Biotechnological Approaches For Improving Biological Nitrogen Fixation

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1 Biotechnological Approaches For Improving Biological Nitrogen Fixation (BNF) in Legumes

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2 Contents…. Introduction Biological Nitrogen fixation Mechanism of Nodulation and Nitrogen Fixation in Legumes Biotechnological Approaches to Improve Biological Nitrogen Fixation. 1. Bacterial Genomics 2. Plant Genomics Conclusion Future thrust

Introduction:

3 Introduction Nitrogen is one of the major nutrient for plant growth & productivity Nitrogen makes up approximately 80% of the elements present in Earth’s atmosphere It is a constituent of chlorophyll, proteins, nucleic acids and other essential nutrients Due to triple bond in the structure (N  N) ,plant & other eukaryotes can’t directly utilized it. So the atmospheric form is converted to available form through various processes ….

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4 NITROGEN FIXATION Burdass. (2002)

Biological Nitrogen Fixation :

5 Biological Nitrogen Fixation Biological Nitrogen Fixation is the enzymatic reduction of dinitrogen (N 2 ) from the atmosphere to ammonia (NH 3 ) through prokaryotes having nitrogenase enzyme system. The symbiotic relationships between the plant and the nitrogen fixing bacteria are such, that the bacteria supply the plant with reduced nitrogen compound and the plant, in return provides carbon as a source of energy.

Basic requirement for the Biological nitrogen fixation are:-:

6 Basic requirement for the Biological nitrogen fixation are:- The enzyme: Nitrogenase - An enzymatic complex which enables fixation of atmospheric nitrogen - It exists in both free living and symbiotic nitrogen fixing bacteria - It is extremely O 2 sensitive A strong reducing agent / Electron donor - reduced ferredoxin - reduced flavodoxin ATP / source of energy - By oxidation of respiratory substrate - Through photophosphorylation - By conversion of pyruvate to acetate Low oxygen tension - cause rapid irreversible oxidation of nitrogenase metal S-centres - also represses synthesis of nitrogenase proteins - Leghaemoglobin protects from O 2

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7 γ γ Component of Nitrogenase Fe protein (  2 subunit ) Also called Component II; Dinitrogenase I; Fraction II etc Mol.wt.- 60,000d Dimer of identical polypeptide chain Function: hydrolysis, electron transfer and binding of Mg, ATP and ADP β α α β Mo-Fe protein (  2  2 subunit ) Also called Component I; Dinitrogenase reductase I; Fraction I etc Mol.wt. -2,40,000d Tetramer of two different polypeptide chain Function: Electron transfer and substrate reduction

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8 Nitrogen fixation

Estimated Average Rates of Biological N2 Fixation:

9 Estimated Average Rates of Biological N 2 Fixation Organism or system N 2 fixed (kg ha -1 y -1 ) Free-living microorganisms Cyanobacteria Azotobacter Clostridium pasteurianum 25 5-25 0.1-0.5 Grass-Bacteria associative symbioses Azospirillum 5-25 Cyanobacterial associations Gunnera Azolla Lichens 10-20 300 40-80 Leguminous plant symbioses with rhizobia Grain legumes ( Glycine, Vigna, Lespedeza, Phaseolus ) Pasture legumes ( Trifolium, Medicago, Lupinus ) 50-100 100-600 Actinorhizal plant symbioses with Frankia Alnus Hippophaë Ceanothus Coriaria Casuarina 40-300 1-150 1-50 50-150 50 (Ladha et al, 1992)

Mechanism of Nodulation and Nitrogen Fixation in Legumes:

10 Mechanism of Nodulation and Nitrogen Fixation in Legumes

Rhizobium-Plant association:

11 Rhizobium-Plant association Rhizobium Host Plant(s) R.meliloti Medicago, melilotus, and Trigonella spp. R. leguminosarum bv.viciae bv. trifolii bv. phaseoli Pisum, vicia, lathyrus and lens spp. Trifolium spp. Phaseolus vulgaris R.loti Lotus spp. R.huakuii Astragalus sinicus R. ciceri Cicer arientinum Rhizobium sp. Strain NGR234 Tropical legumes ,Parasponia spp.(nonlegume) R.tropici Phaseolus vulgaris, Leucaena spp., Macroptilium spp. R.etli Phaseolus vulgaris. R.galegae Galega officinalis, G. orintalis. R.fredii B.japonicium Glycine max, G. sojo, and other legumes B.elkanii Glycine max, G. sojo, and other legumes Bradyrhizobium sp. Strain Parasponia Parasponia spp. A. caulinodans Sesbania spp .(stem nodulating ) Rhijn et al.,( 1995)

