Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley
Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley
复制标题
DOI：
10.1104/pp.105.062810
复制
发表时间：
2005-08-01
期刊：
影响因子：
7.4
通讯作者：
Lacomme, C
中科院分区：
文献类型：
--
作者：
Hein, I;Pacak, MB;Lacomme, C
我们成功地在大麦中实现了病毒诱导的基因沉默(VIGS)，为Mla13介导的对生态型大麦病原菌Blumeria graminis f.sp.的抗性所需的基因进行了功能鉴定。霍尔代伊。最初，通过允许大麦条纹花叶病毒(BSMV)-VIGS载体的复制和系统运动而不会引起过度症状，对大麦品种进行了筛选，以确定其寄主大麦条纹花叶病毒的能力。将导致光漂白的植酸烯去饱和酶沉默作为表型标记，结合逆转录-聚合酶链式反应在分子水平上表征沉默反应。携带Mla13抗性基因的大麦品种Clansman被选为最适合于基于BSMV-VIGS的Rar1、SGT1和Hsp90在MLA介导的白粉病抗性中的功能鉴定的宿主。BSMV诱导了这些候选基因的基因沉默，这些候选基因存在于许多但不是所有小种特有的途径中，被证明是强大的，并且在接种后21d可以在mRNA和蛋白质水平上检测到。系统沉默不仅存在于主茎的新叶中，也存在于腋芽中。通过检测携带同源Avr13基因的不亲和霉菌株在Rar1、SGT1和Hsp90沉默的BSMV植株上的真菌发育，观察到了抗性打破的表型，而感染BSMV对照构建体的植株仍然具有抗性。我们证明Hsp90是Mla13介导的小种特异性抗性所必需的组分，而BSMV诱导的VIGS是鉴定大麦抗病相关基因的有力工具。
We successfully implemented virus-induced gene silencing (VIGS) in barley ( Hordeum vulgare) for the functional characterization of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially, barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes involved in pathogen resistance in barley.