植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种
Summary
在这里, 我们提出了一个测试植物致病力的植物病原菌稻瘟病的协议.这份报告将有助于大规模筛选真菌分离株的致病型, 作为了解植物在分子育种过程中耐药性机制的良好出发点。
Abstract
植物拥有强大的系统来抵御致病真菌的潜在威胁。然而, 对于农业上重要的植物来说, 目前防治这种病原体的措施过于保守, 因而没有足够的效率, 而且可能造成环境风险。因此, 通过对抗病种质的鉴定、抗性基因的分离和鉴定以及分子育种, 确定寄主抗性因子, 以帮助自然控制植物病害是非常必要的。抗病品种。在这方面, 需要建立一个准确、快速、大规模的接种方法来培育和发展植物抗病基因。稻瘟病真菌病原菌引起严重的疾病症状和产量损失。近年来, m . 菌已成为研究植物真菌病原体相互作用机制的模型有机体。因此, 我们报告了植物毒性试验方法的发展, 这是特定的m. 菌。这种方法提供两种喷雾接种与孢子悬浮和伤人接种与菌丝立方体或孢子悬浮液滴。分离水稻叶片伤后接种方法的关键步骤是在植物叶片上制造伤口, 避免因寄主侵彻阻力而引起的干扰。这种喷雾/伤后的协议有助于快速, 准确, 大规模筛选致病型菌的分离株. 这种综合和系统的植物感染方法将作为一个良好的出发点, 以获得广泛的观点, 植物病理学的问题。
Introduction
水稻稻瘟病是1、2世界水稻品种中最严重的病害之一. 附着胞菌感染寄主植物的过程包括孢子的产生和表面附着、孢子萌发和的形成、渗透 peg 的形成和传染性菌丝分化以及疾病的传播。3. 所有这些阶段在许多其他植物病原真菌中很常见, 事实上, 任何单一阶段的封锁都可以防止寄主植物的感染。由于其经济重要性和遗传驯良, m . 菌已成为研究植物-真菌病原体相互作用机制1,4的模型有机体。因此, 研究m . 菌的这些发育阶段的分子基础将有助于阐明真菌致病性的分子机制和候选靶基因的鉴定, 以筛选和设计新的杀菌剂5。
最近有关m . 菌感染的报告着重于前侵染阶段的分子机制, 特别是 conidiation、附着胞形成、穿透钉和传染性生长3,6. 因此, 必须制定一项详细的议定书, 以测试m . 菌感染。在此, 我们提出了一个感染测试的详细方法, 利用喷雾介导的感染化验与孢子悬浮和接种的伤口, 菌丝插头的m。在本报告中, 该议定书的重点是菌株的培养, conidiation 溶液的制备, 以及菌丝栓介导的植物与m. 菌的接种。下面详细介绍了这些步骤, 并分别显示了图 1和表2中显示方法的整个工作流和典型病变的示意图视图。
Protocol
Representative Results
Discussion
植物抗病基因在预防病原体感染方面起着至关重要的作用, 包括真菌病原体1、12。稻瘟病已被用来作为一个模型, 以了解病原体种群结构的性质, 并确定植物抗病基因4。因此, 有必要对主要品种的抗病基因型和无毒基因型进行大规模的检测, 以确定可持续栽培的抗病植物。此外, 无毒基因型在田间病原体中的优势, 使得寄主品种<sup class="xr…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了北京农业大学 (YQ201603) 的专项科研项目和北京教育委员会 (KM201610020005) 科学项目的支持。
Materials
| Agar | AOBOX Biotechnology（China） | 01-023 | |
| Filter paper | GE Healthcare brand(Sweden) | 10311387 | |
| 50-mL tube | CORNING(Amercia) | 430290 | |
| Centrifuge | Eppendorf(Amercia) | 5804R | |
| Tween-20 | Coolaber（China） | CT11551-100ml | |
| Culture dish | Thermofisher(Amercia) | 150326 | |
| 0.5-5 mL pipette | Eppendorf | 4920000105 | |
| 100-1000uL pipette | Eppendorf | 4920000083 | |
| Vacuum pump | Leybold | D25B | |
| Dissection needle | FST | 26000-35 | |
| Incubator | MEMMERT | PYX313 | |
| Inoculation ring | Greiner Bio One | 731175 |
References
- Li, W. T., et al. A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell. 170 (1), 114-126 (2017).
- Chi, M. H., Park, S. Y., Kim, S., Lee, Y. H. A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host. PLoS Pathogens. 5 (4), 1000401 (2009).
- Jia, Y., Valent, B., Lee, F. N. Determination of host responses to Magnaporthe grisea.on detached rice leaves using a spot inoculation method. Plant Disease. 87 (2), 129-133 (2003).
- Ebbole, D. J. Magnaporthe as a model for understanding host-pathogen interactions. Annual Review of Phytopathology. 45, 437-456 (2007).
- Hamer, J. E., Talbot, N. J. Infection-related development in the rice blast fungus Magnaporthe grisea. Current Opinion in Microbiology. 1 (6), 693-697 (1998).
- Howard, R. J., Valent, B. Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annual Review of Microbiology. 50, 491-512 (1996).
- Chen, X. L., et al. N-Glycosylation of Effector Proteins by an α-1,3- Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity. The Plant Cell. 26 (3), 1360-1376 (2014).
- Zhang, Y., et al. M.ARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae. Microbiological Research. 180, 11-22 (2015).
- Du, Y. X., et al. A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae. Current Genetics. 59 (1-2), 33-41 (2013).
- Yang, J., et al. A novel protein com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae. Molecular Plant-Microbe Interactions. 23 (1), 112-123 (2010).
- Cao, Z. J., et al. An ash1-like protein MoKMT2H null mutant is delayed for conidium germination and pathogenesis in Magnaporthe oryzae. BioMed Research International. 2016, 1575430 (2016).
- Bryan, G. T., et al. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. The Plant Cell. 12 (11), 2033-2045 (2000).
- Zhou, J. M. Plant pathology: a life and death struggle in rice blast disease. Current Biology. 26 (18), 843-845 (2016).
- Guo, M., et al. MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae. Applied Microbiology and Biotechnology. 99 (19), 8075-8088 (2015).
- Talbot, N. J. On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. Annual Review of Microbiology. 57, 177-202 (2009).
- Wilson, R. A., Talbot, N. J. Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nature Reviews Microbiology. 7, 185-195 (2009).
- Jia, Y. L., Lee, F. N., McClung, A. 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. Plant Disease. 93, 639-644 (2009).
- Peng, Y. L., Shishiyama, J. Temporal sequence of cytological events in rice leaves infected with Pyricularia oryzae. Canadian Journal of Botany. 66 (4), 730-735 (1988).