Nodulation in Legumes:

12 Nodulation in Legumes Attachment Root hair curling Infection thread Cortical cell differentiation Rhizobia released into cytoplasm and Bacterioid differentiation (symbiosome formation) Induction of nodulins and formation of nodules

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13

NOD Factors & their Functions :

14 NOD Factors & their Functions Lipochitin Oligosacchorides Root nodule formation and infection process. Determination of host specificity. Exopolysaccharides Protection against environmental factors Attachment to surface and osmo-regulation Lipopolysaccharides Play role during infection process, later stages of root nodule invasion,release from the infection thread, symbiosome development K-Antigens Induce the transcription of isoflavonoid Cyclic Glucans Solubility of legume derived flavonoids and Nod factors is greatly increased in the presence of Cyclic Glucans. Specific Suppressors and plant defense response

Functions and properties of nod/nol genes :

15 Functions and properties of nod/nol genes nod Function nod Function A,B Nod factor production and deactylase S Mehthyltransferase C Chitin synthases T Transit Seqquences D Transcriptional activator, LysR family V Sensor, two-component regulatory family E Host specific; homology to -ketoacyl synthases W Regulator, two-component regulatory family F Host specific;homology to acyl carrier protein X Acidic exopolysaccharide encoded by exoz G Host specific;homology to Alcohol dehydrogenase, -ketoacyl reductase K,N,U, Y,Z Identified but no sequence homology to other published gene product (s) and proposed function (s). H Host specific; Sulfotransferase nol A DNA- binding protein IJ Capsular polysaccharide secretion proteins C Heat shock protein L Acetyltransferase K Sugar epimerase M D-Glucosamine synthase hemolysine R DNA-binding protein O Hemolysine T Hrp genes of pathogenic bacteria P ATP- Sulfurylase B,E,F,G,H,I,J,M,N,O,P,U,V,W,X,Y,Z. Identified but no sequence homology to other published gene product and proposed function. Q ATP- sulfurylase and APS Kinase Rhijn et.al.,( 1995)

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16 Solid arrow- common nod gene, hatched arrow - host specific nod gene, white arrow - nodD gene, stippled arrow - nol gene and other nod loci. R. leguminosarum bv. viciae R. leguminosarum bv. trifoli R. meliloti B. japonicum Genetic Organization of nod genes

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17

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18 b a a b Fe Protein Fe-Mo Protein Regulation NtrC-RNA polymerase Activation of NifA

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19 b a a b Reduced Fe Protein Fe-Mo Protein Regulation Electron Transport Ferredoxin ATP Reduction of Electrons Donated to N2 Formation of NH3

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20 b a a b Fe Protein Fe-Mo Protein Regulation Oxygen Presence Activation of NifL Suppression of NifA

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21 A Model system for Legume- Rhizobium Symbiosis 25

Table 1 : Variability among the Rhizobial strains and cultivars of Chickpea nitrogen fixing efficiency :

22 Table 1 : Variability among the Rhizobial strains and cultivars of Chickpea nitrogen fixing efficiency Cultivar Treatment AGDM Mg ha -1 Seed yield Mg ha -1 Nitrogen yield Kg ha -1 Ndfa % N fixed Kg ha -1 ILC 195 control 4.04 1.53 61 61 37 CP31 4.54 1.58 68 72 49 CP39 4.27 1.61 65 69 45 ILC482 control 4.68 1.95 74 66 49 CP31 5.84 2.67 103 64 66 CP39 6.55 2.86 112 71 80 ILC3279 control 4.96 1.70 76 64 49 CP31 5.63 1.96 90 72 65 CP39 5.65 2.95 85 67 57 ILC5396 control 5.85 1.92 83 25 21 CP31 6.53 2.31 96 65 62 CP39 6.67 2.10 99 71 70 LSD 0.05 0.77 0.25 14 18 23 ICRISAT ( Patancheru, A.P.) Beak (1992)

Table 2 : Variability among the different chickpea varieties for nodulation:

