A Toolbox for Nodule Development Studies in Chickpea: a Hairy-Root Transformation Protocol and an Efficient Laboratory Strain Of
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bioRxiv preprint doi: https://doi.org/10.1101/362947; this version posted July 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A toolbox for nodule development studies in chickpea: a hairy-root 2 transformation protocol and an efficient laboratory strain of Mesorhizobium sp. 3 Drishti Mandal1, and Senjuti Sinharoy1* 4 Affiliation: 1National Institute of Plant Genome Research, New Delhi – 110067. India 5 *Corresponding author: E-mail: [email protected] 6 Running title: Toolbox for Chickpea root nodule symbiosis study 7 Abstract: Mesorhizobium sp. produces root nodules in chickpea. Chickpea and model 8 legume Medicago truncatula are members of inverted repeat lacking clade (IRLC). The 9 rhizobia after internalization inside plant cell called ‘bacteroid’. Nodule Specific 10 Cysteine-rich (NCR) peptides in IRLC legumes guide bacteroids to a ‘terminally 11 differentiated swollen (TDS)’ form. Bacteroids in chickpea are less TDS than those in 12 Medicago. Nodule development in chickpea indicates recent evolutionary diversification 13 and merits further study. A hairy root transformation protocol and an efficient laboratory 14 strain are prerequisites for performing any genetic study on nodulation. We have 15 standardized a protocol for composite plant generation in chickpea with a transformation 16 frequency above 50%, as shown by fluorescent markers. This protocol also works well 17 in different ecotypes of chickpea. Localization of subcellular markers in these 18 transformed roots is similar to Medicago. When checked inside transformed nodules, 19 peroxisomes were concentrated along the periphery of the nodules, while ER and golgi 20 bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated 21 for their ability to initiate nodule development, and efficiency of nitrogen fixation. 1 bioRxiv preprint doi: https://doi.org/10.1101/362947; this version posted July 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Inoculation with different strains resulted in different shapes of TDS bacteroids with 23 variable nitrogen fixation. Our study provides a toolbox to study nodule development in 24 the crop legume chickpea. 25 Introduction: 26 Root nodule symbiosis (RNS) is the most successful metabolism-dependent symbiosis 27 on earth. Leguminous plants get reduced nitrogen directly from RNS, at the expense of 28 photosynthate (Werner et al., 2015). The staple crop chickpea (Cicer arietinum) is 29 world’s second largest cultivated legume. Chickpea seeds are a valuable source of 30 dietary protein in lower socio-economic class. Additionally, due to the symbiotic 31 interaction with Mesorhizobium sp., chickpea can be cultivated in a sustainable way 32 (Jain et al., 2013; Varshney et al., 2013). Research on nodule development has been 33 centered upon model legumes Medicago truncatula and Lotus japonicus. Today, model 34 legumes are in the forefront of legume biology in terms of both available knowledge and 35 resources. The only disadvantage is that model legumes are not crop species. In spite 36 of being the most important grain legume in tropical and sub-tropical countries (Jukanti 37 et al., 2012), literature on chickpea nodule development is scarce. Genome sequencing 38 and the establishment of transcriptomic and proteomic resources have laid the pillars for 39 making chickpea a model amongst crop legumes (Jain et al., 2013; Varshney et al., 40 2013; Ramalingam et al., 2015; Pandey et al., 2018). 41 Chickpea belongs to the inverted repeat lacking clade (IRLC), which diverged from 42 model legume Medicago ~10-20 million years ago (Jain et al., 2013; Varshney et al., 43 2013). IRLC legumes develop indeterminate nodules, with a gradient of cells at different 2 bioRxiv preprint doi: https://doi.org/10.1101/362947; this version posted July 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 44 stages of development can be seen from the distal to the proximal part of the nodule. A 45 persistent meristem (zone I) is present at the distal end of the nodule. Bacterial 46 endocytosis and colonization takes place in the postmeristematic cells of the infection 47 zone (zone II), where plant membrane-bound bacterial units are formed. These units 48 are considered as an ammonium-exporting organelle called symbiosome (Roth and 49 Stacey, 1989). The rhizobia inside the symbiosomes are called bacteroids. The 50 bacteroids divide inside the symbiosome and gradually colonize the whole cell. In the 51 nitrogen fixation zone (zone III) bacteroids differentiation is terminated, ammonium 52 assimilation genes are repressed, and nitrogen fixation genes are induced (Oldroyd, 53 2013; Udvardi and Poole, 2013). 