ACTA BOT. CROAT. 78 (1), 2019 1 Acta Bot. Croat. 78 (1), 1–8, 2019 CODEN: ABCRA 25 DOI: 10.2478/botcro-2019-0008 ISSN 0365-0588 eISSN 1847-8476 Expression of small GTPases in the roots and nodules of Medicago truncatula cv. Jemalong Abdul Razaque Memon1*, Christiane Katja Schwager2, Karsten Niehaus3 1 Department of Molecular Biology and Genetics, Faculty of Science and Arts, Usak University, Usak, Turkey 2 Department of Genome Research, Faculty of Biology, Bielefeld University, Bielefeld, Germany 3 Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, Bielefeld, Germany Abstract – In this study we used Medicago truncatula, to identify and analyze the expression of small GTP-bind- ing proteins (Arf1, Arl1, Sar1, Rabs, Rop/Rac) and their interacting partners in the infection process in the roots and nodules. A real-time polymerase chain reaction analysis was carried out and our results showed that Arf1 (AtArfB1c-like), MtSar1, AtRabA1e-like, AtRabC1-like, MsRab11-like and AtRop7-like genes were highly ex- pressed in the nodules of rhizobium inoculated plants compared to the non-inoculated ones. On the contrary, AtRabA3 like, AtRab5c and MsRac1-like genes were highly expressed in non-infected nitrogen supplied roots of M. truncatula. Other Rab genes (AtRabA4a, AtRabA4c and AtRabG3a-like genes) were nearly equally expressed in both treatments. Interestingly, RbohB (a respiratory burst NADPH oxidase homologue) was more highly ex- pressed in rhizobium infected than in non-infected roots and nodules. Our data show a differential expression pattern of small GTP-binding proteins in roots and nodules of the plants. This study demonstrates an important role of small GTP-binding proteins in symbiosome biogenesis and root nodule development in legumes. Keywords: gene expression, Medicago truncatula, root nodules, small GTP-binding proteins * Corresponding author, e-mail: armemon@usak.edu.tr, abdulrezzak.memon@gmail.com Introduction Intracellular membrane trafficking plays a major role in plant growth and development including root hair forma- tion, rhizobium infection process and nodule formation in legumes (Oldroyd and Downie 2008). It is evident from the literature that small GTP-binding proteins perform a critical role in different aspects of root nodule development (Blanco et al. 2009). For example several research groups have shown the importance of Rab1, Rab7 and Rab11F in peribacteroid membrane formation in different legume species like Gly- cine max, Vigna aconitifolia, Medicago sativa and Medica- go truncatula (Cheon et al. 1993, Son et al. 2003, Schiene et al. 2004, Limpens et al. 2009). During rhizobium infec- tion, RabA2 is reported to participate in the polar growth of root hairs of Phaseolus vulgaris (Dalla Via et al. 2017). These authors showed that RabA2 is localized to mobile vesicles around the infection thread (IT) and is required to main- tain the integrity of the membrane during IT progression. Rab7 is reported to be required in vesicular fusion during symbiosome membrane transport in soybean (Cheon et al. 1993). Furthermore, a total of 33 genes of small GTP-bind- ing proteins were identified from a Lotus japonicus genome and among them, some of the Rabs like Rab1, Rab2, Rab5, Rab7, and Rac were predominantly expressed in root nod- ules of these plants (Borg et al. 1997). Likewise, Schiene et al. (2004) have shown that RabF1 is preponderantly expressed in the nodules and possibly involved in membrane vesicular trafficking and symbiosome formation in M. sativa and M. truncatula. Interestingly Rab7 has been shown to be recruit- ed by symbiosome as an endosomal marker when they stop dividing and possibly to regulate the symbiosome matura- tion process (Clarke et al. 2015). Rab GTPases are the largest member of GTPase family in most of the eukaryotes. They work in combination with Rab effector proteins and regu- late nearly all steps of membrane traffic from vesicle bud- ding to vesicle fusion (Pfeffer 2017, Biver et al. 2011). Arf and Sar1 are known to be the key GTP-binding proteins in the regulation of membrane trafficking between ER and GA in plant, yeast and mammalian cells (Pimpl et al. 2000, Ju- MEMON A. R., SCHWAGER C. K., NIEHAUS K. 2 ACTA BOT. CROAT. 