23 Table 2 : Variability among the different chickpea varieties for nodulation Chickpea line Nodule mass at DAS (mg plant -1 ) Grain yield (t ha -1 ) 0 N 50 N 0 N 50 N ICC 435 324 283 3.57 2.58 ICC 435M 0 0 2.24 3.02 ICC 4918 211 139 2.91 3.25 ICC 4918M 0 0 1.56 2.36 ICC 4993 163 120 2.55 2.40 ICC 4993M 0 0 2.06 2.33 ICC 5003 370 191 3.70 2.69 ICC 5003M 0 0 2.69 3.32 ICC 640 222 188 2.98 3.53 PM 233 0 0 1.87 2.36 LSD (0.05) 65 0.47 ICARDA (Aleppo),Syria Rupela (1992)

NEED OF BIOTECHNOLOGICAL APPROACHES IN BNF:

24 NEED OF BIOTECHNOLOGICAL APPROACHES IN BNF To understand the the complexity of biological nitrogen fixation, the role of both bacterial (microsymbiont ) and the host plant (macrosymbiont). To develop efficient Rhizobium strains for fixing nitrogen which is able to compete the existing Rhizobium spp. To develop legume cultivars having good nodulation and nitrogen fixing efficiency.

Biotechnological Approaches for RHIZOBIAL (Microsymbiont) GENOMICS:

25 Biotechnological Approaches for RHIZOBIAL (Microsymbiont) GENOMICS Identification and classification of Rhizobium spp. Complete genome sequencing of Rhizobium spp. To develop Genetically modified Rhizobium strains. Hydrogen uptake system (hup). pSym plasmid transfer. Transfer of nod gene to the nod gene lacking Rhizobium strain. Introduction insecticidal toxin gene in to Rhizobium strain

Classification of Rhizobium spp.:

26 Classification of R hizobium spp. Sequence comparison of 16s rRNA genes. PCR-RFLP of 16s rRNA genes Multilocus enzyme electrophoresis. DNA-DNA Homology. LMW RNA Profile. Whole-Cell Protein profiles. Plasmid profiles. Sequence comparison of Internally transcribed (ITS) spacer region sequences.

Genome Sequencing of Rhizobium spp.:

27 Genome Sequencing of Rhizobium spp. Strains Rhizobium etli CFN42 Chromosome (5mb) & six plasmids : p42a-(0.2mb), p42b-(0.15mb),p42c(0.27mb), p42d-(0.37mb),p42e-(0.5mb),p42f(0.7mb) Sinorhizobium meliloti 1021 Chromosome(3654kb) & pSyma (1354kb), pSymb (1683kb) Mesorhizobium Loti strain MAFF303099 Chromosome (7036 kb) & Two Plasmids (352kb,208kb) Bradyrhizobium sp. strain BTAi ( 9.1 Mbp genome) Rhizobium leguminosarum bv. viciae 3841 Rhizobium sp. NGR234 (ANU 265) Rhizobium tropici PRF 81

EcoR I restriction map of B.japonicum DNA in cosmids pHU1,pHU52,and pHU53. :

28 EcoR I restriction map of B.japonicum DNA in cosmids pHU1,pHU52,and pHU53. Oregon Lambert et al.,( 1987)

TABLE 3 : Expession of hydrogenase activity in Rhizobium transconjugants harboring pLAFR1, pHU1, pHU52,and pHU53 in the free-living state:

29 TABLE 3 : Expession of hydrogenase activity in Rhizobium transconjugants harboring pLAFR1, pHU1, pHU52,and pHU53 in the free-living state Parent or recipients strain E.Coli strain Frequency of Tc r colonies b HUP rate of derepressed transconjugant cell c (nmol-mg of protein -1 ) B.Japonicum USDA 123pc HB101(pLAFR1) 1.2 × 10 -3 <2 HB101(pHU1) 9.4 × 10 -3 <2 HB101(pHU52) 1.9 × 10 -3 132 HB101(pHU53) 6.7 × 10 -3 <2 B.Japonicum USDA138 HB101(pLAFR1) 1.2 × 10 -3 <2 HB101(pHU1) 4.1 × 10 -3 <2 HB101(pHU52) 1.1 × 10 - 4 74 HB101(pHU53) 3.5 × 10 - 4 <2 R.meliloti 102F28 HB101(pLAFR1) 1.6 × 10 -1 <2 HB101(pHU1) 3.3 × 10 -1 <2 HB101(pHU52) 2.3 × 10 -2 73 HB101(pHU53) 1.5 × 10 -2 <2 R.trifolii SU794 HB101(pHU52) ND 240 Oregon Lambert et al.,( 1987)

