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Schreiber, T.; Prange, A.; Schäfer, P.; Iwen, T.; Grützner, R.; Marillonnet, S.; Lepage, A.; Javelle, M.; Paul, W.; Tissier, A.; Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR/Cas endonucleases Mol. Plant (2024) DOI: 10.1016/j.molp.2024.03.013

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.
Publikationen in Druck

Grützner, R.; König, K.; Horn, C.; Engler, C.; Laub, A.; Vogt, T.; Marillonnet, S.; A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana Plant Biotechnol. J. (2023) DOI: 10.1111/pbi.14261

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.
Publikation

Nietzschmann, L.; Smolka, U.; Perino, E. H. B.; Gorzolka, K.; Stamm, G.; Marillonnet, S.; Bürstenbinder, K.; Rosahl, S.; The secreted PAMP-induced peptide StPIP1_1 activates immune responses in potato Sci. Rep. 13, 20534, (2023) DOI: 10.1038/s41598-023-47648-x

Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response.
Publikation

Dahiya, P.; Bürstenbinder, K.; The making of a ring: Assembly and regulation of microtubule-associated proteins during preprophase band formation and division plane set-up Curr. Opin. Plant Biol. 73, 102366, (2023) DOI: 10.1016/j.pbi.2023.102366

The preprophase band (PPB) is a transient cytokinetic structure that marks the future division plane at the onset of mitosis. The PPB forms a dense cortical ring of mainly microtubules, actin filaments, endoplasmic reticulum, and associated proteins that encircles the nucleus of mitotic cells. After PPB disassembly, the positional information is preserved by the cortical division zone (CDZ). The formation of the PPB and its contribution to timely CDZ set-up involves activities of functionally distinct microtubule-associated proteins (MAPs) that interact physically and genetically to support robust division plane orientation in plants. Recent studies identified two types of plant-specific MAPs as key regulators of PPB formation, the TON1 RECRUITMENT MOTIF (TRM) and IQ67 DOMAIN (IQD) families. Both families share hallmarks of disordered scaffold proteins. Interactions of IQDs and TRMs with multiple binding partners, including the microtubule severing KATANIN1, may provide a molecular framework to coordinate PPB formation, maturation, and disassembly.
Publikation

Bürstenbinder, K.; Schwarzerová, K.; European Plant Cytoskeletal Club meeting: A vital platform for advancing plant cytoskeleton research Cytoskeleton 80, 397-399, (2023) DOI: 10.1002/cm.21780

This contribution reports on a meeting of plant cytoskeleton scientists-the European Plant Cytoskeletal Club 2023 conference.
Publikation

Bao, Z.; Guo, Y.; Deng, Y.; Zang, J.; Zhang, J.; Deng, Y.; Ouyang, B.; Qu, X.; Bürstenbinder, K.; Wang, P.; Microtubule-associated protein SlMAP70 interacts with IQ67-domain protein SlIQD21a to regulate fruit shape in tomato Plant Cell 35, 4266-4283, (2023) DOI: 10.1093/plcell/koad231

Tomato (Solanum lycopersicum) fruit shape is related to microtubule organization and the activity of microtubule-associated proteins (MAPs). However, insights into the mechanism of fruit shape formation from a cell biology perspective remain limited. Analysis of the tissue expression profiles of different microtubule regulators revealed that functionally distinct classes of MAPs, including members of the plant-specific MICROTUBULE-ASSOCIATED PROTEIN 70 (MAP70) and IQ67 DOMAIN (IQD, also named SUN in tomato) families, are differentially expressed during fruit development. SlMAP70-1–3 and SlIQD21a are highly expressed during fruit initiation, which relates to the dramatic microtubule pattern rearrangements throughout this developmental stage of tomato fruits. Transgenic tomato lines overexpressing SlMAP70-1 or SlIQD21a produced elongated fruits with reduced cell circularity and microtubule anisotropy, while their loss-of-function mutants showed the opposite phenotype, harboring flatter fruits. Fruits were further elongated in plants coexpressing both SlMAP70-1 and SlIQD21a. We demonstrated that SlMAP70s and SlIQD21a physically interact and that the elongated fruit phenotype is likely due to microtubule stabilization induced by the SlMAP70–SlIQD21a interaction. Together, our results identify SlMAP70 proteins and SlIQD21a as important regulators of fruit elongation and demonstrate that manipulating microtubule function during early fruit development provides an effective approach to alter fruit shape.
Bücher und Buchkapitel