78 (1), 2019 rgens 2004, Memon 2004, Memon 2012). Several observa- tions in A. thaliana and tobacco cells indicate that Arf1 not only functions in retrograde protein transport from Golgi to ER but also in some way affects the anterograde pathway (Memon, 2004). Several isoforms of Arf1 have been identi- fied in the M. truncatula genome (Vernoud et al 2003, Yuk- sel and Memon 2008) but their exact function in root and/or nodules has not been defined. It will be interesting to exam- ine the possible involvement of these proteins in intracellular vesicle trafficking in the early steps of symbiotic interactions. Accumulating evidence shows that Rop/Rac acts as a pre- dominant switch in signal transduction in plants and is re- ported to regulate several developmental processes in plants, such as polarized cell growth, cell morphogenesis, hormone signalling, defence and responses to oxygen deprivation (Craddock et al. 2012). In root hairs (Foreman et al. 2003, Ke et al. 2016, Lei et al. 2015) and in pollen tubes (Potocky et al. 2007), these GTP-binding proteins activate NADPH oxidase (Wong et al. 2007) and are responsible for the api- cal accumulation of reactive oxygen species (ROS). ROS is shown to be essential for cell elongation and to play a vital role in root hair deformation and curling during rhizobial infection (Damiani et al. 2016a, Damiani et al. 2016b). Re- cently it has been shown that Rop activates the NADPH oxi- dase homologue of plants termed Rboh (for respiratory burst oxidase) and generates ROS during early microbial infection (Kiirika et al. 2012). Membrane protein targeting in the symbiosome varies depending on the mosaic nature of the symbiosome which includes the properties of both plasmalemma and tonoplast. This indicates that the participation of several small GTP- binding proteins in symbiosome membrane biogenesis is more complex and this process could be studied in detail. To elucidate the mechanism of membrane transport in root nodules, we made an attempt to retrieve the EST databases of two model legume plants, Medicago and Lotus and searched for the nodule-specific expressed transcripts of small GTPas- es (Yuksel and Memon 2008). This analysis showed a group of 10 small GTPases which are likely to be expressed in root nodules and among them, seven belonged to the Rab cladis- tic group and two belonged to the Arf subfamily group (Yuk- sel and Memon 2008). In this study, we made a detailed expression analysis of these GTP-binding proteins in rhizobium inoculated and non-inoculated roots and nodules of M. truncatula with the intention of exploring the role of these proteins in rhizo- bium infection process and symbiosome formation in M. truncatula roots. Materials and methods Plant cultivation Medicago truncatula cv. Jemalong seeds were surface ster- ilized in concentrated HCl (37%) for 10 min. After several washings with sterile water, seeds were spread on agar plates with plant minimal medium adjusted to pH 5.7 (Rolfe et al. 1980) and germinated for two days in the dark at room tem- perature. For nodulation, each seedling was inoculated on agar plates with 0.1 mL Sinorhizobium meliloti 2011 grown to an optical density of 0.6 in YEP medium (5 g L–1 yeast ex- tract, 10 g L–1 peptone, 5 g L–1 NaCl, pH 7.0) supplemented with 600 mg L–1 streptomycin. Mature roots and nodules were harvested four weeks after infection. Plants were grown under the following conditions: 25 °C, 65% humidity, 200 µE m–2 s–1 and 16 h light/8 h dark. Growth conditions for inoculated and non-inoculated plants were the same except that non-inoculated plants were supplied with NH4NO3 as the nitrogen source. Nodules and roots were separated and were used for RNA isolation, cDNA synthesis, and quantita- tive real-time PCR (qPCR). Real-time qPCR Total RNA was isolated from the roots and nodules of M. truncatula grown with and without rhizobium with the use of the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA was quan- tified using a Nanodrop Analyser 1000 (Thermo Scientific, Wilmington, DE, USA) and cDNA was synthesized from 2 µg of extracted RNA as previously described by (Mehrtens et al. 2005). Forward and reverse primers of the targeted genes were designed using Primer 3 software (Rozen and Skaletsky 2000) and are listed in Table 1. Real-time qPCR (RT-qPCR) was performed using the Platinum SYBR Green qPCR Su- perMix UDG Kit (Invitrogen, Karlsruhe, Germany) follow- ing the manufacturer’s instructions on a Rotor-Gene 6000 (Corbett Research; http://www.corbettlifescience.com/) cy- cler. In our preliminary experiments ubiquitin, actin and ri- bosomal 18S genes were used for internal control. Our data showed that ubiquitin (TC102473) was the most suitable for use as an internal control for RT-qPCR under our