Table 4 : Symbiotic expression of hydrogenase activity in Rhizobium transconjugants harboring pLAFR1, pHU1, pHU52 and pHU53 :

30 Table 4 : Symbiotic expression of hydrogenase activity in Rhizobium transconjugants harboring pLAFR1, pHU1, pHU52 and pHU53 Inoculum Host plant H 2 uptake rate of bacteroids (pmol.min -1 mg of protein -1 ) Phenotype of Tc r cells B.japanicum USDA 123Spc G. Max cv. Williams <50 Hup - B.japanicum USDA 123Spc(pLAFR1) G. Max cv. Williams <50 Hup - B.japanicum USDA 123Spc(pHU1) G. Max cv. Williams 10,060 Hup + B.japanicum USDA 123Spc(pHU52) G. Max cv. Williams 4,220 Hup- B.japanicum USDA 123Spc(pHU53) G. Max cv. Williams <50 Hup- R.meliloti102F28 M. Sativa cv. vernal <50 R.meliloti102F28(pLAFR1) M. Sativa cv. vernal <50 Hup - R.meliloti102F28(pHU1) M. Sativa cv. vernal 380 Hup - R.meliloti102F28(pHU52) M. Sativa cv. vernal 210 Hup + R.meliloti102F28(pHU53) M. Sativa cv. vernal <50 Hup - Oregon Lambert et.al.,( 1987)

Table 5 : Hydrogenase activity of pea bacteroids from R. leguminosarum strain and homology of plasmid and total DNA from these strains to R meliloti nif or B. japonicum Hup-specific DNA sequence:

31 Table 5 : Hydrogenase activity of pea bacteroids from R. leguminosarum strain and homology of plasmid and total DNA from these strains to R meliloti nif or B. japonicum Hup-specific DNA sequence Strain Bacteroid Hydrogenase activity a Homology to hup DNA b Total DNA Plasmid DNA 128C53 0.65  0.15 + + 128C30 0.75  0.18 + + 128C23 0.65  0.12 + + 128C13 0.34  0.08 + + 128C56 0.27  0.06 + + 175G15 <0.05 _ _ UML2 <0.05 _ _ UML5 <0.05 _ _ 128C75 <0.05 _ _ 175G11 <0.05 _ _ 92A3 <0.05 _ ND 128C76 <0.05 _ ND 128C78 <0.05 _ ND Spain Leyva et al., (1987) a- Micromole of O 2 –dependant H 2 hydrogen uptake. mg of protein -1 b- 5.9kb HindIII fragment from PHU1 was used as probe.

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32 Physical and genetic map of the hydrogenase gene cluster from R. leguminosarum bv. vicae UPM791 cloned in cosmid pAL618. Spain Brito et al.,( 2000)

Table 7 : Hydrogenase activities induced by cosmid pAL618 in bacteroids of different Rhizobium strain as a function of the addition of nickel to the plant nutrient solution.:

33 Table 7 : Hydrogenase activities induced by cosmid pAL618 in bacteroids of different Rhizobium strain as a function of the addition of nickel to the plant nutrient solution. Recipient strain Hydrogenase activity ( mol of H 2 h -1 .mg of protein -1 ) Cosmid stability (%) No Ni 2+ added 170  M Ni 2+ added O 2 Methylene blue O 2 Methylene blue R. Leguminosarum bv.viciae UPM791 4.50  0.25 7.50  1.15 16.90  0.67 37.90  6.82 82 R. Leguminosarum bv.viciae UML2 1.57  0.13 2.82 0.12 2.20  0.14 3.50  0.26 60 R. Leguminosarum bv.viciae PRE 5.25  1.04 14.10 2.55 9.79  1.29 51.05  7.54 65 M. Loti Y3 2.15  0.35 3.55  0.18 3.01  0.17 6.45  0.85 53 M.loti U226 3.17  0.42 7.32  0.65 9.56  1.05 24.76  2.66 72 R.etli CFN42 5.00  0.36 12.30  0.60 5.30  0.41 13.50  0.83 70 S. Meliloti 102F34 0.07  00.2 0.07  0.2 0.48  0.11 1.32  0.16 64 Spain Brito et al.,( 2000)

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34 Construction of TnHB100 minitransposon Spain Bascones et al., (2000 )

