Predisposition and Redesigning of Genetic Networks of Rice for Accommodating Nitrogen-Fixing Rhizobial Symbiosis

International Dialogue on Perception and Prospects of Designer Rice 245

Predisposition and Redesigning of Genetic Networks of Rice for Accommodating Nitrogen-Fixing Rhizobial Symbiosis Pallavolu M. Reddy, Alma Rosa Altúzar-Molina, Marlene Ortiz-Berrocal, Rigoberto Medina-Andrés, Mariana López-Sámano, Lourdes Martínez-Aguilar and María de Lourdes Velázquez-Hernández Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, México. Email: [email protected]

Abstract

In ecosystems of Asia, Africa and Latin America, rice is the dominant crop in intensive irrigated lowlands, and the

vulnerable uplands. Nitrogen is the nutrient that most frequently limits rice production. Global agriculture now

relies heavily on N fertilizers, which are developed using fossil fuel as the energy supply. It is estimated that nearly

twice as much fixed nitrogen will be required to raise rice production by 2050 to supplement the food requirements of

the increasing human population. Excess N in the global system in its various forms augments greenhouse effects,

diminishes ozone levels, promotes smog, contaminates drinking water, acidifies rain, eutrophicates rivers, and

stresses ecosystems. These problems can be alleviated if rice plants are conferred with the ability to fix nitrogen to

meet their N requirements for growth. A major goal of biological nitrogen fixation (BNF) research has been to extend

the nitrogen-fixing capacity to cereal plants such as rice. If a BNF system could be assembled in the rice plant, it

could increase the potential for nitrogen supply because fixed nitrogen would be available to the plant directly, with

little or no loss. Such a system could also enhance resource conservation and environmental security, besides freeing

farmers from the economic burden of purchasing fertilizer nitrogen for crop production. The strategies of enabling

rice to fix its own nitrogen are complex and long-term in nature. However, the recent remarkable progress made in

our understanding of nitrogen-fixing symbiosis in legumes together with the exciting discoveries that are being made

in plant science will assist in fully realizing the goal of developing nitrogen-fixing rice in foreseeable future. This

review discusses prospects for redesigning genetic networks of rice for accommodating nitrogen-fixing rhizobial

symbiosis.

Keywords: Rice, nitrogen-fixation, legume-Rhizobium symbiosis Introduction Rice is the principle food for more than half of humankind. The productivity of a rice crop is dependent on several variables including weather, soil type, moisture and nutrients. Among the nutrients the most important that affects rice production is nitrogen. The nitrogen requirement for rice production is conventionally met by the application of synthetic nitrogen-rich fertilizer. It is estimated that about 70-80% more nitrogen fertilizer will be required to raise rice production by 2050 to supplement the food requirements of increasing human population (Africare, Oxfam America, WWF-ICRISAT Project 2010). Considering the prevailing practice, it is natural to imagine that the increase in requirement of fixed nitrogen for augmenting crop production will be met by industrially generated chemical nitrogen fertilizer. The

production of chemical fertilizer is a high energy requiring process, which is mainly dependent on fossil fuels, and hence a major contributor to the depletion of natural resources (Ladha and Reddy 1995). In addition, excessive chemical fertilization is deleterious to human health and contributes to environmental degradation. To circumvent these problems the best option for meeting the fertilizer requirement is through biological nitrogen fixation (BNF). BNF is the major contributor to the nitrogen economy of biosphere, and it is propelled by diazotrophic bacteria, which reduce dinitrogen to ammonium using nitrogenase enzyme system. In view of the need for fixed nitrogen and importance of BNF, it is imperative to develop autotrophic nitrogen-

Society for Advancement of Rice Research, Directorate of Rice Research, Hyderabad, India 246

fixing non-legume crops, particularly important cereal crops such as rice (Ladha and Reddy 1995; Beatty and Good 2011). If a BNF system could be assembled in the rice plant, it could increase the potential for nitrogen supply because fixed nitrogen would be available to the plant directly, with little or no loss. Such a system could also enhance resource conservation and environmental security, and is more consistent with the development of sustainable agriculture. Most land plants, including the monocots like rice, are able to establish endosymbiotic associations with endomycorrhizal fungi, to form phosphate-acquiring arbuscular mycorrhizae (AM) (Fig. 1). In contrast, legumes not only are able to establish root endosymbioses with endomycorrhizal fungi, to form phosphate-obtaining AM, but also capable of entering into symbiotic associations with diverse diazotrophic bacteria, to form nitrogen-fixing nodules (Fig. 1). Root nodule symbiosis (RNS) is one of the most evolved and efficient nitrogen-fixing systems, but it is restricted only to four orders including legumes within the Eurosid clade (Soltis et al 1995). Genetic elements that mediate AM symbiosis (AMS) development in legumes are also found to be central for AMS formation in rice (eg, Gutjahr et al 2008). Further, in legumes, these same genetic elements, besides promoting AMS, also participate in mediating RNS development (see

