Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Ottawa Research and Development Centre, Agriculture Canada, 960 Carling Avenue, Ottawa, ON, K1S 5B6, Canada.
Plant J. 2022 Oct;112(2):369-382. doi: 10.1111/tpj.15949. Epub 2022 Sep 8.
Fusarium graminearum is a fungal pathogen that causes Fusarium head blight in cereal crops. The identification of proteins secreted from pathogens to overcome plant defenses and cause disease, collectively known as effectors, can reveal the etiology of a disease process. Proximity-dependent biotin identification (BioID) was used to identify potential effector proteins secreted in planta by F. graminearum during the infection of Arabidopsis. Mass spectrometry analysis of streptavidin affinity-purified proteins revealed over 300 proteins from F. graminearum, of which 62 were candidate effector proteins (CEPs). An independent analysis of secreted proteins from axenic cultures of F. graminearum showed a 42% overlap with CEPs, thereby assuring confidence in the BioID methodology. The analysis also revealed that 19 out of 62 CEPs (approx. 30%) had been previously characterized with virulence function in fungi. The functional characterization of additional CEPs was undertaken through deletion analysis by the CRISPR/Cas9 method, and by overexpression into Triticum aestivum (wheat) leaves by the Ustilago hordei delivery system. Deletion studies of 12 CEPs confirmed the effector function of three previously characterized CEPs and validated the function of another four CEPs on wheat inflorescence or vegetative tissues. Lastly, overexpression in wheat showed that all seven CEPs enhanced resistance against the bacterial pathogen Pseudomonas syringae DC3000.
镰刀菌禾谷孢是一种真菌病原体，可引起谷物作物的镰孢穗枯病。鉴定从病原体分泌以克服植物防御并引起疾病的蛋白质，统称为效应物，可以揭示疾病过程的病因。邻近依赖性生物素鉴定（BioID）用于鉴定禾谷镰刀菌在感染拟南芥时在植物体内分泌的潜在效应蛋白。链霉亲和素亲和纯化蛋白质的质谱分析揭示了来自禾谷镰刀菌的 300 多种蛋白质，其中 62 种是候选效应蛋白（CEPs）。对禾谷镰刀菌无菌培养物分泌蛋白的独立分析显示，CEPs 与 42%重叠，从而保证了 BioID 方法的可信度。分析还表明，62 个 CEPs 中有 19 个（约 30%）以前在真菌中具有毒力功能。通过 CRISPR/Cas9 方法的缺失分析以及通过 Ustilago hordei 递呈系统在小麦中过表达，对其他 CEPs 进行了功能表征。对 12 个 CEPs 的缺失研究证实了三个以前表征的 CEPs 的效应子功能，并验证了另外四个 CEPs 在小麦花序或营养组织上的功能。最后，在小麦中的过表达表明，所有七个 CEPs 都增强了对细菌病原体丁香假单胞菌 DC3000 的抗性。