Table 8 : Hydrogen metabolism in bacteroids and nodules induced by TnHB100-containing rhizobial strains   :

35 Table 8 : Hydrogen metabolism in bacteroids and nodules induced by TnHB100-containing rhizobial strains   Strain Bacteroid hydrogenase activity (nmol/mg of bacteroids per h ) Nodule H2 evolution ( mol of H 2 h -1 .mg of protein -1 ) B. japonicum 752 <10 4.97  3.19 752-H1 4,400  1,250 <0.1 752-H2 3,200  500 < 0.1 744 <10 13.09  1.42 744-H1 4,300  450 < 0.1 744-H11 2,850  250 <0.1 M. Ciceri UPM Ca7 <10 4,43  0.79 UPMCa7-H1 20  10 3.24  0.43 UPMCa7-H1 40  10 4.19  0.23 M.loti U226 <10 5.88  0.53 U226-H3 21,300  2,450 < 0.1 U226-H7 18,075  1,450 <0.1 Continue…….

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36 R.etli CE 3 <10 4.48  2.07 CE3-H2 10,400  3,850 <0.1 CE3-H7 < 10 <0.1 CE3-H71 2,350  500 < 0.1 R leguminosarum bv viciae UPM791 3,000  200 < 0.1 PRE < 10 10.50  3.3 PRE-H4’ 2,350  1,22, < 0.1 PRE-H2 2,550  500 < 0.1 UML2 400  100 5.49  0.19 UML2-H3 400  100 1.85  0.17 UML2-H8 350  100 2.31  0.16 S. meliloti 102F34 < 10 13.24  1.64 102F34-H1 <10 14.24  4.17 102F34-H3 50  20 13.65  1.74 Continue……. Spain Bascones et.al., (2000 )

Table 9 : Symbiotic phenotype in the Rhizobium etli-Phaseolus vulgaris symbiosis expressing E.coli gdhA gene.:

37 Table 9 : Symbiotic phenotype in the Rhizobium etli-Phaseolus vulgaris symbiosis expressing E.coli gdhA gene. Strain dpi a Nodulation b (g) Nitrogenase specific activity ( mol ethylene / h/g nodule ) Yield c (g) CFN42 18 0.020 (  0.005 ) 80 (  10 ) 0234 (  0.048 ) 25 0.040 (  0.005 ) 70 (  8) 0.442 (  0.082 ) CFN42/pTR101 18 0.027 (  0.004 ) 65 (  8) 0.234 (  0.043) 25 0.046 (  0.011 ) 53 (  5) 0.500 (  0.157 ) CFN42/pAM1a 18 0 0 0198 (  0.033) 25 0 0 0.364 (  0.056 ) CFN2012 18 0.024 (  0.004 ) 76 (  12) 0.250 (  0.046 ) 25 0.043 (  0.008 ) 55 (  10) 0.450 (  0.096 ) CFN2012/pTR101 18 0.022 (  0.005 ) 49 (  12) 0297 (  0.051) 25 0.042 (  0.011 ) 51 (  9) 0.545 (  0.065 ) CFN2012pAM1a 18 0.024 (  0.004) 61 (  12) 0.294 (  0.067 ) 25 0.045 (  0.010 ) 54 (  8) 0.529 (  0.082) Uninoculated 18 0 0 0.230 ( 0.058 ) 25 0 0 0.345 (  0.032 ) Mexico Mendoza et al., (1995) pTR101-mini RK-2 vector with par 0.8kb stability locus. pAM1a- pTR101 with E. coli gdhA gene

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38 Japan Mikiko et.al., (1998) The mating procedure between strains 4S, 4S5, and A136.

Table 10 : Induction of nodules and their acetylene reduction activity on white clover inoculation with Rhizobium and Agrobacterium:

39 Table 10 : Induction of nodules and their acetylene reduction activity on white clover inoculation with Rhizobium and Agrobacterium Strain Nodulation b Acetylene reduction c 4S + 0.19 4S5 + 0.25 H1 - 0 H1R1 + 0.37 AT4Sa +++ 0 AT4SB ++ 0 AT4SD ++ 0 AT4SE ++ 0 AT4SG ++ 0 A136 - 0 Japan Mikiko Abe et.al., (1998) b- for nodulation: total 20 to 50 seedling are used. +, < 5 nodules/ plant; ++,5  10 nodules/plant; +++, >10 nodules /plant. C- Actylene reduction (nM /plant/h) was measured at 50 days after inoculation.

