+Pi-ta%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.</a> The Plant Cell. 12 (11), 2033-2045 (2000).</li>
<li>Zhou, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+pathology%3A+a+life+and+death+struggle+in+rice+blast+disease%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Plant pathology: a life and death struggle in rice blast disease.</a> Current Biology. 26 (18), 843-845 (2016).</li>
<li>Guo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MoGrr1%2C+a+novel+F-box+protein%2C+is+involved+in+conidiogenesis+and+cell+wall+integrity+and+is+critical+for+the+full+virulence+of+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae.</a> Applied Microbiology and Biotechnology. 99 (19), 8075-8088 (2015).</li>
<li>Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+trail+of+a+cereal+killer%3A+Exploring+the+biology+of+Magnaporthe+grisea%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea.</a> Annual Review of Microbiology. 57, 177-202 (2009).</li>
<li>Wilson, R. A., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Under+pressure%3A+investigating+the+biology+of+plant+infection+by+Magnaporthe+oryzae%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.</a> Nature Reviews Microbiology. 7, 185-195 (2009).</li>
<li>Jia, Y. L., Lee, F. N., McClung, A. <a target="_blank" href="https://apsjournals.apsnet.org/doi/10.1094/PDIS-93-6-0639">Determination of Resistance Spectra of the Pi-ta and Pi-k Genes to U.S. Races of Magnaporthe oryzae Causing Rice Blast in a Recombinant Inbred Line Population.</a> Plant Disease. 93, 639-644 (2009).</li>
<li>Peng, Y. L., Shishiyama, J. <a target="_blank" href="http://www.nrcresearchpress.com/doi/abs/10.1139/b88-107?journalCode=cjb1#.WvnHgogvzIU">Temporal sequence of cytological events in rice leaves infected with Pyricularia oryzae.</a> Canadian Journal of Botany. 66 (4), 730-735 (1988).</li>
</ol>

저자는 공개 없다.

식물 질병 저항 유전자 곰 팡이 병원 균1,12를 포함 하 여, 병원 균에 의해 감염 방지에 필수적인 역할을 재생 합니다. 쌀 폭발 병원 체 인구 구조의 특성을 이해 하 고 식물 저항 유전자4를 식별 하는 모델로 사용 되었습니다. 따라서, 그것은 질병 저항 유전자를 지속적으로 재배 될 수 있는 질병 저항 식물을 식별 하는 농업 식물의 주요 ...

쌀 폭발, M. grisea를 기인한 쌀 품종 전세계1,2에 대 한 가장 심각한 질병 중 하나입니다. 프로세스는 M. grisea 감염 호스트 식물 conidia 생산 및 표면 부착, conidia 발 아 및 appressorium 형성, 침투 못 감염 hypha 차별화의 형성을 포함 하 고 질병 확산 3. 이러한 모든 단계는 많은 다른 식물 병원 성 곰 팡이에서 일반적이 고, 실제로, 모든 단일 단계 봉쇄 호스트 식물의 감염을 방지. 때문에 그것의 경제 중요성 및 유전 추적성, M. grisea 식물 곰 팡이 병원 체 상호 작용1,4의 메커니즘을 공부에 대 한 모델 생물으로 떠오르고 있다. 따라서, 기본 균 pathogenicity 및 심사 및 디자인 소설 후보 대상 유전자의 식별 분자 메커니즘을 명료 하 도움이 될 것입니다 이러한 발달 단계 M. grisea 에 분자 기초 공부 살 균 제5.
최근 보고서 M. grisea 감염에 관한 사전 침투 단계, 특히에서 conidiation, appressorium 형성, 침투 못 및 전염 성 성장3, 의 분자 메커니즘에 초점을 맞춘합니다 6. 따라서, M. grisea 감염 테스트 상세한 프로토콜을 개발 하는 것이 필수적입니다. 여기, 우리 conidial 서 스 펜 션와 M. grisea의 사체 플러그와 상처의 접종 감염 스프레이 중재 분석을 활용 한 감염 테스트에 대 한 자세한 방법 제시. 이 보고서는 프로토콜, conidiation 솔루션의 준비와 바로 플러그 중재 접종 M. grisea와 식물의 종자, 문화에 중점을 둡니다. 다음이 단계는 아래의 세부 사항 그리고 방법의 전체 워크플로 보여주는 회로도 보기에 설명 하 고 전형적인 병 변은 각각 그림 1 과 2에 표시 됩니다.

