Molecules of microbial origin have the capacity to induce a morphogenetic response in symbiotic eukaryotic hosts (Matsuo et al., 2005; Alegado et al., 2012; Lerouge et al., 1990). Nitrogen-fixing rhizobia produce species-specific decorated chitin-like molecules, Nod factors (Lerouge et al., 1990) that are recognized by LysM receptors in legume roots and initiate two different processes; nodule development and infection thread (IT) formation (Radutoiu et al., 2003; Murakami et al., 2018). In addition to their signalling capacity, Nod factors have been considered morphogens based on their effect on host developmental programs (Long, 1996), but direct measurements of in planta-produced Nod factor levels proved intractable, and therefore limited our knowledge on their concentration-dependent effect on the host (Morieri et al., 2013). ITs are tubular structures used by bacteria as conduits to advance from the root hair tip towards the root cortex. IT passage through cortical layers involves reprogramming of individual cells for microbial infection (van Brussel et al., 1992). Inside nodule primordia, branched IT arrays spread from the main shaft, guiding the confined rhizobia towards competent cortical cells where endocytosis takes place (Monahan-Giovanelli et al., 2006). Although a number of components are known (Xie et al., 2012; Yokota et al., 2009; Yano et al., 2009), the mechanisms governing IT formation are not defined, and even less understood is the control of IT branching inside root nodules. Rhizobia grow and multiply during infection thread progression (Fournier et al., 2008) and consequently bacterial signals accumulate inside the limited space within ITs. Nevertheless, the host determines the compatibility of Nod factors and exopolysaccharide produced by bacteria during root hair infection, via NFR1/NFR5 and EPR3 receptors, respectively (Kawaharada et al., 2015; Hayashi et al., 2014).

In parallel with the signalling events induced by Nod factor recognition, legumes express chitinases with Nod factor cleaving activity (Staehelin et al., 1994; Goormachtig et al., 1998). Plant chitinases are primarily found as components of immune responses induced by chitin producing pathogens (Lipka et al., 2005), but novel functions of these enzymes have evolved in plants (De Jong et al., 1992) and in symbiotic interactions between eukaryotes and microbes (Kremer et al., 2013). The identification of Nod factor-cleaving chitinases induced during legume-rhizobia symbiosis led to a model where these enzymes are required for Nod factor hydrolysis to avoid activation of immune responses induced by these chitin-like molecules during symbiosis (Goormachtig et al., 1998; Savoure et al., 1997; Staehelin et al., 2000). However, unlike chitin, Nod factors do not trigger immune responses in Lotus (Bozsoki et al., 2017) and in this context the function of Nod factor cleaving chitinases remained unclear. This was further confirmed by the Medicago truncatula, Nod factor hydrolase (NFH1) mutants that display delayed infection thread formation and nodule hypertrophy (Cai et al., 2018).