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40 Rhizobium strain 4S Agrobacterium transconjugant strain AT4SG Agrobacterium transconjugant strain AT4SG Rhizobium Meliloti 1021

Table 11 : Nodulation on five kinds of leguminous plant with Rhizobium and Agrobacterium-transconjugant inoculation.:

41 Table 11 : Nodulation on five kinds of leguminous plant with Rhizobium and Agrobacterium -transconjugant inoculation. Plants Strains 4S 4S5 AT4Sa AT4SB AT4SG A136 H1 H1R1 Cont. Trifolium repens + + + + + - - + - Medicago sativa - - - - + - - n.d. - Vicia hirsuta - - + - - - - n.d. - Vigna mungo - - - - - - - n.d. - Glycine max - - - - - - - n.d. - Japan Mikiko Abe et.al., (1998)

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42 Construction of the transposon Tn5nodX and its suicide delivery vehicle pRF14 Canada Fobert et.al., (1991)

Table 12: Root nodules formed on the pea cultivars Novella, Trapper, Traper nn, and Afganistan by field isolates or derivatives of these isolates carrying Tn5nodX:

43 Table 12: Root nodules formed on the pea cultivars Novella, Trapper, Traper nn, and Afganistan by field isolates or derivatives of these isolates carrying Tn5nodX Strain Avg no.of nodules per plant (  SD ) Novella Traper Traper nn Afganistan O14 37 8 25 4 0 0 O14Tn5nodX 42 9 43 6 35 7 22 9 128C53 35 5 42 5 2 0 128C53Tn5nodX 39 4 43 8 32 8 27 9 JIM201 43 11 49 8 3 1 JIM201Tn5nodX 57 9 48 9 46 7 41 5 JISP36 36 7 53 9 0 8 1 JISP36Tn5nodX 42 7 51 9 24 3 49 6 SMY8510 42 8 52 7 2 0 STM8510Tn5nodX 47 12 51 9 37 6 41 5 SMY8513 42 8 32 7 2 0 SMY8513Tn5nodX 43 10 31 4 25 4 31 3 USDA2449 44 7 47 8 9 2 10 2 USDA2449Tn5nodX 51 9 55 6 53 5 47 4 USDA2397 46 11 51 11 9 3 1 USDA2397Tn5nodX 58 11 49 9 55 7 42 6 Canada Fobert et.al., (1991)

Table13 : Ability of a Tn5nodX derivative (014Tn5nodX) of a local R. leguminosarum biovar viciae field isolate (014) to outcompete 014 and occupy root nodules on Trapper nn peas homozygous for the sym-2 mutation.:

44 Table13 : Ability of a Tn5nodX derivative (014Tn5nodX) of a local R. leguminosarum biovar viciae field isolate (014) to outcompete 014 and occupy root nodules on Trapper nn peas homozygous for the sym-2 mutation. Strains in inoculation 014/014Tn5nodX viable cell ratio in inoculam No. of nodules/plant of Trapper nn inoculated 1 2 3 4 5 014 0 0 0 0 0 014Tn5nodX 30 14 20 16 23 014+ 014Tn5nodX 1:1 20 23 14 30 24 10:1 23 29 12 24 22 100:1 14 21 22 23 21 1000:1 20 20 13 16 20 Canada Fobert et.al., (1991)

Table 14 : Biochemical and symbiotic properties of His- mutants of Rhizobium leguminosarum bv. trifoli strain RTH48 with berseem clover.:

45 Table 14 : Biochemical and symbiotic properties of His - mutants of Rhizobium leguminosarum bv. trifoli strain RTH48 with berseem clover. Group Strain Growth on Nodulation ARA * MM MM+His MM+HOL MM+HOL-P N/A RTH48 + + + + + 6-9 1 His-2, - + _ _ + 0 His-12 2 His-17 - + + _ _ 0 3 His-1 to - + + + _ 0 His-21 † *ARA, acetylene reduction activity, mole h -1 plant -1 + = visible growth = no visible growth † except His-2, His-12 and His-17 Hisar Yadav et al., (1998)

Table 15 : Effect of cloned nod genes on nodulation of cv. Afghanistan peas:

