Transient reprogramming of crop plants for agronomic performance

Abstract

The development of a new crop variety is a time-consuming and costly process due to plant breeding's reliance on gene shuffling to introduce desired genes into elite germplasm followed by backcrossing. We propose alternative technology that transiently targets various regulatory circuits within a plant, leading to operator-specified alterations of agronomic traits, such as time of flowering, vernalization requirement, plant height or drought tolerance. We redesigned techniques of gene delivery, amplification and expression around RNA viral transfection methods that can be implemented on an industrial scale and with multiple crop plants. The process does not involve genetic modification of the plant genome and is thus limited to a single plant generation, is broadly applicable, fast, tunable, versatile, and can be used throughout much of the crop cultivation cycle. The RNA-based reprogramming may be especially useful in case of major plant pathogen pandemics, but also for commercial seed production and for rapid adaptation of orphan crops.3 Modern plant breeding relies on recombination to introduce novel useful genes/alleles into elite germplasm. Development of a new variety is time-consuming and expensive, even with a use of most advanced technologies such as genome editing. We sought to design a flexible, rapid and industrially scalable alternative platform to alter hormonal and other regulatory circuits within a plant, by rebuilding the known techniques of transient gene expression around gene delivery methods that can be performed on an industrial scale, and that can be practiced with multiple crop plants. Our approach focused on two types of vectors commonly used in laboratory science; namely, Agrobacterium as the primary DNA vector, and RNA viral amplicons as secondary/primary vectors and amplifiers of information molecules. We and others have successfully used Agrobacterium-based transfection to design industrial-scale manufacturing processes for producing recombinant proteins in plants [1][2][3][4] , including biopharmaceuticals, vaccines and biomaterials 5 . This earlier-generation transient reprogramming focused on a single plant species, Nicotiana benthamiana. The method required vacuum-assisted infiltration of bacteria into the intercellular leaf space and, by design, ignored the general agronomic performance of the plant other than the high-level expression of heterologous recombinant proteins that were almost exclusively of non-plant origin. A few attempts to modify agronomic traits, namely viral induction of flowering, were also previously reported, but were limited to research-scale experiments [6][7][8][9][10][11] .We report here that multiple economically important crop plants can be induced to exhibit desirable agronomic performance traits, by simply spraying them with agrobacteria carrying viral replicons to express plant genes. Moreover, we also demonstrate that most of the agronomic traits can also be engineered by spraying plants with packaged RNA viral vectors thus 4 eliminating DNA release...

Custom-made design of metabolite composition inN. benthamianaleaves using CRISPR activators

“…Transient delivery of genetic information in the form of T-DNA or RNA is becoming increasingly popular in plant biotechnology. Recently, transient reprogramming of crop plants was shown using RNA virus-based delivery systems, either as viral particles or mediated by Agrobacterium (Torti et al 2021). Agroinfiltration has become not only a widely used experimental procedure (Norkunas et al 2018;Grosse-Holz et al 2018), but also a potent and scalable plant biomanufacturing strategy as recently demonstrated with the production of plantmade vaccines against influenza and SARS-CoV2 (D'Aoust et al 2008;Diego-Martin et al 2020).…”

Reprogramming plant specialized metabolism by manipulating protein kinases

“…However, as stable transformation protocol is not available for many medicinal plants, transient-based reprogramming (cite the new mol plant paper), hairy roots, cell cultures, or modified Agrobacterium -mediated gene delivery and virus-based gene silencing are effective alternatives (Ma et al 2020 ; Maher et al 2020 ). A recent study demonstrates a versatile and efficient Agrobacterium and RNA viral replicon-based transient expression system that can be used to reprogram developmental and hormone signaling pathways in a wide range of plant species (Torti et al 2021 ). Such an approach can potentially be used for PKs to reprogram specialized metabolism in plants.…”

RNA Viral Vectors for Accelerating Plant Synthetic Biology

“…For example, Llorente et al (2020) were able to establish that loss of photosynthetic competence and enhanced carotenoid production were required for chromoplast development via overexpression of the crtB protein from a plant RNA viral vector. Recent work by Torti et al (2021) demonstrated that single genes could be delivered to a range of plants via RNA viral vectors to create several different agronomically important traits including dwarfing and flowering time. They used a combination of whole plant delivery techniques via high pressure spraying, modified versions of potato virus X that included additional silencing machinery, and modified cargos with higher GC content to achieve these results.…”

“…As encapisdation plays a role in viral genome stability and effective systemic movement for some viruses ( Lee et al, 2011 ), designing novel strategies to reengineer cargo sequences to make them more “viral-like” could be another way to improve expression from viral vectors. The efficacy of increasing the GC content of cargos to stabilize them in viral vectors was recently demonstrated ( Torti et al, 2021 ). The success of refactoring a cargo sequences by including strong introns and higher GC content indicates there might be more promising strategies to improve cargo stability and expression through sequence modification.…”