We identified chit5-1, chit5-2 and chit5-3 as fix minus Gifu mutant alleles from screens aimed at discovering symbiotic defective mutations following inoculation with Mesorhizobium loti (Sandal et al., 2006) (Figure 1A). The impaired symbiotic association was reflected in significantly reduced shoot growth (Figure 1—figure supplement 1) and nitrogen deficiency symptoms when compared to wild type (Figure 1A). This defective phenotype was alleviated by the addition of nitrogen (10 mM KNO₃) to the growth substrate (Figure 1—figure supplement 2). Detailed analysis of the symbiotic phenotype manifested by the three alleles revealed that nodule organogenesis was initiated in the presence of M. loti (Figure 1—source data 1), but most nodules remained arrested at the primordia stage with only a few developing into large, white or pink-spotted nodules (Figure 1B,C). Assessment of bacterial nitrogenase activity through acetylene reduction assays revealed that mutant nodules, despite their occasional functional appearance (pink-spotted), had significantly reduced nitrogenase activity compared to wild-type (Figure 1D). The limited nitrogen fixation capacity is reflected in the observed plant growth and nodule phenotypes. This was further supported by reduced transcriptional activation of leghemoglobin (Lb3) and sulfate transporter (Sst1) genes which are prominent molecular markers for mature nodules and successful nitrogen-fixing symbiosis (Ott et al., 2005; Krusell et al., 2005) (Figure 1—figure supplement 3). In contrast, transcripts associated with early rhizobial infection, early nodulin N6 (Mathis et al., 1999), pectate lyase Npl1 (Xie et al., 2012) and transcription factor Ern1 (Kawaharada et al., 2017a), were induced to higher levels in the chit5 mutants compared to wild-type plants (Figure 1—figure supplement 3). No morphological signatures associated with activation of plant defense responses, such as the onset of early senescence, previously reported for some fix minus mutants (Bourcy et al., 2013) was observed in chit5 plants (Figure 1B). Mpk3, Wrky29 and Wrky33, marker genes upregulated during plant immunity (Lohmann et al., 2010), were found to have a wild-type level of expression in both uninoculated and 21-dpi whole mutant roots (Figure 1—figure supplement 3 and Figure 1—source data 2). Recent transcriptome analyses of Lotus (Kelly et al., 2018a) revealed that progression of symbiosis in wild-type roots and formation of the symbiotic nodules (21 dpi) is accompanied by downregulation of these immunity marker genes (Figure 1—figure supplement 4A). We have analysed the expression of these marker genes in 21dpi nodules of wild-type and chit5 mutants. We found a higher level of the Npl1, Mpk3, Wrky29 and Wrky33 transcripts in chit5 mutant nodules compared to wild-type nodules (Figure 1—figure supplement 4). The differences in the expression levels of the early symbiotic and defense markers detected between wild-type and chit5 mutants indicate a deregulated symbiotic and immune signalling, seemingly arresting nodules at an early infection stage. We investigated whether rhizobial infection was affected and found that a normal number of ITs were formed in chit5 mutant root hairs (Figure 2A), and these proceeded in a manner similar to wild-type plants (Figure 2—figure supplement 1). Together, these results demonstrate that the early programs initiated by Nod factor signalling, nodule organogenesis and formation of root hair ITs, operate normally in chit5 mutants.

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Figure 1—source data 1

Bacterial strains used in this study.

https://doi.org/10.7554/eLife.38874.007

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Figure 1—source data 2

Primers used for RT-qPCR analyses.

https://doi.org/10.7554/eLife.38874.008

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In determinate nodules, like those formed by L. japonicus, ITs are present inside the cortex of mature nodules and can be quantified (Madsen et al., 2010). We have performed detailed investigations of IT phenotypes in wild-type and chit5 mutant nodule sections (n = 30) using light and electron microscopy. Counting of the cortical ITs formed inside wild-type and infected mutant nodules revealed a severe and significant reduction in their number in the mutant nodules (Figure 2B). Mutants displayed an impaired symbiotic occupancy of the nodule central tissue, with only scattered infected cells that formed inside the pink-spotted nodules (Figure 2C). Bacteria accumulated in the intercellular spaces between nodule cells (Figure 2C and D), giving rise to fewer, localized intracellular infections (Figure 2D). Symbiosomes present in the infected mutant cells contained multiple bacteroids of irregular shape and of atypical appearance (accumulation of white matrix) (Figure 2D). These phenotypes are likely a result of symbiotic and defense genes deregulation inside chit5 nodules. These results demonstrate that CHIT5 functions during cortical IT development and branching within nodules, and is required for the establishment of fully functional nitrogen-fixing symbiosis.

