Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri
Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri
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DOI：
10.1111/j.1467-7652.2010.00555.x
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发表时间：
2011-04-01
影响因子：
13.8
通讯作者：
Rosa Marano, Maria
中科院分区：
文献类型：
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作者：
Enrique, Ramon;Siciliano, Florencia;Rosa Marano, Maria
柑橘是一种重要的经济作物，柑橘溃烂病是由柑橘黄单胞菌引起的一种严重病害。Citri(Xcc)GenBank拥有大量的表达序列标签(EST)，这些EST富含在对这种病原体的防御反应中产生的转录本；然而，目前在柑橘中还没有策略来评估候选基因的功能。这极大地限制了研究，因为防御信号基因通常涉及多个途径。在这项研究中，我们证明了RNA干扰(RNAi)作为一种功能基因组学工具的有效性，以评估参与柑橘柠檬对柑橘溃烂病菌防御反应的候选基因的功能。将编码柑橘寄主基因发夹RNA的双链RNA表达载体通过转化农杆菌瞬时渗入柠檬叶片。作为原理的证明，我们已经建立了柑橘植酸烯脱氢酶(PDS)和胼胝质合成酶(CalS1)基因的沉默。表型和分子分析表明，沉默载体不仅在柠檬植物中具有功能，而且在芸香科其他物种中也具有功能。利用沉默的Ca/S1，我们已经证明了植物细胞壁相关的防御是柑橘植株抵御黄单胞菌侵染的主要初始屏障。此外，我们在这里提出的结果表明，在病程中被来自Xcc的黄原胶抑制的过氧化氢积累，有助于抑制野生型或CalS1沉默的植物中缺乏黄原胶的Xcc突变株的生长。通过这项工作，我们证明了在柑橘中进行高通量反向遗传分析是可行的。
Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by the bacterial phytopathogen, Xanthomonas citri subsp. citri (Xcc). GenBank houses a large collection of Expressed Sequence Tags (ESTs) enriched with transcripts generated during the defence response against this pathogen; however, there are currently no strategies in citrus to assess the function of candidate genes. This has greatly limited research as defence signalling genes are often involved in multiple pathways. In this study, we demonstrate the efficacy of RNA interference (RNAi) as a functional genomics tool to assess the function of candidate genes involved in the defence response of Citrus limon against the citrus canker pathogen. Double-stranded RNA expression vectors, encoding hairpin RNAs for citrus host genes, were delivered to lemon leaves by transient infiltration with transformed Agrobacterium. As proof of principle, we have established silencing of citrus phytoene desaturase (PDS) and callose synthase (CalS1) genes. Phenotypic and molecular analyses showed that silencing vectors were functional not only in lemon plants but also in other species of the Rutaceae family. Using silencing of Ca/S1, we have demonstrated that plant cell wall-associated defence is the principal initial barrier against Xanthomonas infection in citrus plants. Additionally, we present here results that suggest that H2O2 accumulation, which is suppressed by xanthan from Xcc during pathogenesis, contributes to inhibition of xanthan-deficient Xcc mutant growth either in wild-type or CalS1-silenced plants. With this work, we have demonstrated that high-throughput reverse genetic analysis is feasible in citrus.