exper- imental conditions. Therefore, in our further experiments we used ubiquitin as an internal control in data evaluation. Raw data were analyzed with the CAmpER software tool implementing an algorithm based on a four parameter logis- tic model (FPLM) (Tichopad et al. 2003) of the fluorescence curve. The FPLM algorithm was used to calculate efficien- cy-corrected crossing points (cCPs) that were subsequently analyzed using the REST (Relative Expression Software Tool) software (Tichopad et al. 2003, Bucciarelli et al. 2006). Each experiment was replicated 3 times and each sample was rep- licated 3 times for real-time qPCR analysis. Statistical analysis All the analyses were performed in triplicate and the results were recorded as mean ± standard deviation (SD). Visualizations were created using GraphPad Prism 7.5 for windows (GraphPad Software, San Diego USA). Analysis of variance (ANOVA) and post-hoc test (Tukey) was car- ried out by using IBM SPSS statistics 23 Software. Tukey test was used to evaluate the significance of different treatment groups. SMALL GTP-BINDING PROTEINS IN THE ROOT NODULES ACTA BOT. CROAT. 78 (1), 2019 3 Results To obtain a global view of the gene expression of small GTP-binding proteins in roots and nodules of M. truncatula, RT-qPCR experiments were carried out with plants inoculated with S. meliloti for 4 weeks. The efficiency of cDNA synthesized was assessed by end- point RT-qPCR using constitutive gene-specific prim- ers encoding ubiquitin as a reference gene. Expression patterns of MtRab, MtArf, MtArl, MtSar, MtRop7, MtRac1, MtRbohB and MtCOP in nodules and rhizo- bia inoculated and non-inoculated roots of M. trun- catula are shown in Figs. 1, 2, and 3. The RT-qPCR experiments with these genes showed that AtRabA1e and AtRabC1 homologues and MSRab11 were highly expressed in the nodules com- pared to the roots of non-inoculated plants (see Figs. 1a, f, h). On the other hand, the expression pattern of homologues of AtRabA3, AtRabA4a, AtRabA5c and AtRabG3a was significantly higher in the roots of non- inoculated plants compared to the nodules and roots of inoculated plants (see Figs. 1b, c, e, g). In contrast, ho- mologues of AtRabA4c was predominantly expressed both in the roots of inoculated and non-inoculated plants (Fig. 1d). Inoculation of M. truncatula roots with S. melilo- ti caused a strong induction of the MtSar1 transcript (see Fig. 2c). We found that MtSar1 expression was in- duced around 6 times more in inoculated than in the roots of non-inoculated nodules (Fig. 2). Post-hoc test showed that MtSar1 expression in nodules was statis- tically highly significant (P < 0.05) as compared to the expression in control roots and roots treated with S. meliloti. Interestingly ArfB1c transcript accumulation was significantly reduced in the roots of non-inoculat- Tab. 1. Primer pairs used for quantitative real-time PCR. Primer3 software was used to design the primers of the each gene. Gene Forward Primer Sequence (5’–3’) Reverse Primer Sequence (5’–3’) AtRabA1e CGAATCTTCAGTTGAAACCGCTC GCTGGTCAAGAAAGGTACCGAG AtRabA3 ACCCTCCACATTCACACCAG GAGGTGCATTAGGAGCAATGC AtRabA4a GGCCAAGAACGATATAGAGCAG TGCATCCTCTGTGGGTACTG AtRabA4c GTGCGTATTACCGTGGAGCTG GAGCATCTTCAGTAGGTACTGCC AtRabA5c GCTGGACAAGAAAGGTTTAGAGC CCCACCAACATTCTCGCAAC AtRabC1 GTTCAGGGCAGCAAGAATTCGA TCCCAAATGGCAAGCTTAAGC AtRAbG3a CACTTGATAACTGGCACGAGGAG GGAATGCAGCATCAACATTGAGG MSRab11 GTGCTGATTGGTGATTCTGG CTTGATCGCCTCGTTATGTC AtARFB1c CATCGCTCCTCTCAGGTCTTG GAGGACACTCTGGAGGCAC AtArlA1d GAGGACATGATTCCTACAGTGGG CACTCAATGATGGCTTGCTCAG MtSar1 CGCTCAGGTGGATGCTGTAGTC CATGAACACCTCCAAAGGACG MtArf1 CTGGATGTAGTGGCGCTGT CCAAGACAAGATTCGTCCACTG Mt-z-COP-2 CTCCTTCCAAACTCGAGTTG GCTCCAGATTCTGTACTATTAC MSRac1 GCTTGACCTTCGGGATGATAAG GACAACTCTTATGGCTGCGTC MtRbohB GCTCGCTCTGCTCTTATTGC TGCGCTTGTAGACACTACGC AthRop7 CTCTTCACCCTGGGCAGTAG GGCCATTGAGCTATAGAGGAGC Ubiquitin GCAGATAGACACGCTGGGA AACTCTTGGGCAGGCAATAA Fig. 1. Expression levels of Rab group GTP binding proteins (at mRNA level) in the nodules and roots (roots-SM) of Medicago truncatula in- oculated with rhizobium Sinorhizobium meliloti. Ubiquitin was used as internal control. Control roots (control N+) were harvested from un-inoculated plants which were supplied with nitrogen fertilizer. Bars represent standard deviation of three replicates. Different letters show significant differences between groups (P < 0.05) based on Tukey test. MEMON A. R., SCHWAGER C. K., NIEHAUS K. 4 ACTA BOT. CROAT. 