Markmann and Parniske 2008). Thus, recent advances in understanding RN and AM symbiotic processes in legumes in conjunction with AM symbiosis in rice at the molecular level have created an excellent framework to investigate possibilities for extending the mycorrhizal symbiotic genetic network of rice to accommodate rhizobial symbiosis for nitrogen fixation.

Legume-Rhizobium symbiosis as a model for developing rhizobial symbiosis in rice RNS between legumes and soil Gram-negative bacteria collectively called rhizobia is achieved through two highly synchronized programs, namely bacterial infection (entry) and the development of a novel organ called nodule for housing rhizobia (Oldroyd and Downie 2008; Kouchi et al 2010; Popp and Ott 2011). In nodules, rhizobia fix atmospheric nitrogen and provide it to their respective host plant thereby effectively rendering the host legume independent of exogenous nitrogen supplies. Legume-rhizobia initial interactions A comparison with the depiction of similarities in signal exchange and infection processes during interaction of mycorrhizal fungi with plant roots, and rhizobia with the legumes is presented in Fig. 2. Nodule development in legumes is triggered due to interactions between the host plant and rhizobia (Pawloski and Bisseling 1996).

Figure 1. Types of root endosymbioses in rice and legumes. Legumes are able to form symbioses with both phosphate-acquiring arbuscular mycorrhizal fungi and nitrogen-fixing rhizobia, while rice is capable of developing symbiosis only with arbuscular mycorrhizal fungi. a = arbuscule; h= fungal hyphae; ic = rhizobia-infected nodule cell; n = nodule; s = germinating fungal spore


Figure 2. Plant-microbe interactions during the development of arbuscular mycorrhizal and root nodule symbioses. A = Plant-Glomus; B= legume-Rhizobium signal exchange and infection. Rhizobia interaction with leguminous plants begins with the secretion of flavonoids from roots and consequent flavonoid-triggered nod gene expression in the bacterium leading to the production of lipochitooligosaccharide nodulation signal molecules, commonly known as Nodfactors (NFs) (Fig. 2B; Denarie et al 1996, Reddy et al 2007). NFs play a pivotal role in the development of RNS. Perception of nodulation signals Systematic analysis of phenotypes of the mutants of model legumes (Lotus japonicus and Medicago truncatula) arrested at various stages of early symbiotic responses facilitated discovery and sequential ordering of the plant genes responsible for NF perception and initial signal transduction (Fig 3 and Table 1; Oldroyd and Downie 2004). One of the most critical discoveries is the identification of the genes that code for the proteins that perceive rhizobial Nod signals. Root expressed plant receptor-like kinases (RLKs) possessing LysM modules in their extracellular domains (LysM-RLKs) were found to be the key elements responsible for the recognition of NFs. NF receptors have been identified in several nodulating legumes: NFR5/NFR1 in L. japonicus (Madsen et al 2003, Radutoiu et al 2003)

and in Gycine max (Indrasumunar et al 2010), LYK3/NFP in M. truncatula (Limpens et al 2003, Arrighi et al 2006) and SYM37/SYM10 in Pisum sativum (Zhukov et al 2008). Perception of NFs by LysM-RLKssets off a cascade of symbiotic events initiating transcriptional activation of symbiosis-related genes that trigger physiological and developmental changes (such as Ca2+ fluxes, Ca2+ spiking, root hair deformation, curling, infection thread formation, cortical cell divisions, etc.) within the root to permit bacterial infection and nodule organogenesis (Fig. 3; see Kouchi et al 2010; Oldroyd et al 2011). The NF perceiving LysM-RLKs are unique to nodulating legumes, and play a vital role in triggering the signaling for the development of RNS.

