Plant Functional Genomics in A Few Days: Laser-Assisted Delivery of Double-Stranded RNA to Higher Plants.

Nabil Killiny,Siddarame Gowda,Pedro Gonzalez-Blanco,Ed Etxeberria,Xavier Martini

doi:10.3390/plants10010093

Abstract

The technology of transgenic plants is challenging and time consuming, especially for higher plants and trees such as citrus. Double-stranded RNA (dsRNA) delivery via a plant virus is an alternative method to create transgenic plants by suppressing the expression of plant endogenous genes. Citrus tristeza virus-based vector has been constructed specifically for use in citrus trees. However, this is time-consuming, as it can take up to nine months to produce the desired phenotype. Here we describe a much faster method for the study of gene function in citrus trees. In the current study, we used laser light for the delivery of dsRNA to citrus leaves. We targeted the endogenous reporter gene phytoene desaturase (PDS) and obtained the classical phenotype (leaf bleaching) in only three days after the laser-assisted delivery. Interestingly, the phenotype response was systemic, which indicates the movement of dsRNA and/or ssRNA within the plants. In addition, dsRNAs were taken up by phloem cells and the bleaching phenotype was clear around the main veins. In conclusion, the delivery of dsRNA to plants through laser treatment may provide a fast and more specific tool to study the gene function in higher plants and trees.

Highlights

Creating transgenic plants for plant improvement and resistance to diseases is slow and expensive
To increase penetration of double-stranded RNA (dsRNA), and accelerate the gene silencing, we examined the use of laser light to puncture microscopic holes within the lipidized leaf surface without extensive damage to the leaf tissue
We introduced laser light micro-perforation as a fast and reliable alternative method of introducing dsRNA into citrus leaves

Summary

Introduction

Creating transgenic plants for plant improvement and resistance to diseases is slow and expensive. The traditional method of making transgenic plants requires many steps: defining and locating the desired genetic traits, isolating and multiplying the DNA fragments for insertion into the recipient plants, and growing and evaluating several generations of the modified plants to determine if the new genes have been passed on and are expressing the targeted traits. RNAi uses exogenous double-stranded RNA (dsRNA) to stop or reduce production of a targeted protein by knocking down the complementary endogenous messenger RNA (mRNA) before translation. The enzyme Dicer cleaves the dsRNA into short double-stranded RNA fragments called small interfering RNA (siRNAs) that guide a large protein complex that silence mRNA [1,2]

Methods

Results

Conclusion