Map-based cloning and whole genome sequencing revealed that all three alleles are defective in one of the three chitinase-coding genes present at the identified genomic location (Figure 3A, Figure 3—figure supplement 1, and Figure 3—figure supplement 2), while the remaining two paralogs are pseudogenes with premature stop codons (Figure 3—figure supplement 2).The chit5-1 and chit5-3 mutants were found to have large genomic deletions encompassing the chitinase gene, while chit5-2 carries a point mutation (CCA to CTA), that results in a proline to leucine (P₁₆₈-L) transition in the predicted protein sequence (Figure 3A and Figure 3—figure supplement 2). The symbiotic defective phenotype of all three alleles was restored by genetic complementation with the wild-type Chit5 gene expressed from its native promoter (2072 bp) and terminator (2108 bp) regions, or from the LjUbiquitin promoter and Nos terminator (Figure 3—figure supplement 3). Analyses of Chit5 expression using transcriptional reporters (Chit5-tYFPnls and Chit5-GUS) revealed promoter activity in all cells of uninoculated roots, in infected root hairs (Figure 3B), and nodule primordia (Figure 3—figure supplement 4). Chit5 promoter activity decreased inside fully functional nodules, indicating a reduced requirement for Chit5 at later stages of a functional symbiosis (Figure 3B, Figure 3—figure supplement 4).

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Figure 3—source data 1

HPLC-MS analysis of Nod factor isolated after exposure to roots of wild-type Gifu or chit5 mutants.

https://doi.org/10.7554/eLife.38874.017

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The CHIT5 protein consists of a predicted signal peptide at the amino terminus followed by a class V (glycosyl hydrolase 18) domain with a conserved catalytic DxDxE motif (Figure 3—figure supplement 5). The proline residue mutated in chit5-2 allele is located immediately after the catalytic site, and is conserved across GH18-type chitinases, indicating a crucial role for the function of this protein (Figure 3—figure supplement 5). CHIT5 shares a high level of similarity to Medicago truncatula NFH1 (Zhang et al., 2016), including the presence of A and B loops and the proline residue (P₂₉₄), previously reported as essential for Nod factor hydrolysis (Zhang et al., 2016). Furthermore, we found that a version of CHIT5 mutated in the DxDxE motif (E₁₆₆ to K) was no longer able to complement the symbiotic defective phenotype, suggesting that enzymatic activity is required for CHIT5 function in planta (Figure 3—figure supplement 6). To investigate the enzymatic properties of CHIT5 in planta, we took advantage of its predicted secretion (Figure 3—figure supplement 5) and ubiquitous expression in Lotus roots (Figure 3B) and analysed the hydrolytic capacities of wild-type and mutant root exudates on chitin hexamers (hexa-N-acetylchitohexaose, abbreviated CO-VI) and on R7A Nod factor (NodMI-V(C18:1, Me, Cb, FucAc), abbreviated LCO-V).

For testing CHIT5-dependent hydrolysis of CO-VI by root exudates we made use of a physiological test based on the differential capacity of CO-VI, CO-III/CO-II and Nod factor elicitors to induce ROS production in Lotus (Bozsoki et al., 2017). CO-VI elicitors present in the control samples (not exposed to plant root exudates) induced ROS production, while those exposed to wild-type or mutant roots lost this capacity (Figure 3C). This shows that CO-VI hydrolysis took place during incubation with plant roots irrespective of the genotype and that CHIT5 is thus not solely required for CO-VI hydrolysis in the Lotus rhizosphere. Similar analyses performed with LCO-V showed no ROS production, confirming that intact R7A Nod factor molecules (present in the control samples) or their possible hydrolysis products resulting from exposure to plant roots, lack the ability to induce ROS in Lotus roots (Figure 3D) (Bozsoki et al., 2017). Analyses of butanol-extracted fractions from LCO-V samples determined that hydrolysis of the R7A Nod factor into NodMI-II (C18:1, Me, Cb, abbreviated LCO-II) was induced by exudates from roots (Figure 3E). Exudates from all chit5 mutants induced lower production of LCO-II than wild-type exudates, and there was a corresponding reduction in LCO-V degradation by mutants compared with wild-type (Figure 3E and Figure 3—source data 1). Since LCO-II was produced after exposure to chit5 exudates, additional plant chitinases expressed in Lotus roots (Figure 3—figure supplement 7) may contribute to Nod factor hydrolysis in the Lotus rhizosphere. Our in planta assays demonstrate that secreted CHIT5 contributes to hydrolysis of R7A Nod factor. Nevertheless, CHIT5 and unknown hydrolase(s) induce only a limited hydrolysis of the fully compatible Nod factor, most likely to ensure that symbiotic signalling occurs, as shown in wild-type plants.

