Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato

Abstract

Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.

Figure 1. Red fluorescence derived from DsRED1 six days after agroinfiltration into potato leaves.

(A) Red fluorescence from a single infiltration site on Katahdin. (B) The same infiltration site as (A) under bright field. (C) Red fluorescence from a single infiltration site on Atlantic. (D) The same infiltration site as (C) under bright field. (E) Red fluorescence from a single infiltration site on USW1. No transgenic cells were detected on this image. The strong red fluorescence signals in this infiltration site were derived from autofluorescence associated with the necrotic tissue. (F) The same infiltration site as (E) under bright field. All bars are 10 mm.

Figure 2. Laser-scanning confocal micrographs showing GFP fluorescence from agroinfiltrated leaf cells.

Katahdin leaves were agroinfiltrated with (A) pK7FWG2 empty vector; (B) 35S::GFP; (C) StRAR1::GFP; (D) StGS2::GFP; (E) StV-INV::GFP; and (F) MtDMI3::GFP. The background fluorescence derived from plastids is in blue color. All the scale bars represent 10 µm. Arrows point to the nucleus in the cells.

Figure 3. RT-PCR analysis of transient silencing of the potato Rar1 gene in two independent potato leaves (A and B).

Leaf samples around the infiltrated spots were collected at days 1, 2, 5 and 6 dpi. Lane 1: 100 bp DNA ladder marker; Lane 2: leaf sample from un-infiltrated control; Lane 3: leaf from infiltrated site 1 dpi; Lane 4: leaf from infiltrated site 2 dpi; Lane 5: leaf from infiltrated site 5 dpi; Lane 6: leaf from infiltrated site 6 dpi. Actin was amplified as a control for the amount of template. The amplified Rar1 and Actin transcripts are 339 bp and 360 bp, respectively.

Figure 4. A double agroinfiltration procedure to test candidate genes associated with potato late blight resistance mediated by the RB gene.

All the pictures were taken at 10 days post infiltration and bars represent 2 cm. (A) Infiltration with Agrobacterium carrying pGR106-IpiO1 and HR response was observed around the infiltrated site. (B) Infiltration with Agrobacterium containing pHellsgate-8 silencing construct. (C) Double agroinfiltration with Agrobacterium carrying Sgt1-RNAi construct followed with pGR106-IpiO1. No HR was observed around the infiltrated site. (D) Double agroinfiltration with Agrobacterium carrying Rar1-RNAi construct followed with pGR106-IpiO1. HR response was observed around the infiltrated site.