About the VIGS Library

NbME library:

The Nicotiana benthamiana Mixed Elicitor cDNA (NbME) library was made from N. benthamiana plant leaves treated with different abiotic and biotic stress elicitors listed below:

An analog of salicylic acid (INA CGA41396 from CIBA 1.3 mM; samples collected 4h and 24h after), Jasmonic acid (100 μM from Sigma; samples collected 4h and 12h after), ethylene (50 ppm in sealed chamber incubation; samples collected 2 and 12 h after)

Pto/avrPto-mediated HR (by leaf infiltration of a mixture of Agrobacterium cultures carrying 35S::AvrPto and 35S::Pto transgenes; samples collected 24h and 48h after), Pseudomonas syringae pv. tomato T1-mediated non-host HR (titer 108 CFU/ml; samples collected 4 and 18 h after), disease caused by P. syringae pv. tabaci (titer 106 CFU/ml; samples collected 24 and 72 h after)

Control untreated leaf tissue was included as well. Equal amounts of total RNA were pooled for mRNA purification using polyATract (Promega Co., Madison, WI, U.S.A.). The cDNA library was constructed following the instruction of PCR-Select kit (BD Bioscience-Clontech, Palo Alto, CA, U.S.A.) with some modifications. The cDNA population was normalized instead of subtraction (driver and tester were the same), size selected (0.1 to 1.0 kb) and the inserts were made compatible with the GATEWAY cloning system by adding attB1 and attB2 sequences (Invitrogen) to Nested PCR primer 1 and Nested PCR primer 2R respectively (PCR-Select kit, BD Bioscience-Clontech).

The library was cloned via a GATEWAY reaction (Invitrogen) into the destination vector pTRV2. The library was transformed into Agrobacterium strain GV2260. The culture of individual clones were arrayed in 96 well plates. The library is stored in -800C.

References:

Anand A, Vaghchhipawala Z, Ryu C-M, Kang L, Wang K, del-Pozo O, Martin GB, Mysore KS: Identification and Characterization of Plant Genes Involved in Agrobacterium-Mediated Plant Transformation by Virus-Induced Gene Silencing. Molecular Plant-Microbe Interactions 2007, 20(1):41-52.

Chakravarthy S, Velásquez AC, Ekengren SK, Collmer A, Martin GB: Identification of Nicotiana benthamiana Genes Involved in Pathogen-Associated Molecular Pattern–Triggered Immunity. Molecular Plant-Microbe Interactions 2010, 23(6):715-726.

NbTI library:

Tobacco mosaic virus-induced (TI) cDNA library was constructed from the N. benthamiana leaves (NbTI) inoculated with TMV. This library was cloned into pTRV2 vector. The cDNA pool was transformed to Agrobacterium strain GV2260 and the cultures were arrayed in 96 well plates. The library is stored in -800C.

Source: Dinesh-Kumar Laboratory UC Davis

Brief VIGS protocol and phenotype observations:

The Agrobacterium strain GV2260 containing pTRV1 was grown at 28 oC in LB medium containing appropriate antibiotics. After 24 h, the cells were harvested and re-suspended in the infiltration buffer (10 mM MES pH 5.5; 200 mM acetosyringone) to a final absorbance (OD at 600 nm) and incubated for approx. 3 h with mild shaking at room temperature. Agrobacterium strain containing pTRV1 was suspended in MES buffer pH 5.5 and infiltrated into lower leaves using 1 ml needle-less syringe. Later, pTRV2 [pTRV2 or pTRV2::GFP*] carrying bacterial colony from LB-agar plate was inoculated on to the leaf using tooth picks. The inoculated plants were maintained under the temperature range of 20-23oC for effective viral infection and spread. Phenotype observations and silencing confirmation were done from 2 to 4 weeks after VIGS vector inoculation.

*Green fluorescent protein (GFP) is a non-plant gene and a partial fragment was cloned into pTRV2 vector in order for development of vector control plants.

Reference:

Senthil-Kumar, M., Lee, H.K., Mysore, K.S. 2013. VIGS-mediated forward genetics screening for identification of genes involved in nonhost resistance. Journal of Visualized Experiments, 78, e51033