M. loti NodD1 and NodD2 transcriptional regulators control the expression of Nod factor biosynthesis genes and are preferentially active at distinct stages of M. loti-Lotus symbiosis (Kelly et al., 2018b). NodD1 is primarily active inside root hair ITs, while NodD2 activates the transcription of Nod factor biosynthesis genes within nodules. NodD1 and NodD2 were found to differ significantly in their capacity to induce Nod factor biosynthesis genes transcription on Lotus roots and coordinated activity between them ensures an intermediate gene expression in wild-type R7A (Kelly et al., 2018b). Our analysis of chit5 mutants revealed a requirement of CHIT5 for cortical IT extension leading to effective colonization of nitrogen-fixing nodules (Figure 2). This provided us with the opportunity to further investigate the necessity of CHIT5 for monitoring Nod factor levels in planta, by assessing the phenotypes of nodD1 (D1^-/D2⁺) and nodD2 (D1⁺/D2^-) bacterial mutants. chit5 mutants inoculated with nodD1, that induces a 3-fold higher level of Nod factor biosynthesis gene transcription compared to wild-type R7A in response to Lotus root exudates (Kelly et al., 2018b), displayed a severe nitrogen-deficient phenotype (Figure 4A). Comparable root hair IT formation was observed on wild-type and chit5 plants (Figure 4—figure supplement 1) yet nodules induced by nodD1 on chit5 mutants were even more severely impaired than those induced by R7A. Nodules were rarely or superficially infected and were found to be severely ineffective in acetylene reduction assays (Figure 4 and Figure 4—figure supplement 1). In contrast, inoculation with nodD2, that induces a 4-fold lower level of Nod factor biosynthesis gene transcription compared to wild-type R7A (Kelly et al., 2018b), resulted in full restoration of chit5 defective phenotypes (Figure 4 and Figure 4—figure supplement 1). A similar number of pink nodules were formed on wild-type and chit5 mutant roots and plants were no longer nitrogen-deficient. Fully colonized nodules with efficient nitrogen-fixing symbiosomes were formed (Figure 4—figure supplement 1). These contrasting phenotypes of chit5 mutants in response to M. loti strains affected in the regulation of Nod factor biosynthesis indicate that CHIT5-dependent modulation of Nod factor levels within nodules is critical for cortical IT development and complete transition to nitrogen-fixing symbiosis.

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Nod factors are perceived by the NFR1/NFR5 receptors (Radutoiu et al., 2003). The expression of Chit5 is similar to that of Nfr1/Nfr5 (Kawaharada et al., 2017b) and follows the spatio-temporal development of symbiosis in the root and nodules (Figure 3B and Figure 3—figure supplement 4). The observed requirement for tight modulation of Nod factor levels inside nodules (Figure 4A and Figure 4—figure supplement 1) prompted us to investigate whether the chit5 phenotype in the presence of wild-type M. loti could reflect a bottleneck in Nod factor signalling due to limited/tightly controlled NFR-dependent signalling. We tested this possibility by overexpressing Nfr1 and Nfr5 receptors in chit5 mutants, but observed no change in their symbiotic phenotype after inoculation with M. loti R7A (Figure 4—figure supplement 2). This indicates that increased and ectopic expression of Nod factor receptors is insufficient to restore the chit5 defective phenotype.

