HAU16008-02-4 - The Plant Journal(2004 40 322331 doi...

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TECHNICAL ADVANCE Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species Choong-Min Ryu , Ajith Anand, Li Kang and Kirankumar S. Mysore * Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Pkwy., Ardmore, OK 73402, USA Received 15 March 2004; revised 15 July 2004; accepted 23 July 2004. * For correspondence (fax þ 580 224 6692; e-mail: [email protected]). Present address: Laboratory of Microbial Genomics, Korea Research Institute of Bioscience & Biotechnology (KRIBB), S2 Oun-dong, Yusong-gu, Taejon, 305-333, S. Korea. Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called ‘agrodrench’, where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase ( PDS ), a 20S proteasome subunit ( PB7 ) or Mg-protoporphyrin chelatase ( Chl H ) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods. Keywords: agrodrench, virus-induced gene silencing, agroinoculation, Tobacco rattle virus , Solanaceae. Introduction Virus-induced gene silencing (VIGS) is an RNA-mediated post-transcriptional gene-silencing mechanism that pro- tects plants against foreign gene invasion (Baulcombe, 1999). In addition to allowing a better understanding of how plants defend themselves against plant viruses, VIGS has emerged as a functional genomics tool for knocking out gene expression of target plant genes in some plants (Ekengren et al. , 2003; Holzberg et al. , 2002; Liu et al. , 2002a; Peart et al. , 2002; Sharma et al. , 2003; Yoshioka et al. , 2003). VIGS is also being used as a forward genetics tool to identify a desired phenotype (Lu et al. , 2003b). Plant virus-based vectors carrying plant sequences homologous to endogenous plant genes trigger gene silencing through a homology-dependent RNA degradation mechanism commonly referred to as RNA silencing. Small interfering
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