Klemm, S.; Buhl, J.; Möller, B.; Bürstenbinder, K.; Quantitative analysis of microtubule organization in leaf epidermis pavement cells (Hussey, P.J., Wang, P.). The Plant Cytoskeleton 2604, 43-61, (2023) ISBN: 978-1-0716-2866-9 DOI: 10.1007/978-1-0716-2867-6_4

Leaf epidermis pavement cells form highly complex shapes with interlocking lobes and necks at their anticlinal walls. The microtubule cytoskeleton plays essential roles in pavement cell morphogenesis, in particular at necks. Vice versa, shape generates stress patterns that regulate microtubule organization. Genetic or pharmacological perturbations that affect pavement cell shape often affect microtubule organization. Pavement cell shape and microtubule organization are therefore closely interconnected. Here, we present commonly used approaches for the quantitative analysis of pavement cell shape characteristics and of microtubule organization. In combination with ablation experiments, these methods can be applied to investigate how different genotypes (or treatments) affect the organization and stress responsiveness of the microtubule cytoskeleton.
Preprints

Schreiber, T.; Tripathee, S.; Iwen, T.; Prange, A.; Vahabi, K.; Grützner, R.; Horn, C.; Marillonnet, S.; Tissier, A.; DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta bioRxiv (2022) DOI: 10.1101/2022.05.11.491484

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.
Preprints

Bao, Z.; Guo, Y.; Deng, Y.; Zang, J.; Zhang, J.; Ouyang, B.; Qu, X.; Bürstenbinder, K.; Wang, P.; The microtubule-associated protein SlMAP70 interacts with SlIQD21 and regulates fruit shape formation in tomato (2022) DOI: 10.1101/2022.08.08.503161

The shape of tomato fruits is closely correlated to microtubule organization and the activity of microtubule associated proteins (MAP), but insights into the mechanism from a cell biology perspective are still largely elusive. Analysis of tissue expression profiles of different microtubule regulators revealed that functionally distinct classes of MAPs are highly expressed during fruit development. Among these, several members of the plant-specific MAP70 family are preferably expressed at the initiation stage of fruit development. Transgenic tomato lines overexpressing SlMAP70 produced elongated fruits that show reduced cell circularity and microtubule anisotropy, while SlMAP70 loss-of-function mutant showed an opposite effect with flatter fruits. Microtubule anisotropy of fruit endodermis cells exhibited dramatic rearrangement during tomato fruit development, and SlMAP70-1 is likely implicated in cortical microtubule organization and fruit elongation throughout this stage by interacting with SUN10/SlIQD21a. The expression of SlMAP70 (or co-expression of SlMAP70 and SUN10/SlIQD21a) induces microtubule stabilization and prevents its dynamic rearrangement, both activities are essential for fruit shape establishment after anthesis. Together, our results identify SlMAP70 as a novel regulator of fruit elongation, and demonstrate that manipulating microtubule stability and organization at the early fruit developmental stage has a strong impact on fruit shape.
Publikation

Yang, B.; Stamm, G.; Bürstenbinder, K.; Voiniciuc, C.; Microtubule‐associated IQD9 orchestrates cellulose patterning in seed mucilage New Phytol. 235, 1096-1110, (2022) DOI: 10.1111/nph.18188

Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes are guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis. Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles in cell wall polysaccharide biosynthesis. Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. IQD9 as well as a previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein act to maintain cellulose synthase velocity. Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in cell wall biosynthesis. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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