Abstract
Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.
Highlights
Virus-induced gene silencing (VIGS) is a useful tool for plant functional genomics because it permits knockdown of genes-of-interest and observation of elicited phenotypes within 3 to 4 weeks
To permit more effective use of Barley stripe mosaic virus (BSMV) VIGS for functional genomics experiments, we have developed an approach with Agrobacterium tumefaciens strains harboring the BSMV a, b, and c cDNAs in Ti plasmids for initiation of BSMV infections upon infiltration of N. benthamiana leaves
Our results demonstrate that agroinfiltrated leaves of N. benthamiana are excellent sources of BSMV VIGS inoculum for high throughput dissection of wheat, barley and B. distachyon genes involved in disease development and pathogenesis
Summary
Virus-induced gene silencing (VIGS) is a useful tool for plant functional genomics because it permits knockdown of genes-of-interest and observation of elicited phenotypes within 3 to 4 weeks. The VIGS principle is based on antiviral responses that target RNAs for degradation and is triggered by accumulation of double-stranded RNAs (dsRNA) appearing in the infection cycle [5,6]. ‘‘shock’’, phase of infection intense symptoms develop and high levels of virus accumulate. As host defense mechanisms are activated, infections usually transit into a protracted chronic phase characterized by modulated symptoms and low virus abundance. By inserting sequence fragments derived from ‘‘genes-of-interest’’ into VIGS vectors, the corresponding mRNAs are selectively degraded during virus infection to result in transient silencing of the targeted gene
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