Agrobacterium-Mediated Transient Gene Expression and Silencing: A Rapid Tool for Functional Gene Assay in Potato

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

Sgt1, but not Rar1, is essential for the RB-mediated broad-spectrum resistance to potato late blight

<p>Abstract</p> <p>Background</p> <p>Late blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes.</p> <p>Results</p> <p>We previously cloned a late blight resistance gene, <it>RB</it>, from a diploid wild potato species <it>Solanum bulbocastanum</it>. Transgenic potato lines containing a single <it>RB </it>gene showed a rate-limiting resistance against all known races of <it>Phytophthora infestans</it>, the late blight pathogen. To better understand the <it>RB</it>-mediated resistance we silenced the potato <it>Rar1 </it>and <it>Sgt1 </it>genes that have been implicated in mediating disease resistance responses against various plant pathogens and pests. The <it>Rar1 </it>and <it>Sgt1 </it>genes of a <it>RB</it>-containing potato clone were silenced using a RNA interference (RNAi)-based approach. All of the silenced potato plants displayed phenotypically normal growth. The late blight resistance of the <it>Rar1 </it>and <it>Sgt1 </it>silenced lines were evaluated by a traditional greenhouse inoculation method and quantified using a GFP-tagged <it>P. infestans </it>strain. The resistance of the <it>Rar1</it>-silenced plants was not affected. However, silencing of the <it>Sgt1 </it>gene abolished the <it>RB</it>-mediated resistance.</p> <p>Conclusion</p> <p>Our study shows that silencing of the <it>Sgt1 </it>gene in potato does not result in lethality. However, the <it>Sgt1 </it>gene is essential for the <it>RB</it>-mediated late blight resistance. In contrast, the <it>Rar1 </it>gene is not required for <it>RB</it>-mediated resistance. These results provide additional evidence for the universal role of the <it>Sgt1 </it>gene in various <it>R </it>gene-mediated plant defense responses.</p

Genomic heterogeneity and structural variation in soybean near isogenic lines

Near-isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the genetic heterogeneity extant among NIL sub-lines is an unaddressed research topic that might have implications for how genomic and phenotypic data from NILs are utilized. In this study, a recently developed high-resolution comparative genomic hybridization (CGH) platform was used to investigate the structure and diversity of genetic introgressions in two classical soybean NIL populations, respectively varying in protein content and iron deficiency chlorosis (IDC) susceptibility. There were three objectives: assess the capacity for CGH to resolve genomic introgressions, identify introgressions that are heterogeneous among NIL sub-lines, and associate heterogeneous introgressions with susceptibility to IDC. Using the CGH approach, introgression boundaries were refined and previously unknown introgressions were revealed. Furthermore, heterogeneous introgressions were identified within seven sub-lines of the IDC NIL ‘IsoClark’. This included three distinct introgression haplotypes linked to the major iron susceptible locus on chromosome 03. A phenotypic assessment of the seven sub-lines did not reveal any differences in IDC susceptibility, indicating that the genetic heterogeneity among the lines does not have a significant impact on the primary NIL phenotype

RT-PCR analysis of transient silencing of the potato <i>Rar1</i> gene in two independent potato leaves (A and B).

<p>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. <i>Actin</i> was amplified as a control for the amount of template. The amplified <i>Rar1</i> and <i>Actin</i> transcripts are 339 bp and 360 bp, respectively.</p

A double agroinfiltration procedure to test candidate genes associated with potato late blight resistance mediated by the <i>RB</i> gene.

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

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

<p>Katahdin leaves were agroinfiltrated with (A) pK7FWG2 empty vector; (B) 35S::GFP; (C) <i>St</i>RAR1::GFP; (D) <i>St</i>GS2::GFP; (E) <i>St</i>V-INV::GFP; and (F) <i>Mt</i>DMI3::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.</p

Red fluorescence derived from <i>Ds</i>RED1 six days after agroinfiltration into potato leaves.

<p>(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.</p

Identification of miniature inverted-repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: New functional implications for MITEs

Small RNAs regulate the genome by guiding transcriptional and post-transcriptional silencing machinery to specific target sequences, including genes and transposable elements (TEs). Although miniature inverted-repeat transposable elements (MITEs) are closely associated with euchromatic genes, the broader functional impact of these short TE insertions in genes is largely unknown. We identified 22 families of MITEs in the Solanaceae (MiS1–MiS22) and found abundant MiS insertions in Solanaceae genomic DNA and expressed sequence tags (EST). Several Solanaceae MITEs generate genome changes that potentially affect gene function and regulation, most notably, a MiS insertion that provides a functionally indispensable alternative exon in the tobacco mosaic virus N resistance gene. We show that MITEs generate small RNAs that are primarily 24 nt in length, as detected by Northern blot hybridization and by sequencing small RNAs of Solanum demissum, Nicotiana glutinosa, and Nicotiana benthamiana. Additionally, we show that stable RNAi lines silencing DICER-LIKE3 (DCL3) in tobacco and RNA-dependent RNA polymerase 2 (RDR2) in potato cause a reduction in 24-nt MITE siRNAs, suggesting that, as in Arabidopsis, TE-derived siRNA biogenesis is DCL3 and RDR2 dependent. We provide evidence that DICER-LIKE4 (DCL4) may also play a role in MITE siRNA generation in the Solanaceae