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

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.

Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF‐YA1 transcription factors as a core genetic network in nitrogen‐fixing nodule symbioses

“…46966 as template (Nekrasov et al ., ). Constructs for CRISPR/Cas9‐mediated mutagenesis were assembled as described previously (van Zeijl et al ., ; Wardhani et al ., ). To allow golden gate cloning of β‐glucuronidase (GUS) reporter constructs, the Bpi I and Bsa I restriction sites in putative promoter sequences of PanNF‐YA1, PanNF‐YA3 and PanNF‐YA6 were mutated by introducing single nucleotide substitutions (Engler et al ., ).…”

Genome Editing in Trees: From Multiple Repair Pathways to Long-Term Stability

“…CRISPR/Cas9-induced albino mutants have been reported in poplar ( Fan et al, 2015 ), citrus ( Jia and Wang, 2014 ; Zhang et al, 2017 ), apple ( Nishitani et al, 2016 ), grape ( Nakajima et al, 2017 ), cassava ( Odipio et al, 2017 ), coffee ( Breitler et al, 2018 ), and kiwifruit ( Wang Z. et al, 2018 ). Successful implementation of CRISPR has also been demonstrated by targeting potential developmental and biosynthesis pathway genes in grape ( Ren et al, 2016 ) and the tropical tree Parasponia andersonii ( van Zeijl et al, 2018 ; Table 1 ). New CRISPR reagents have been developed to expand genome editing capabilities.…”

Efficient CRISPR/Cas9-Mediated Gene Editing in an Interspecific Hybrid Poplar With a Highly Heterozygous Genome

“…Between 2013 and 2019, CRISPR/Cas9 technology has been successfully used to modify plant species from 45 genera across 24 families ( Shan et al., 2020 ). However, the application of the CRISPR/Cas9 system to genetically alter woody perennials has been limited to only the following 10 species: apple ( Nishitani et al., 2016 ), cacao ( Fister et al., 2018 ), cassava ( Odipio et al., 2017 ), citrus ( Jia et al., 2017 ), coffee ( Breitler et al., 2018 ), grape ( Nakajima et al., 2017 ), kiwifruit ( Wang et al., 2018 ), Parasponia andersonii ( Van Zeijl et al., 2018 ), pomegranate ( Chang et al., 2019 ), and poplar ( Fan et al., 2015 ; Zhou et al., 2015 ). Recalcitrance to transformation and/or difficulties in regeneration are two predominant issues preventing the more widespread application of CRISPR in woody plants ( Bewg et al., 2018 ).…”