Signaling components downstream of the Nod factor receptors are common for both mycorrhizal and rhizobial symbioses In legumes, acting downstream of the Nod factor receptors are a conserved set of gene products that participate in the development of both RNS and the more widespread and ancient AMS (Oldroyd and Downie 2004).


Figure 3. A current model representing the common symbiosis pathway and the gene cascades involved in the development of AMS in legumes and rice, and the nodule organogenesis and bacterial infection processes in RNS in legumes

Thus, the signaling pathway mediated by these genes is termed as ‘common symbiosis pathway’ (CSP; Markmann and Parniske 2008) (Fig. 3). Since AMS is more ancient in origin (~ 400 million years old) than RNS (~ 70 mys) and because the genetic program is shared between the RNS and the AMS, it is hypothesized that during evolution, several of the genetic constituents that participate in AMS formation are coopted for the development of RNS, and hence serve as a conserved common genetic framework for the development both types of root symbioses. Studies with the model legumes L.japonicas and M. truncatula revealed that these conserved set of CSP genes encode the plasma membrane receptor kinase LjSYMRK [MtDMI2 in M. truncatula] (Endre et al 2002; Stracke et al 2002), two predicted cation channels LjCASTOR and LjPOLLUX [MtDMI1] (Ane et al 2004; Imaizumi-Anraku et al 2005), three nuclear pore protein components [LjNUP85 (Saito et al 2007), LjNUP133 (Kanamori et al 2006) and LjNENA (Groth

et al 2010)], the calcium calmodulin dependent kinase LjCCaMK [MtDMI3] (Lévy et al 2004; Mitra et al 2004; Tirichine et al 2006) and the CCaMK-interacting nuclear-localized protein LjCYCLOPS [MtIPD3] (Messinese et al 2007; Yano et al 2008). In L. japonicus, following the perception of NFs by LysM-RLKs, biphasic Ca2+ signaling is induced (a rapid influx of Ca2+ followed by periodical oscillations/spiking of cytosolic Ca2+ at the perinuclear region) in epidermal cells in a SYMRK, CASTOR& POLLUX, NUP85, NUP133 and NENA-dependent manner (Stracke et al 2002; Imaizumi-Anraku et al 2005; Saito et al 2007; Kanamori et al 2006; Groth et al 2010). Ca2+ spiking is critical for both AMS and RNS (Kosuta et al 2008). CCaMK and CYCLOPS are positioned downstream of Ca2+ spiking as they were found to be not associated with the generation of Ca2+ spiking (Gleason et al 2006; Tirichine et al 2006; Messinese et al 2007; Yano et al 2008; Sieberer et al 2012).


Table 1. Genes involved in early signal transduction in legume symbioses, and summary results depicting the role of symbiosis related genes on AMS and RNS in Lotus japonicus, Medicago truncatula and Oryza sativa, and the complementation of legume symbiosis mutants with the O. sativa gene orthologues/homologues Lotus japonicas/Medicago trancatula

Oryza sativa orthologue/ homologue

Gene Lj/Mt mutant phenotype

Gene product

Ref

Accession Os mutant phenol

type Myc

Percent AA Identity with Lj/Mt proteins and remarks

Complemetation of Lj/Mt mutants with Os orthologues/ homologues

Nod

Myc

Myc Nod Ref

LjNFR1/MtLYK3 - + LysM receptor kinase

1, 2

AP004464 52/53; No TM or EC domains

LjNFR5/MtNFP - + LysM receptor kinase

3, 4

AK072207 41/39 (EC: 40/38; IC: 42/41) 3 LysMs like in Lj/Mt

LjSYMRK/MtDMI2 © - - LRR receptor kinase

5, 6

AK099778 36/36 (EC: 13/13; IC: 66/66) Only 2 LRRs and a short N-terminal sequence in Os in comparison to 3LRRs and a long N-terminal region in Lj/Mt

