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

Potential regulatory phosphorylation sites in a Medicago truncatula plasma membrane proton pump implicated during early symbiotic signaling in roots

AbstractIn plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells

A role for the mevalonate pathway in early plant symbiotic signaling

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system

Ca2+-regulated Ca2+ channels with an RCK gating ring control plant symbiotic associations

A family of plant nuclear ion channels, including DMI1 (Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca2+ spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K+) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca2+-gated K+ (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca2+ sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca2+ binding sites to the physiological function of CASTOR as well as DMI1

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

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

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

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

Metabolomic profiling reveals suppression of oxylipin biosynthesis during the early stages of legume–rhizobia symbiosis

AbstractThe establishment of symbiosis between leguminous plants and rhizobial bacteria requires rapid metabolic changes in both partners. We utilized untargeted quantitative mass spectrometry to perform metabolomic profiling of small molecules in extracts of the model legume Medicago truncatula treated with rhizobial Nod factors. One metabolite closely resembling the 9(R)-HODE class of oxylipins reproducibly showed a decrease in concentration within the first hour of in planta nod factor treatment. Oxylipins are precursors of the jasmonic acid biosynthetic pathway and we showed that both this metabolite and jasmonic acid inhibit Nod factor signaling. Since, oxylipins have been implicated as antimicrobial compounds produced by plants, these observations suggest that the oxylipin pathway may play multiple roles in facilitating Nod factor signaling during the early stages of symbiosis