54 Terminally differentiated, enlarged bacteroids with different morphotypes are a typical 55 feature of the IRLC legumes. The major determining factor behind these irreversibly 56 differentiated bacteroids is nodule specific cysteine-rich (NCR) peptides (Montiel et al., 57 2016; Montiel et al., 2017). The molecular mechanism of NCR peptide regulated-endo- 58 reduplication of the symbiont genome has been worked out in model legume Medicago. 59 Medicago genome encodes more than 700 NCR genes. At least 138 NCR peptides get 60 processed in the endoplasmic reticulum (ER) and targeted towards symbiosomes 61 (Wang et al., 2010; Durgo et al., 2015). NCR peptides force endo-reduplication of the 62 symbiont genome. As a result, the bacteria (now bacteroid) lose their ability to divide 63 and re-grow on culture media, but their size increases up to ~10-fold (Mergaert et al., 64 2006; Young et al., 2011; Sinharoy et al., 2013). NCR gene family evolved in Medicago 65 by a recent local gene duplication (Alunni et al., 2007). NCRs have been identified in 66 several IRLC legumes, including chickpea (Kant et al., 2016; Montiel et al., 2016; 3 bioRxiv preprint doi: https://doi.org/10.1101/362947; this version posted July 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 67 Montiel et al., 2017). The number of NCR genes among different IRLC legumes varies 68 greatly. This results in different morphotypes of bacteroids in different legumes, such as 69 swollen/spherical, elongated, and elongated-branched (Montiel et al., 2017). 70 Mesorhizobium TAL620-induced nodules in chickpea express only 63 NCR genes, 71 while its’ bacteroids endo-reduplicated up to 4 fold (Kant et al., 2016; Montiel et al., 72 2017). Chickpea and Medicago NCRs share less than 80% identity. Chickpea NCR 73 peptides have more identity with Glycyrrhiza uralensis, Onobrychis vicifolia, and 74 Astragalus canadensis, while phylogenetically chickpea is closer to Medicago (Montiel 75 et al., 2016; Montiel et al., 2017). Chickpea’s swollen/ spherical bacteroids are basal to 76 the evolution of NCR-guided morphogenesis (Montiel et al., 2017). In contrast to 77 chickpea, Medicago has more than 700 NCR genes produce elongated-branched 78 bacteroids depicting an advanced stage of this trait. Interestingly, this morphogenesis of 79 bacteroids and the genome endo-reduplication is thought to determine the efficiency of 80 nitrogen fixation in respective legumes (Oono and Denison, 2010). Thus, comparative 81 investigation of nodule development in chickpea and Medicago will enhance our 82 knowledge on the evolutionary link between variable nitrogen fixation efficiencies 83 amongst different legumes. 84 An efficient hairy-root transformation protocol is an indispensable tool to understand 85 nodule biology, enabling us to study the localization of any protein (fluorescent tag), 86 activities of promoters (GUS fusion), and the effects of over-expression or knock-down 87 of certain genes. We have undertaken an effort to establish hairy root transformation in 88 chickpea and study nodule development. Unlike A. tumefaciens, A. rhizogenes 89 generates transformed roots from the site of infection. A. rhizogenes contains root locus 4 bioRxiv preprint doi: https://doi.org/10.1101/362947; this version posted July 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 90 (rol) genes in the Ri-plasmid, which promotes the formation of genetically transformed 91 adventitious hairy roots. A. rhizogenes carrying a recombinant Ri plasmid can generate 92 composite plants. These plants contain untransformed shoot and transform root 93 (Georgiev et al., 2012). When such an A. rhizogenes additionally carrying a gene of 94 interest in a binary vector is used for transformation, a certain percentage of co- 95 transformed roots are obtained. Both overexpression and downregulation of a specific 96 gene can be achieved by these co-transformed roots (Limpens et al., 2004; Sinharoy 97 and DasGupta, 2009; Sinharoy et al., 2015). Recently, it has been reported that 98 CRISPR/Cas9 mediated gene knock out is also possible in hairy roots (Ron et al., 2014; 99 Cai et al., 2015; Wang et al., 2016). Till date, all the successful protocol for hairy root 100 generation in other legumes have shown the transformed roots to be biologically similar 101 to untransformed roots, with no difference in normal nodule development (Stougaard et 102 al., 1987; Quandt et al., 1993; Stiller et al., 1997; Boisson-Dernier et al., 2001; Van-de- 103 Velde et al., 2003; Limpens et al., 2004; Estrada-Navarrete et al., 2006; Kereszt et al., 104 2007; Sinharoy et al., 2009; Bonaldi et al., 2010; Imanishi et al., 2011; Brijwal and 105 Tamta, 2015; Thilip et al., 2015; Habibi et al., 2016; Thwe et al., 2016). A. rhizogenes‐ 106 mediated hairy-root transformation is an efficient and less time-consuming alternative 107 method for the functional validation of genes.