CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.

Arjan Van Zeijl,Marijke Hartog,Titis A K Wardhani,Wouter Kohlen,Elena E Fedorova,Maryam Seifi Kalhor,Ton Bisseling,Fengjiao Bu,Rene Geurts,Luuk Rutten,Sidney Linders

doi:10.3389/fpls.2018.00284

Abstract

Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.

Highlights

Parasponia are tropical tree species belonging to the Cannabis family (Cannabaceae) and are known as the only non-legume plants that can establish a nitrogen-fixing endosymbiosis with rhizobium (Clason, 1936; Trinick, 1973; Akkermans et al, 1978)
We show that using Agrobacterium tumefaciens-mediated transformation, stable transgenic lines of P. andersonii can be obtained in ∼3–4 months
We aimed to establish a reverse genetics platform for P. andersonii based on CRISPR/Cas9 genome editing

Summary

Introduction

Parasponia are tropical tree species belonging to the Cannabis family (Cannabaceae) and are known as the only non-legume plants that can establish a nitrogen-fixing endosymbiosis with rhizobium (Clason, 1936; Trinick, 1973; Akkermans et al, 1978). Parasponia spp. are typical fast-growing pioneer plants, capable of covering nitrogen-poor eroded soils in a relatively short time span (Becking, 1992). Young Parasponia trees can grow at speeds exceeding 45 centimeters per month, and fix up to 850 kg N ha−1 year−1 in association with rhizobium (Trinick, 1980, 1981; Trinick and Hadobas, 1989). As Parasponia is the only non-legume that can establish rhizobium symbiosis, it may represent a valuable model to study the core genetic networks underlying this symbiosis (Geurts et al, 2012, 2016; Behm et al, 2014)

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