Phytogene therapy development with RNAi mediated gene silencing in crops for disease control without GM

目的利用反义寡脱氧核苷酸（ODNs）或硫代磷酸酯（PTOs）特异性沉默植物和真菌致病基因。预测的结果是发展表观遗传策略，以保护大麦和小麦免受主要谷物病原体的侵害。背景利用小分子asRNA介导的RNAi技术沉默基因已广泛应用于动物科学领域，但在植物科学领域的应用尚不多见。小的合成特异性asODN或其化学修饰的硫代磷酸酯（PTO）正被成功地用于植物中的靶向基因沉默，通过木质部或通过气孔直接渗透递送“裸”18-19聚体ODN/PTO。利用寄主诱导基因沉默技术（HIGS）对大麦白粉病（Blumeria graminis f. sp. hordei; Bgh）的毒力因子（效应因子）进行了沉默。HIGS涉及将靶基因克隆为正义/发夹构建体，用于生物射弹转化和植物表达。在HIGS中，只有一部分细胞被转化，因此不可能将感染表型与靶基因表达降低联系起来。此外，HIGS也可能沉默“脱靶”基因，不允许实际应用。初步数据-我们证明了荧光ODNs的细胞在大麦叶（切割/浸润）的成功摄取。- asPTO比asODN更有效地降低感染成功率。针对特定真菌或植物基因的asPTO减少了Bgh对大麦的感染。成功的靶标包括Blumeria效应子BEC 1011（RNA酶样）、通用金属蛋白酶样BEC 1019和植物易感基因Blufensin-1（BLN-1）。初步qRT-PCR数据表明，减少的感染表型与减少的基因表达相关。工作计划和目标-优化PTO和其他ODNs化学，以及从离体叶片或分蘖/穗到整株植物的递送方法。- 将PTO-基因沉默技术转移到控制小麦病原体如B上。禾谷镰刀菌（Fusarium graminearum）（Fg）和三裂孢菌（Zymoseptoria triangulae）（Zt）。- 在Bgt、Fg和Zt中沉默和查询BEC 1019通用性。这将通过使用敲除构建体来删除Fg和Zt中的BEC 1019同源物来证实。设计和测试新的asPTO，用于沉默其他真菌效应基因或一般毒力因子，促进真菌感染的植物易感基因。成功的asPTO随后将在先正达（Stein Crop Protection，CH）的农用工业杀真菌剂筛选设施中进行测试。

AimThe aim of this project is to use small antisense (as) oligodeoxynucleotides (ODNs) or as phosphorothioates (PTOs) to specifically silence plant and fungal genes that are necessary for the establishment of fungal disease in cereals. The predicted outcome is the development of epigenetic strategies to protect barley and wheat against major cereal pathogens. BackgroundDelivery of small asRNA for RNAi-based gene silencing is extensively used in animal science, but only emerging in plant sciences. Small synthetic specific asODNs or their chemically modified phophorothioates (PTOs) are being used successfully for targeted gene silencing in plants, delivering "naked" 18-19 mers ODNs/PTOs via xylem or by direct infiltration through stomata. Virulence factors (effectors) of barley powdery mildew (Blumeria graminis f.sp. hordei; Bgh) were silenced in planta by Host Induced Gene Silencing (HIGS). HIGS, involves cloning the target gene in a sense/as hairpin construct for biolistic transformation and in planta expression. In HIGS, only a fraction of the cells are transformed, thus it was not possible to link infection phenotype to decreased expression of the targeted gene. Moreover HIGS might also silence "off-target" genes and does not allow practical applications. Preliminary data-We demonstrated successful uptake of fluorescent ODNs by cells in barley leaves (cut/ infiltrated). -asPTOs were more effective than asODNs to decrease infection success rate.-asPTOs targeting specific fungal or plant genes reduced barley infection by Bgh. Successful targets included Blumeria effectors BEC1011 (RNAse like), the universal metalloprotease like BEC1019 and the plant susceptibility gene Blufensin-1 (BLN-1).-Preliminary qRT-PCR data indicated that the reduced infection phenotypes correlated with reduced gene expression.-Recent patents suggest successful uptake from spray application for disease control.Workplan and objectives -Optimising PTOs and other ODNs chemistries, and delivery methods from excised leaves or tillers/ ears to entire plants. -Transferring the PTO-gene silencing technology to control wheat pathogens such as B. graminis f.sp tritici (Bgt), Fusarium graminearum (Fg) and Zymoseptoria tritici (Zt). -Silencing and querying BEC1019 universality in Bgt, Fg and Zt. This will be confirmed by using knock out constructs to delete the BEC1019 homolog in Fg and Zt.-Designing and testing of new asPTOs for silencing other fungal genes effectors or general virulence factors, plant susceptibility genes facilitating fungal infection. Successful asPTOs will then be tested in agro-industry fungicide screening facilities at Syngenta, Stein Crop Protection, CH.