R NR 24

LjCASTOR © - - Ca2+-gated ion channel protein

7 AK068216 - 69/- R R 25, 26, 27

LjPOLLUX/MtDMI1© - - Ca2+-gated ion channel protein

7, 8

AK072312 - 63/63 NR NR 25, 26, 27

LjNUP85 © - - Nucleoporin 9 AK072636 + 60/- ND ND 25 LjNUP133 © - - Nucleoporin 10 AK073981 + 49/- ND ND 25 LjNENA © - - WD40

domain containing nucleoporin

11 NP001043597 59/- ND ND

LjCCaMK/MtDMI3 © - - Ca2+/CaM-dependent kinase

12, 13, 14

AK070533 - 72/70 R R 25, 26, 28

LjCYCLOPS/MtIPD3 ©

- - NLS & CC domain containing protein

15, 16

EF569223 - 48/46 R R 26, 29, 30

LjLHK/MtCRE1 - + Histidine kinase

17, 18

BR000246 58/58

LjNSP1/MtNSP1 - + GRAS family TF

19, 20

NT079921 41/41 R 31

LjNSP2/MtNSP2 - + GRAS family TF

19, 21

NT107191 48/46 R 31

LjNIN/MtNIN - + RWP-RK TF

22, 23

AK100046 38/36 NR 31

© Genes of common symbiotic pathway; EC: Extracellular; IC: Intracellular; Nod: Nodulation; Myc: Mycorrhization; -: Defective; +: Effective; R: Restored; NR: Not restored; ND: Not determined. 1. Radutoiu et al 2003; 2. Limpens et al 2003; 3. Madsen et al 2003; 4. Arrighi et al 2006; 5. Stracke et al 2002; 6. Endre et al 2002; 7. Imaizumi-Anraku et al 2005 ; 8. Ané et al 2004; 9. Saito et al 2007 ; 10. Kanamori et al 2006; 11. Groth et al 2010; 12. Tirichine et al 2006 ; 13. Lévy et al 2004; 14. Mitra et al 2004 ; 15. Yano et al 2008 ; 16. Messinese et al 2007; 17. Tirichine et al 2007; 18. Gonzalez-Rizzo et al 2006; 19. Heckmann et al 2006; 20. Smit et al 2005; 21. Kalo et al 2005; 22. Schauser et al 1999; 23. Marsh et al 2007; 24. Markmann et al 2008; 25. Banba et al 2008; 26. Gutjahr et al 2008; 27.Chen et al 2009; 28.Chen et al 2007; 29.Chen et al 2008; 30.Yano et al 2008; 31. Yakota et al 2010.


Instead, the nuclear localizing calmodulin/calcium-binding CCaMK is proposed to be a prime candidate to decode calcium signature toset off a cascade of signaling events paving a way for the initiation of either AMS or RNS (Kosuta et al 2008). CYCLOPS, a coiled coil domain containing protein, complexes with CCaMK and enables signal transduction for promoting fungal and bacterial infection in AMS and RNS, respectively (Yano et al 2008). Infection process and nodule organogenesis are highly synchronized and interdependent processes In legumes, rhizobial infection occurs via root hairs or the cracks formed at the sites of lateral root emergence. Although entry of rhizobia through root hairs is the predominant form of infection in most legumes, in a smaller percentage of plants (largely basal legumes) the bacterial infections occur through epidermal cracks. Nevertheless, both these infection processes are Nod factor-dependent, and hence reliant upon the participation of LysM-RLKs. Studies with L. japonicus showed the occurrence of yet a third type of bacterial invasion mechanism through intercellular infection even in the absence of functional NFR1 and NFR5 receptors, implicating a Nod factor-independent mode of entry (Madsen et al 2010). Thus, intercellular single cell infection is suggested to be the ground state of rhizobial infection of roots, followed by the crack entry and root hair infection modes. Infection through root hairs is considered to be the most sophisticated and stringently regulated mode of entry. In order to facilitate efficient rhizobial incorporation, many legumes have evolved a structural pathway referred to as infection thread (IT) for invasion (Sprent 2007). Initiation of ITs requires attachment of bacteria on the root hairs and curling of root hairs to entrap bacteria. Root hair curl provides an enclosure for the attached bacterial infection pocket, where bacteria proliferate, colonize and express cell wall-degrading enzymes to penetrate to reach the root hair plasma membrane to form IT, an inward growing tubular structure (Murray 2011; Popp and Ott 2011). Signal transduction through CSP following NF recognition is a prerequisite for promoting the coordinated development of IT and nodule organogenesis in RNS (Kouchi et al 2010). In parallel with IT formation in the root hair, the cells in the root cortex are triggered todivide [cortical cell division (CCD)] to initiate the development of a nodule meristem (Fig. 2B). This synchronized nodule organogenesis simultaneously with IT formation is believed to be crucial for successful nitrogen-fixing nodule formation (Oldroyd and Downie 2008). Rhizobia