Bacterial genetics coupled with in planta phenotypic studies and chemical analyses of Nod factors produced by rhizobial mutants have determined that various Nod factor decorations are required for initiation and progression of symbiosis (Ehrhardt et al., 1995; Rodpothong et al., 2009). We investigated the importance of Nod factor decorations for CHIT5 activity in Lotus by assessing the phenotypes induced by R7A nolL and nodZ mutants that produce Nod factors lacking acetyl (LC(C18:1, Me, Cb, Fuc)) and acetylated-fucosyl decorations (LCO-V(18:1, Me, Cb)), respectively on the reducing-end of the Nod factor (Rodpothong et al., 2009). Compared to wild-type M. loti, these bacterial mutants are delayed in initiating nodule organogenesis and form fewer infected nodules (Figure 4—figure supplement 3), a phenotype similar to the one induced by M. loti nodD1 mutant (Kelly et al., 2018b). Interestingly, we found that these bacterial mutants infected chit5 mutants and wild-type similarly and that, compared to wild-type or M. loti nodD1, no significant differences in nodule number, infection rate and nitrogen fixing ability were observed between plant genotypes (Figure 4—figure supplement 3). This suggests that cortical infection and onset of nitrogen fixation in the symbiotic organ might require tight modulation of Nod factor levels in a structure-dependent manner. Decorations have been shown to be important for determining the rate of Nod factor degradation by different chitinases (Ovtsyna et al., 2000). Nod factors produced by nodZ and nolL mutants may become accessible to other chitinases present in the chit5 mutants for degradation. Alternatively, Nod factor signalling induced by nodZ and nolL mutants may be reduced compared to wild-type bacteria and therefore not requiring CHIT5-dependent modulation. These findings based on analyses of plant and bacterial mutants in binary interactions questioned the functional relevance of CHIT5 in the context of natural environments where a diverse rhizobial population, presumably producing Nod factors with various structures at various levels, is present. We investigated this by analysing the phenotypes of chit5 mutant plants grown in soil and exposed to the natural rhizobial population compared with wild-type and nfr5-2 plants. We found that both chit5 and nfr5 mutant plants were clearly distinctive from wild-type, as they were nitrogen starved in spite of numerous nodule primordia formed on chit5 plants (Figure 4—figure supplement 4). This indicates, that at least in the presence of the tested soil rhizobial population, CHIT5 is a major determinant for the onset of nitrogen-fixing symbiosis in the nodules whose formation is controlled by NFR5.

Our study revealed that in L. japonicus, CHIT5-dependent hydrolysis of the Nod factor morphogen plays a crucial role during primordia infection and for full-transition to symbiotic nitrogen fixation. Chit5 in L. japonicus and Nfh1 in M. truncatula are highly similar, however the mutant phenotypes in the two model-legumes are very different (Cai et al., 2018). Unlike in the nfh1 mutant (Cai et al., 2018), the number and appearance of root hair ITs and the size of nodule primordia appear not to be affected in chit5 mutants, indicating that in Lotus other mechanisms independent of CHIT5 control these early stages of symbiosis. Chit5 and Nfh1 are part of genomic clusters with closely-located paralogs (Figure 3—figure supplement 1, Figure 4—figure supplements 5 and 6). In Lotus, two of the three paralogs evolved as pseudogenes (Figure 3—figure supplement 2 and Figure 4—figure supplement 7), while in Medicago all are functional genes and expressed (Figure 4—figure supplement 5). This may contribute to the differential phenotypes observed in the two model legumes, and may explain the strong phenotype displayed by Chit5 mutants in Lotus. Alternatively, Chit5 and Nfh1 might be highly similar but non-orthologous genes. Based on our observations from Lotus we propose a likely scenario explaining CHIT5 function (Figure 4D). Bacteria maintain a low level of Nod factors within root hair-traversing ITs by preferential activity of NodD1 (Kelly et al., 2018b). Bacterial amplification inside primordia-elongating ITs, coupled with the switch for preferential activity of NodD2 (Kelly et al., 2018b) leads to higher levels of fully decorated pentameric Nod factors (LCO-V) that unbalance the symbiotic and immune signalling in the cortical cells. CHIT5 hydrolytic activity on LCO-V is crucial for maintaining balanced symbiotic and defense signalling in the root developmental field and is required for cortical IT extension inside primordia, efficient bacterial endocytosis and ultimately, development of the nitrogen-fixing organ. In the absence of Chit5, IT elongation is impaired leading to bacteria accumulation in the intercellular space and sparse intracellular infection (Figure 4D). However, we cannot exclude that a CHIT5-dependent product derived from Nod-factor hydrolysis plays a role during nodule infection. Interestingly, similar but less frequent infections, as observed in chit5 mutants inoculated with wild-type M. loti, was reported for spontaneous nodules formed on nfr1snf1 plants when infected by an M. loti nodC mutant lacking the ability to produce Nod factors. Surprisingly, the symbiosomes formed by the M. loti nodC mutant were reported to have normal appearance and the plants to be nitrogen proficient (Madsen et al., 2010). These results suggest that Nod factors, or their hydrolytic products, are dispensable for nitrogen fixation within infected cells. Based on these results and our findings from symbiotic gene expression in chit5 mutants and their phenotype in the presence of mutant rhizobia, we consider the scenario proposed in Figure 4D as the most likely framework for CHIT5 function. Future studies will likely reveal which molecular determinants are responsible for decoding the CHIT5 output into nitrogen-fixing state inside infected cells.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Anna Malolepszy