<table><tbody><tr><td>Filter paper</td><td>GE Healthcare brand(Sweden)&amp;nbsp;&amp;nbsp;</td><td>10311387</td><td></td></tr><tr><td>50-mL tube</td><td>CORNING(Amercia)</td><td>430290</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf(Amercia)</td><td>5804R</td><td></td></tr><tr><td>Culture dish</td><td>Thermofisher(Amercia)</td><td>150326</td><td></td></tr><tr><td>0.5-5 mL pipette</td><td>Eppendorf</td><td>&amp;nbsp;4920000105</td><td></td></tr><tr><td>100-1000uL pipette</td><td>Eppendorf</td><td>4920000083</td><td></td></tr><tr><td>Vacuum pump</td><td>Leybold</td><td>D25B</td><td></td></tr><tr><td>Dissection needle</td><td>FST</td><td>26000-35</td><td></td></tr><tr><td>Incubator</td><td>MEMMERT</td><td>PYX313</td><td></td></tr><tr><td>Inoculation ring</td><td>Greiner Bio One</td><td>731175</td><td></td></tr></tbody></table>

the plant infection test: spray and wound-mediated inoculation with the plant pathogen magnaporthe grisea

식물 병원 성 곰 팡이 의해 잠재적인 위협 으로부터 방어 하는 강력한 시스템을가지고 있습니다. 그러나 농업으로 중요 한 식물에 대 한, 현재 조치 같은 병원 체에 대처 하기 위해 너무 보수적인 증명 그리고, 따라서, 충분 하지 효과적인, 그리고 그들은 수 있습니다 잠재적으로 포즈 환경 위험. 따라서, 그것은 매우 내성 germplasm의 식별, 격리 및 저항 유전자의 특성 및 분자 사육을 통해 자연스럽 게 제어 식물 질병을 호스트 저항 요소를 식별 하는 데 필요한 저항 하는 재배 품 정. 이와 관련, 번 식 하 고 식물 저항 유전자를 개발, 신속, 정확 하 고 대규모 접종 메서드를 설정할 필요가 있다. 쌀 버섯 모양 병원 체 Magnaporthe grisea 원인 심각한 질병 증상을 폭발 하 고 손실을 항복. 최근에, M. grisea 식물 곰 팡이 병원 체 상호 작용의 메커니즘을 공부에 대 한 모델 생물으로 떠오르고 있다. 따라서, 우리 보고 M. grisea위해 특정 식물 독성 시험 방법의 개발. 이 메서드는 conidial 현 탁 액을 분무 접종 및 균 사체 큐브 또는 conidial 서 스 펜 션의 방울 부상 접종에 대 한 제공합니다. 분리 된 쌀 잎 부상 접종 방법의 주요 단계는 호스트 침투 저항으로 인 한 간섭 방지 식물 잎에 상처를 만드는 것입니다. 이 M. grisea 격리의 pathotypes의, 정확, 신속 하 고 대규모 심사에 기여 스프레이/부상 프로토콜. 이 통합 하 고 체계적인 식물 감염 방법 식물 병리학에서 문제의 광범위 한 관점을 확보에 대 한 훌륭한 출발점 될 것입니다.

기술에 대 한 전체 워크플로 그림 1에 표시 됩니다. 14 일 된 취약 밥 묘 종에 식물 감염 분석 수행 (O. sativa 이력서 공동-39) 취약 7-하루-오래 된 보 리 잎 또는 (H. vulgare 이력서 골든 약속)7,,89. 쌀 잎에 감염에 대 한 테스트, M. grisea 야생-타입 변형과 P131 Com1 삭제 ?...