need to enter the plant root for promoting RNS. The LysM RLK LYK3 with high stringency for NF structure has been identified as an entry receptor in M. truncatula (Limpens et al 2003). This recognition triggers the IT development and its progression towards nodule primordia. IT initiation/progression and release of bacteria into nodule cortical cells are contingent upon the constituents of NF perception and signaling through CSP (Oldroyd et al 2011). In addition to these components, the gene products shown to be essential for microcolony formation, IT initiation and progression in legumes include: NSP1, NSP2 and NIN (TFs) for microcolony formation (Schauser et al 1999; Oldroyd and Long 2003; Kalo et al 2005; Smit et al 2005; Heckmann et al 2006); CYCLOPS/IPD3 (coiled coil protein), ERN1 (TF), FLOT2, FLOT4 (lipid raft-associated proteins) and CERBERUS/LIN (a putative E3 ubiquitin ligase) for IT initiation (Middleton et al 2007; Kiss et al 2009; Yano et al 2009; Haney and Long 2010); PIR/NAP (two members of SCAR/WAVE complex involved in actin rearrangement), RPG (nucleus localizing coiled coil protein), VAPYRIN (an ankyrin domain containing cytoplasmic major sperm protein), SYMREM1 (remorin), nsRING andPUB1 (two putative E3 ubiquitin ligases) are necessary for IT progression (Shimomura et al 2006; Arrighi et al 2008; Yakota et al 2009; Lefebvre et al 2010; Mbengue et al 2010; Miyahara et al 2010; Pumplin et al 2010; Murray et al 2011) (Fig. 3). Furthermore, CRE1/LHK (cytokinin receptor; Murray et al 2007) and SICKLE (EIN2 ethylene response mediator; Penmetsa and Cook 1997; Penmetsa et al 2008) were also shown to participate in infection processes. Among the infection related genes, VAPYRIN was found to have infection functions in both rhizobial and mycorrhizal colonization in legumes (Pumplin et al 2010; Murray et al 2011). This finding supports the notion that during the evolution of RNS, not only genes from CSP, but also the components of the infection machinery were recruited from the pre-existing mycorrhizal symbiosis (Charpentier and Oldroyd 2010). In addition to IT development and nodule organogenesis, bacterial release into the nodule cortical cells is also stringently reliant upon NF-mediated signaling through the constituents of CSP. It was found that SYMRK, a component of CSP, also plays a critical role in promoting the release of bacteria into the cortical cells of nodules where rhizobia are housed and fix nitrogen (Capoen et al 2005; Limpens et al 2005). CCaMK is highly conserved in both nodulating and non-nodulating plants. Recently it was found that a gain-of-function CCaMKT265D suppresses loss-of-function mutations of the common symbiosis genes


SYMRK, POLLUX, CASTOR and NUP85 required for the generation of Ca2+ spiking, not only for nodule organogenesis but also for successful infection by rhizobia and AM fungi, demonstrating that the CSP genes upstream of Ca2+ spiking are required solely to activate CCaMK (Hayashi et al 2010). In other words, fundamental role of the CSP is to activate CCaMK to elicit downstream signaling events specific for promoting the development of either AMS or RNS, and hence any loss-of-function mutations in the upstream CSP genes is deleterious to both infection and nodulation/mycorrhization. In RNS, the activated CCaMK is central for triggering the cascade of nodulation events via cytokinin activity through MtCRE1/LHK1 (histidine kinase), and the activation of downstream genes by the action of nodulation-specific NSP1, NSP2, ERN1 and NIN transcription factors (Fig. 3; Gleason et al 2006; Tirichine et al 2006, 2007; Gonzalez-Rizzo et al 2006;Marsh et al 2007;Murray et al 2007) leading to root hair curling, infection thread formation and cortical cell divisions resulting in nodule development (Fig. 2B).