   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Present address

   Salk Institute for Biological Studies, La Jolla, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft

   Contributed equally with

   Simon Kelly

   Competing interests

   No competing interests declared

2. Simon Kelly

   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft

   Contributed equally with

   Anna Malolepszy

   Competing interests

   No competing interests declared

3. Kasper Kildegaard Sørensen

   Department of Chemistry, University of Copenhagen, Frederiksberg, Denmark

   Contribution

   Data curation, Formal analysis, Visualization

   Competing interests

   No competing interests declared

4. Euan Kevin James

   The James Hutton Institute, Scotland, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7969-6570

5. Christina Kalisch

   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Present address

   Novozymes, Kalundborg, Denmark

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   affiliated with Novozymes. The author has no other competing interests to declare

6. Zoltan Bozsoki

   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4267-9969

7. Michael Panting

   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

8. Stig U Andersen

   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Contribution

   Data curation, Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1096-1468

9. Shusei Sato

   Kazusa DNA Research Institute, Kisarazu, Japan

   Present address

   Graduate School of Life Sciences, Tohoku University, Miyagi, Japan

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

10. Ke Tao

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Data curation, Formal analysis, Investigation, Methodology

    Competing interests

    No competing interests declared

11. Dorthe Bødker Jensen

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Formal analysis, Investigation

    Competing interests

    No competing interests declared

12. Maria Vinther

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

13. Noor de Jong

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

14. Lene Heegaard Madsen

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Formal analysis, Investigation, Visualization, Methodology

    Competing interests

    No competing interests declared

15. Yosuke Umehara

    Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan

    Contribution

    Formal analysis, Investigation

    Competing interests

    No competing interests declared

16. Kira Gysel

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Data curation, Investigation, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4245-9998

17. Mette U Berentsen

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Present address

    Eurofins Steins Laboratorium A-S, Vejen, Denmark

    Contribution

    Formal analysis, Investigation

    Competing interests

    affiliated with Eurofins Steins Laboratorium A-S. The author has no other competing interests to declare

18. Mickael Blaise

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Present address

    Institut de Recherche en Infectiologie de Montpellier-IRIM, University of Montpellier, Montpellier, France

    Contribution

    Conceptualization, Data curation, Supervision, Methodology

    Competing interests

    No competing interests declared

19. Knud Jørgen Jensen

    Department of Chemistry, University of Copenhagen, Frederiksberg, Denmark

    Contribution

    Conceptualization, Formal analysis, Supervision, Validation, Methodology

    Competing interests

    No competing interests declared

20. Mikkel B Thygesen

    Department of Chemistry, University of Copenhagen, Frederiksberg, Denmark

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Visualization, Methodology, Writing—original draft

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-0158-2802

21. Niels Sandal

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Data curation, Formal analysis, Supervision, Validation, Investigation

    Competing interests

    No competing interests declared

22. Kasper Røjkjær Andersen

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Conceptualization, Data curation, Validation, Visualization, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4415-8067

23. Simona Radutoiu

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration

    For correspondence

    radutoiu@mbg.au.dk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8841-1415

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