78 (1), 2019 ed plants (Fig. 2a). This clearly suggests that ArfB1c is pos- sibly nodule specific and expressed in host plant roots after rhizobium infection, indicating its importance in nodule de- velopment. Intriguingly, another MtArf1(MtrGB57486383) transcript was equally expressed in both roots treated with S. meliloti and nodules of inoculated plants (Fig. 2d) but its expression was increased in non-inoculated roots suggest- ing its general role in vesicular trafficking. Our data show that one of the interacting partners of Arf1, a z-subunit of MtCOP2 (a component of COP1 ves- icles) was predominantly expressed in rhizobia inoculated roots (Fig. 2e) and this expression pattern was quite differ- ent than that of Arf1 expression in roots and nodules. Fig. 2b shows that AtArlA1d was highly expressed in both nod- ules and roots of non-inoculated plants and this expression pattern of At ArlA1d was different than that of the expres- sion pattern of the other genes in the ARF/Sar1 group (see Figs. 2 a, b, c, d). To assess the role of MtRop in the infection process, MtRop gene expression, and their relative transcriptional levels were analyzed in nodules and roots of inoculated and non-inoculated plants. The RT-qPCR experiments showed that indeed our previously identified transcript (homologues to A. thaliana Rop7 and A8IK57_MedtrRop 8) is predomi- nantly expressed in root nodules (Fig. 3c), indicating its role in nodule biogenesis. There are few data available related to the participation of NADPH oxidases in Rhizobium and symbiotic interactions in legumes. This prompted us to investigate the Rac and RBOHS role during the initial stages of nodulation (Figs. 3a, b). Our results showed that the expression of MtRbohB transcript was significantly increased in roots and nodules of inoculated as compared with non-inoculated plants (see Fig. 3b). Discussion The cDNA array analysis performed with M. truncatula has identified the genes involved in nitrogen and carbon me- tabolism (Tesfaye et al. 2006). However, very little is known about the genes and proteins involved in vesicular trafficking which is reported to increase in root cells during the infec- tion process (Navazio et al. 2007, Yuksel and Memon 2008). The symbiotic interaction between rhizobia and roots of le- gumes results in the formation of an infection thread followed by the formation of a bacteria containing compartment, the symbiosome, which continuously fuses with numerous ves- icles and finally gives rise to plant cells colonized by a huge number of bacteroids within mature nitrogen-fixing nodules (Wang et al. 2010, Ivanov et al. 2010). Since a huge amount of membrane material is needed to be transported from ER to the GA for symbiosome formation (Oldroyd and Downie 2008), it is assumed that the transport of the symbiotic mem- branes should be dramatically enhanced due to rhizobium in- fection. Consequently, we carried out a differential expression analysis, which provided a comprehensive view of the rate of Fig. 2. Expression levels of Arf/Sar group GTP- binding proteins (at mRNA level) in nodules and roots (roots SM) of Medicago trun- catula inoculated with rhizobium Sinorhizobium meliloti. Ubiquitin was used as internal control. Control roots (control N+) were har- vested from un-inoculated plants which were supplied with nitro- gen fertilizer. Bars represent standard deviation of three replicates. Different letters show significant differences between groups (P < 0.05) based on Tukey test. Fig. 3. Expression level of Rop/Rac group GTP binding proteins and Mt Rboh gene in nodules and roots (roots SM) of Medicago truncatula inoculated with rhizobium Sinorhizobium meliloti. Ubiquitin was used as internal control. Control roots (control N+) were harvested from un-inoculated plants which were supplied with nitrogen fertilizer. Bars represent standard deviation of three replicates. Different letters show significant differences between groups (P < 0.05) based on Tukey test. SMALL GTP-BINDING PROTEINS IN THE ROOT NODULES ACTA BOT. CROAT. 