Ratiometric reporter systems enable comparisons of the abundance of a protein of interest, or "target," relative to a reference protein Both proteins are encoded on a single transcript but are separated during translation This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes We generated a set of 18 Gateway-compatible vectors termed pRATIO that combine a variety of promoters, fluorescent, and bioluminescent reporters, and 2A "self-cleaving" peptides These constructs are easily modified to produce additional combinations or introduce new reporter proteins We found that mScarlet-I provides the best signal-to-noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana Firefly and Gaussia luciferase also produce high signal-to-noise in N benthamiana As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization KAI2 is normally found in the cytoplasm and the nucleus of plant cells In N benthamiana, karrikin-induced degradation of KAI2 was only observed when it was retained in the nucleus These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli

A series of dual-reporter vectors for ratiometric analysis of protein abundance in plants.

Rice is the model plant system for monocots and the sequencing of its genome has led to the identification of a vast array of genes for characterization. The tedious and time-consuming effort of raising rice transgenics has significantly delayed the pace of rice research. The lack of highly efficient transient assay protocol for rice has only added to the woes which could have otherwise helped in rapid deciphering of the functions of genes. Here, we describe a technique for efficient transient gene expression in rice seedlings. It makes use of co-cultivation of 6-day-old rice seedlings with Agrobacterium in the presence of a medium containing Silwet® L-77, acetosyringone and glucose. Seedlings can be visualized 9 days after co-cultivation for transient expression. The use of young seedlings helps in significantly reducing the duration of the experiment and facilitates the visualization of rice cells under the microscope as young leaves are thinner than mature rice leaves. Further, growth of seedlings at low temperature, and the use of surfactant along with wounding and vacuum infiltration steps significantly increases the efficiency of this protocol and helps in bypassing the natural barriers in rice leaves, which hinders Agrobacterium-based transformation in this plant. This technique, therefore, provides a shorter, efficient and cost-effective way to study transient gene function in intact rice seedling without the need for a specialized device like particle gun.

/pdf/a-rapid-and-highly-efficient-method-for-transient-gene-2q1oasxksj.pdf

A Rapid and Highly Efficient Method for Transient Gene Expression in Rice Plants.

Agrobacterium mediated transformation in rice

Summary In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue‐types and/or at specific times often precludes co‐expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator‐like effectors (dTALEs) and cognate synthetic TALE‐activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1‐STAP1 and dTALE2‐STAP2—can activate STAP‐driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell‐types, requiring empirical evaluation in each case. dTALE expression leads to off‐target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE‐STAP system thus provides a powerful approach to fine‐tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

/pdf/a-single-promoter-tale-system-for-tissue-specific-and-18bl7x0l.pdf

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

Abstract C 4 photosynthesis is used by the most productive plants on the planet, and compared with the ancestral C 3 pathway, it confers a 50% increase in efficiency 1 . In more than 60 C 4 lineages, CO 2 fixation is compartmentalized between tissues, and bundle-sheath cells become photosynthetically activated 2 . How the bundle sheath acquires this alternate identity that allows efficient photosynthesis is unclear. Here we show that changes to bundle-sheath gene expression in C 4 leaves are associated with the gain of a pre-existing cis -code found in the C 3 leaf. From single-nucleus gene-expression and chromatin-accessibility atlases, we uncover DNA binding with one finger (DOF) motifs that define bundle-sheath identity in the major crops C 3 rice and C 4 sorghum. Photosynthesis genes that are rewired to be strongly expressed in the bundle-sheath cells of C 4 sorghum acquire cis -elements that are recognized by DOFs. Our findings are consistent with a simple model in which C 4 photosynthesis is based on the recruitment of an ancestral cis -code associated with bundle-sheath identity. Gain of such elements harnessed a stable patterning of transcription factors between cell types that are found in both C 3 and C 4 leaves to activate photosynthesis in the bundle sheath. Our findings provide molecular insights into the evolution of the complex C 4 pathway, and might also guide the rational engineering of C 4 photosynthesis in C 3 crops to improve crop productivity and resilience 3,4 . 

Exaptation of ancestral cell-identity networks enables C4 photosynthesis

Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed analysis of processes ranging from gene expression and protein localization through to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualise fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee, and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins, mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato that are robustly expressed and visible in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolour imaging of different sub-cellular compartments. Overall, we conclude that mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato are suitable for high resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.

https://www.biorxiv.org/content/biorxiv/early/2019/05/30/649160.full.pdf

Fluorescent reporters for functional analysis in rice leaves

Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed the analysis of processes ranging from gene expression and protein localization to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualize fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins (mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato) that are robustly expressed and detectable in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolor imaging of different subcellular compartments. Overall, we conclude that mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato are suitable for high-resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.

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Fluorescent reporters for functional analysis in rice leaves

Fluorescent reporters for functional analysis in rice leaves