Watch this Scientific Journal Video about The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea at JoVE.com

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

식물 감염 테스트: 스프레이 식물 병원 체 Magnaporthe Grisea 와 상처-중재 접종

여기, 우리 식물 병원 체와 식물 독성 테스트 프로토콜을 제시 Magnaporthe grisea. 이 보고서 균 분리의 pathotypes의 대규모 검열을 하 고 분자 번 식 하는 동안 식물의 저항 메커니즘을 이해 하기 위한 훌륭한 시작 지점으로 제공.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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Universidad San Sebastian

Research

JoVE Journal

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The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

12.6K 조회수.  Beijing University of Agriculture. 여기, 우리 식물 병원 체와 식물 독성 테스트 프로토콜을 제시 Magnaporthe grisea. 이 보고서 균 분리의 pathotypes의 대규모 검열을 하 고 분자 번 식 하는 동안 식물의 저항 메커니즘을 이해 하기 위한 훌륭한 시작 지점으로 제공.

비디오: The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

병원체 - 관련 분자 패턴 (PAMP) 트리거된 식물의 내성 (PTI)에 대한 분석

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

연구

면역학 및 감염병학

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

옥수수와 테오신테 라인에 우스틸라고 성 실증의 병원성을 평가하기위한 신속하고 효율적인 방법

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

환경과학

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

Endophytes로 곰팡이 Entomopathogens 구축 : Endophytic 생물학적 방제 향해

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

생체공학

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

검은 후추 식물에 대한 Phytophthora capsici에 대한 효과적인 접종 방법

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

유전학

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

식물 뿌리를 토양 매개 미생물로 감염시키는 접종 전략

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

을 사용해서 식물 방어 응답의 미세 특성에 대한 세균성 잎 침투 분석<em&gt; 애기 장대-슈도모나스 시린 개 (Pseudomonas syringae)</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

기본 현미경과 스마트폰을 사용하여 보리(Hordeum vulgare)에 벼 돌풍병(Magnaporthe oryzae)의 초기 감염 부위 시각화

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more ...

발생학

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

애기장대 및 기타 식물의 흰가루병 감염 표현형을 정확하게 평가하기 위한 쉽고 유연한 접종 방법

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

생물학

옥수수 껍질과 균류 사이의 세포 상호 작용의 시각화

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

저자 스포트라이트: Investigating Fungal Pathogenicity Mechanisms in Maize 

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

곰팡이 잎 옥수수 병원체에 의한 감염의 살아있는 세포 이미징을 위한 분리된 옥수수 덮개

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

식물 감염 테스트: 스프레이 식물 병원 체 Magnaporthe Grisea 와 상처-중재 접종

요약

더 많은 비디오 탐색

병원체 - 관련 분자 패턴 (PAMP) 트리거된 식물의 내성 (PTI)에 대한 분석

옥수수와 테오신테 라인에 우스틸라고 성 실증의 병원성을 평가하기위한 신속하고 효율적인 방법

Endophytes로 곰팡이 Entomopathogens 구축 : Endophytic 생물학적 방제 향해

검은 후추 식물에 대한 Phytophthora capsici에 대한 효과적인 접종 방법

식물 뿌리를 토양 매개 미생물로 감염시키는 접종 전략

을 사용해서 식물 방어 응답의 미세 특성에 대한 세균성 잎 침투 분석

기본 현미경과 스마트폰을 사용하여 보리(Hordeum vulgare)에 벼 돌풍병(Magnaporthe oryzae)의 초기 감염 부위 시각화

애기장대 및 기타 식물의 흰가루병 감염 표현형을 정확하게 평가하기 위한 쉽고 유연한 접종 방법

곰팡이 잎 옥수수 병원체에 의한 감염의 살아있는 세포 이미징을 위한 분리된 옥수수 덮개

곰팡이 잎 옥수수 병원체에 의한 감염의 살아있는 세포 이미징을 위한 분리된 옥수수 덮개