78 (1), 2019 5 transcription of small GTP-binding proteins during nodule development in M. truncatula. Our previous research conducted on databases of two model legume plants (Medicago and Lotus) containing ESTs of genes expressed in legume-rhizobium infection process have enabled the identification of 14–15 genes of GTP-bind- ing proteins possibly involved in root hair formation and nodule development in M. truncatula (Yuksel and Memon 2008). Analyses of sequence similarities and of the pres- ence of specific family and subfamily conserved motifs in their primary sequence allowed the identification of clos- est homologues from Arabidopsis and the assignment of around 8 Medicago and lotus sequences to the Rab family, 4 to Arf/Arl/Sar family and 2 to Rop/Rac family involved in root hair formation and nodule development (Yuksel and Memon 2008). Rab proteins have been functionally linked to the development of nodules in legumes (Limpens et al. 2009). For example, Rab1 and Rab7 proteins are known to be part of symbiosome membrane biogenesis (Cheon et al. 1993). Within the Medicago Rab family, the most abundant subclass is the RabA subclade and this is consistent with the significant expansion of the RabA group reported in Arabi- dopsis (Woollard and Moore 2008). Two Rab gene homo- logues to AtRabA1e and AtRabC1 and one gene homologue to MsRab11 were shown to be dominantly expressed in nod- ules of rhizobium inoculated plants (Figs. 1a, f, h). Although other Rabs (RabA3, RabA4a, RabA4c, RabA5c RabG3a) were also expressed in nodules and roots of inoculated plants their expression level was not significantly increased compared to the roots of un-inoculated plants (Figs. 1 b,c,d,e,g). The pre- cise role of these Rabs at different stages of infection process cannot be clarified at this stage; however, our gene expression data clearly indicates that these proteins play an important role in symbiosome formation. In Arabidopsis most of the genes in the RabA4 group were spatially expressed in root tissues except AtRabA4d which was specifically expressed in stamen and pollen (Yuksel and Memon 2008, Szumlanski and Nielsen 2009). A subclade of Rab GTPase is reported to be involved in Golgi to plasma membrane trafficking in both plant and mammalian cells (Woolward and Moore 2008), therefore it could fulfil the complementary role in different steps of the exocytic pathway in root cells. Evidence from dif- ferent sources supports the involvement of RabA GTPases in post Golgi trafficking (Lycett 2008) and could play a sub- stantial role in nodule development. Another RabG3 class (RabG3a) is related to Rab7 (Mazel et al. 2004) and has been highly expressed in roots and nodules. This Rab GTPase has been reported an endosomal marker and acquired by symbi- osomes when they stop dividing and that Rab7 regulates the maturation of the symbiosomes into nitrogen fixing organ- elles in M. truncatula (Son et al. 2003, Limpens et al. 2009). The level of ArfB1c expression in the nodules of inocu- lated plants was significantly higher than in the roots of non- inoculated ones. Indeed, very little ArfB1c transcript accu- mulation has been observed in the roots of non-inoculated plants (Fig. 2a). This indicates that in Medicago truncatula, this Arf is possibly a nodule specific and expressed in the host plant roots after rhizobium infection. Intriguingly another MtArf (MtrGB57486383) transcript was expressed both in roots and nodules of inoculated and non- inoculated plants suggesting its general role in vesicular trafficking in plant cells (Fig 2d). In our previous experiment with Arf1 protein expression, we observed a significant increase in Arf1 pro- tein expression with rhizobial infection and the major pool of the Arf1 in both nodules and roots is possibly present in active form (Memon 2012). It is interesting to note that Sar1, which is required in vesicular trafficking in early anterograde trafficking from ER to cis-Golgi, has shown a very high ex- pression in nodules as compared to roots in rhizobium in- oculated plants (Fig. 2c). These observations indicate that the genes of these proteins involved in early secretory pathway may play a central role in nodule development and biogen- esis (Memon 2012, Oldroyd and Downie 2008). The expres- sion of AtArl A1d was more or less similar in both nodules and non-inoculated roots (Fig. 2b). This suggests the dual role of this Arl in both the early and late secretory pathway. Interestingly the expression pattern of MtzCOP-2 was shown to be different than that of AtArfB1c (Figs. 2a, e). These dif- ferences in expression could be due to the fact that Arf1is not only required in the retrograde pathway from GA to ER transport but also involved in late secretory transport from trans-Golgi to endosomes and to the plasma membrane (Memon 2004, Matheson et al. 2007, Park and Jurgens 2012). Rop GTP-binding proteins work as signalling molecules to control the polar growth of root hairs, and the formation of