Advances in resolving genetic predisposition of rice for symbiotic nitrogen fixation A possibility for constructing nitrogen-fixing rice is to integrate legume-like nitrogen-fixing symbiotic system in this crop plant. Earlier research on determining genetic predisposition of rice for forming symbiosis with rhizobia evidenced that rice possesses some developmental subprograms in its genome, which are similar to those that lead to the development of symbioses in legumes (Reddy et al 2002). For example, the homologues of many of the early nodulin (ENOD) genes that participate nodule organogenesis in legumes are conserved in Oryza species (Reddy et al 1998a, 1999; Kouchi et al 1999; Zhu et al2006; rice genome sequence). Like legumes, rice roots exude compounds that are able to induce the transcription of the nod genes in rhizobia (Reddy et al 2000b; Sreevidya et al 2006). In turn, rhizobial Nod factors are able to elicit the expression of symbiotically responsive legume ENOD12 gene promoter in a rice cellular background, indicating that rice is able to recognize Nod factors (Reddy et al 1998b). Also, similar to that in legumes, the expression of legume ENOD40 promoter is elicited only in the vascular tissues in rice (Reddy et al 2003). In addition, it was also observed that the expression of legume ENOD40, a critical gene that provokes nodule organogenesis, is able to elicit cortical cell divisions in rice roots (Reddy et al 2000a, 2003), and the expression of symbiosis-related legume lectin genes enabled rhizobial attachment at root hair tips and promoted inter- and intra-cellular colonization in root endodermis

and cortex, and occasionally resulted in the formation of infection pegs (Sreevidya et al 2005). The participation of genes common for both bacterial and fungal symbioses in legumes prompted the hypothesis that pre-existing AMS genes were recruited during the evolution of RNS (Markmann and Parniske 2008). Although rice does not develop a symbiotic association with rhizobia, it is able to enter into symbiotic associations with mycorrhizal fungi. Since it has been shown that part of the genetic program is shared between the RNS and the AMS and because both legumes and rice are able to establish AMS with the same fungal species, it is reasonable to assume that rice possesses common symbiotic genes. In recent past, it was discovered that the rice lines mutated in the orthologs of CASTOR/POLLUX, CCaMK or CYCLOPS genes are indeed unable to establish symbiosis with AM fungi (Table 1; Chen et al 2007, 2008, 2009; Banba et al 2008; Gutjahr et al 2008, Yano et al 2008), and transgenic introduction of the rice orthologs SYMRK, CASTOR, CCaMK and CYCLOPS into corresponding legume mutants restored AM formation (Chen et al 2007, 2009; Banba et al 2008; Markmann et al 2008; Yano et al 2008) (Fig. 4; Table 1). Moreover, the gene orthologs OsCASTOR, OsCCAMKand OsCYCLOPS are also able to fully complement RNS and restor functional nodules when transformed into the legume mutants deficient in the respective genes (Fig. 4; Banba et al 2008; Yano et al 2008). These findings clearly demonstrated that some of the CSP gene orthologs of rice are fully functionally conserved, and even suitable for promoting RNS.

In legumes, among the critical genes which are shown to be required for RNS, but not for AMS, are two GRAS-domain transcription factors, NSP1 and NSP2 (Oldroyd and Long 2003; Kalo et al 2005; Smit et al 2005; Heckmann et al 2006) and a RWP-RK containing transcription factor, NIN (Schauser et al 1999; Marsh et al 2007). Recently, Yokota et al (2010) demonstrated that the even the homologs of NSP1 and NSP2 from rice are able to fully rescue the RNS-defective phenotypes of the mutants of corresponding genes in the model legume L.japonicas (Fig. 4). In contrast, OsNLP1 (NIN-like protein 1) failed to complement the nodulation minus Ljnin mutant. These findings clearly demonstrated that even the orthologues of nodulation-specific TFs such as NSP1 and NSP2are functionally conserved in rice.

Above findings evidence that the genetic elementsthat promote AMS (together with some other critical genetic constituents) are conservedacross legumes and rice, and they can function as potential buildings blocks for extending symbiotic networks to accommodate RNS in rice.


Figure 4. Comparison of exon structures of the orthologous symbiotic genes of Lotus japonicus and rice, and restoration of AMS/RNS in the L. japonicus mutants defective in symbioses with the rice orthologs. CaM-BD = calmodulin binding domain; CC = coiled coil domain; CEC = conserved extracellular region; EF = helix-loop-helix calcium binding domain; LRR = leucine-rich repeats; NEC = N-terminal extracellular region; PK = protein kinase domain; RCK = regulator of the conductance of K+; SP = signal peptide; TM = transmembrane domain. LHRI, LHRII (leucine heptad repeat regionsI and II), VHIID, PFYRE and SAW are conserved motifs in GRASS domain-containing transcription factors. Myc+ = arbuscular mycorrhizae; Nod+ = nodulation; Empty boxes depict no complementation. (1. Markmann et al 2008; 2. Banba et al 2008; 3. Yano et al 2008; 4. Yokota et al 2010).