the interdigitated pattern and leaf pavement and pollen tube growth in Arabidopsis (Fu et al. 2005, Gu et al. 2005). Recent data show that the spatiotemporal dynamics of Rop proteins, the cytoskeleton, endocytosis, and exocytosis are intertwined (Yalovsky et al. 2008). The expression of some Rops in roots of M. truncatula is altered in response to rhizobia infection (Liu et al. 2010). Previously we identified a single Rop gene LjaTC 11919 from Lotus which was predominantly expressed in nodules (Yuksel and Memon 2008) and is closely associated with a gene from M. truncatula (Mtr IMGACC140545-16.1). This gene is homologous to AtRop7 and MtrRop8. Our real- time qPCR results showed about 3 times more expression of AthRop7 transcript in the nodules of inoculated plants than in those of non-inoculated one (Fig. 3c), indicating its role in nodule biogenesis. Some reports have shown the increased expression of MtRop5 and MtRop6 after rhizobium inocula- tion (Riely et al. 2011). It is assumed that these genes could be involved in cytoskeleton reorganization of the host cell dur- ing rhizobium infection process. There is increasing evidence that Rop/Rac proteins are involved in the generation of ROS during the early stag- es of microbial infection via the activation of respiratory burst NADPH oxidase homologues of plants (RBOH) (Ke et al. 2016). The ROS are reported to be involved in signal- ling in response to rhizobia–legume interaction (Pauly et al. 2006, Jamet et al. 2007) and it appears that ROS produc- tion is essential for optimal symbiosis formation. Rac and RBOHs seem to play an important role in ROS production MEMON A. R., SCHWAGER C. K., NIEHAUS K. 6 ACTA BOT. CROAT. 78 (1), 2019 during symbiotic interaction (Marino et al. 2011, Montiel et al. 2012, Arthikala et al. 2014). Interestingly the down- regulation of RbohB through RNAi in Phaseolus vulgaris roots reduced ROS production and lateral root density and greatly impaired nodulation (Montiel et al. 2012). On the contrary, over-expression of RbohB in P. vulgaris signifi- cantly enhanced the ROS production, infection thread for- mation and increased the density of symbiosomes in nod- ules, and the density and size of bacteroids in symbiosomes (Arthikala et al. 2014). Our data in Fig. 3b showed signifi- cantly high expression of MtRbohB in rhizobium inoculat- ed roots compared to non-inoculated ones. These observa- tions illustrate the importance of the NADPH oxidases and their interacting GTPases (Rops) in Rhizobium infection process and show that is possibly plays an important role in the early infection process. Rac1 is an orthologue to small GTPase MtROP9 in M. truncatula (Kiirika et al. 2012) and our data show that it was expressed in both roots and nod- ules but its expression was significantly higher in the roots of non-inoculated plants (Fig 3a). This implies that Rac1 is not only involved in ROS-mediated early infection process as reported earlier (Kiirika et al 2012) but also involved in the activation of plant defence response. The early defence response of Rac/Rop-like GTPases in plants has been previ- ously reported (Wong et al. 2007). For example Rac1 in rice activates Rboh mediated ROS signalling and is reported to be a positive regulator in disease resistance. This work provides important experimental evidence for the involvement of small GTP-binding proteins and the re- spiratory burst oxidase homologue (RBOH) in Rhizobium infection process and nodule development in the roots of M. truncatula. Conclusion In this communication, we described the role of small GTP-binding proteins (Arf1, Arl1, Sar1, Rabs, Rop/Rac) and some of their interacting components in the biogenesis of the symbiosome membranes in the root nodules of M. truncatu- la. Our gene expression data showed that several Rabs, Rop, Arf and Sar proteins are involved in early and late secretory pathways during rhizobium-legume interaction and nodule development. This study gives an important clue that in ad- dition to the proteins involved in the late secretory pathway (like Rab7, Rab11), the GTP-binding proteins of the early secretory pathway (Sar1, ArfB1c RabA1e, RabC1) are also more highly expressed in rhizobium inoculated roots and nodules than in non-inoculated roots. This suggests that the activation of multiple GTP-binding proteins (both in ear- ly and late secretory pathway) is required for symbiosome membrane biogenesis and root nodule development. 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