Future steps Discussion in preceding sections suggests that rice, although it does not develop a symbiotic association with rhizobia, possesses at least some cellular programs similar to those that promote RNS in legumes. Rice forms symbiosis with arbuscular mycorrhizal fungi and possesses CSP genes (as well as other genetic constituents) that were shown to have the ability to support RNS when transferred to legume mutants defective in respective genes. These findings indicate that partial genetic network that could potentially support RNS is already in place in rice. Nevertheless, in addition to the components of CSP, the elements that enable perception and transmittance of Nod signal are vital for eliciting symbiotic responses leading to the

formation of RNS, since signal transduction through CSP following NF recognition is a prerequisite for promoting this coordinated development of infection and nodulation. Hence, a molecular approach to make rice amenable to both AMS and RNS symbioses is to genetically alter rice plants to integrate signalling pathways that is able to facilitate the formation of both phosphate-acquiring AM and the nitrogen-fixing rhizobial symbiosis. So the coordinated expression of legume Nod factor receptor kinases together with the nodulation-specific transcription factors in rice may facilitate novel specificity for Nod signal recognition and aid in subsequent signal transduction required for promoting RNS responses in rice roots (see Fig. 3). At this stage it is difficult to foresee what will be the


outcome of such an extension of symbiotic genetic framework beyond CSP in rice. Nevertheless, if successful, this research will provide valuable information about the practicality of the construction of nodulating rice and provide insights as to what additional requirements will be necessary to bring this idea to the stage of agricultural use. Since long it is known that rhizobia readily colonize rhizosphere of rice, and frequently are able to enter inside the roots to form endophytic associations (Reddy et al 1997; Chaintreuil et al 2000; Dazzo et al 2000). A primary mode of rhizobial invasion of rice roots is through cracks in the epidermis and fissures created during emergence of lateral roots. This infection process, unlike in legumes, is Nod factor-independent and does not promote the formation of infection threads through root hairs or the development of intricate network of infection threads in root cortex. It is possible that cereals such as rice do not possess certain critical genetic traits to trigger the formation of sophisticated infection threads in roots (Reddy and Ladha 1995).For rhizobial infection in rice, development of a sophisticated infection-system favoring bacterial entry through root hair may not be necessary. So long as Nod factor-responsive symbiotic machinery is in place to support RNS in rice, a process of Rhizobium infection through primitive "crack-entry" mode may, in fact, facilitate bypassing the cellular machinery needed for sophisticated type of infection through root hair. Such a "crack-entry" by rhizobia is a common mode of infection in several aquatic nodulating legumes such as Sesbania rostrata. Finally, it is important to mention here that although rhizobia have the ability to invade and colonize the rice roots they evoke a localized plant-defense response culminating host cell death (Reddy et al 1997). In contrast, rhizobial infection and colonization in compatible legume host plants do not elicit any significant defense response in the host plant. The cross-communication between rhizobial and legume plant partners is fine-tuned not to evoke plant defense responses during the course of RNS development. This is largely because of the compartmentalization or sequestering of the infecting microsymbiont from host cells by a plant-derived membrane interface (Ivanov et al 2010). Rice is able to synthesize an interface layer around the infecting fungus in mycorrhizal symbiosis. So the challenge is to convert this ability to trigger the synthesis of peribacterial interface to shield invading rhizobia from plant defense responses and enable the plant to enter into N2-fixing RNS. ACKNOWLEDGEMENTS Rice research program in the authors’ lab is funded by CONACyT, Mexico(No. 128135) to PMR.

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Citation: Reddy PM, Altúzar-Molina AR, Ortiz-Berrocal M, Medina-Andrés R, López-Sámano M, Martínez-Aguilar L and Velázquez-Hernández MDL. 2013. Predisposition and redesigning of genetic networks of rice for accommodating nitrogen-fixing rhizobial symbiosis. In: Muralidharan K and Siddiq EA, eds. 2013. International Dialogue on Perception and Prospects of Designer Rice. Society for Advancement of Rice Research, Directorate of Rice Research, Hyderabad 500030, India, pp 245-257.

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Predisposition and Redesigning of Genetic Networks of Rice for Accommodating Nitrogen-Fixing Rhizobial Symbiosis