46 Table 15 : Effect of cloned nod genes on nodulation of cv. Afghanistan peas Strain Nodule number after 24 days cv. Afghanistan Wisconsin Perfection 8401 0 0 8401/pIJ1095 1  1 74  15 8401/pIJ7244 11  3 10  2 TOM 62  9 89  11 TOM/pIJ1095 4  2 61  7 TOM/pIJ7244 43  9 87  12 TOM/pIJ1597 8  2 59  8 A34 0 78  13 A34/oIJ1357 34  4 79  12 A195 0 0 Netherlands Hogg et al., (2001)

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47 Construction of pSM4 Control : IC3554, Treated : IC3554(pSM4) Canada Nambiar et al., (1990)

Table 16 : Comparison of the ability of Rivellia angulata Larvae to infest and damage root nodules of induced by Bradyrhizobium sp. Strain IC3554 and strain IC3554(pSM4) carrying the entomocidal gene -endotoxin from bt. sp. israelensis :

48 Table 16 : Comparison of the ability of Rivellia angulata Larvae to infest and damage root nodules of induced by Bradyrhizobium sp. Strain IC3554 and strain IC3554(pSM4) carrying the entomocidal gene -endotoxin from bt. sp. israelensis Bacterial inoculant Average no.of nodules per pot % of nodules damaged % of N levels at 40 days after plants were removed from the insect cage Plants exposed to caged insects None 148 90 (72) 1.2 Strain IC3554 234 86 (68) 1.4 Strain IC3554(pKT230) 189 79 (61) 1.2 Strain IC3554(pSM4) 207 46 (42) 2.9 Plants not exposed to caged insects:all 3 inoculants 257 0 3.2 Canada Nambiar et al., (1990)

Table 17 : Comparative growth of different isolates of Mesorhizobium ciceri on NF and NF supplemented medium :

49 Table 17 : Comparative growth of different isolates of Mesorhizobium ciceri on NF and NF supplemented medium S. No Isolates no / strain no NF NF+ NaN 3 NF+(NH 4 ) 2 SO 4 NF+KNO 3 NF+ KClO 3 Presence of ACC deaminase a 1 MC-2 + + ++ +++ + 2 MC-18-7 +++++ +++++ +++++ +++++ +++++ + 3 MC-25 + + ++ +++ + ND 4 MC-31 + ++ ++ ++ + ND 5 MC-47 + ++ ++ ++ + ND 6 MC-49 + + ++ ++ + ND 7 MC-51 + ++ ++ ++ + ND 8 MC-59 +++++ +++++ +++++ +++++ +++++ ND 9 MC-408 + +++++ +++++++ ++++++ + ND 10 W-5 +++++ +++++ ++++++ ++++++ +++++ + NRCPB,(New Delhi) Anonymous (2005) a- presence of ACC (1-aminocyclopropane-1-carboxylic acid)examined by PCR using acdS gene sequences as primers.

Highlights of running project on BNF in INDIA:

50 Highlights of running project on BNF in INDIA Research on Institutes Developing of transgenic Rhizobium strain for pulse crops Division of Microbiology, IARI, New Delhi. Improving Fertilizer use efficiency and BNF BARC, Trombay. Construction of transgenic Rhizobium for Groundnut and Pigeon Pea by cloning and expressing of genomic region of iron siderophore receptor. M. S. University, Baroda. Developing of tools for molecular typing of native and transgenic Cynobacteria and Rhizobium strains NEHU, Shilong .

THE HOST PLANT (Macrosymbiont) GENOMICS:

51 THE HOST PLANT (Macrosymbiont) GENOMICS To date, about 45 genes that control nodulation & nitrogen fixation have been studied in eight legume species, but only one i.e. Nin (initiation of nodule) has been isolated. (Stougaard 2000) Two major type of nodulin genes have been defined by their pattern of expression and function 1) Early nodulins 2) Late nodulins

Early nodulins:

52 Nodulin expressed before beginning of N 2 fixation are called “ early nodulins” ( Enod ) These genes mainly responsible for nodule morphogenesis i.e. cortical cell division, meristem establishment, etc Some of them are induced by Nod factor (Enod12) While others are activated at later stage (Enod2) Early nodulins Genes expressed just before or during N 2 fixation are called “ late nodulin” Genes are involved mainly in different metabolic activities necessary for functioning of nodules Late nodulins include :- leghaemoglobin (Lb), glutamine synthates (GS), uricase, sucrose synthase etc. 2. Late nodulins

Table 18 : Nodulation response of Nod- chickpea genotypes and their F1, F2, and backcross progenies to rhizobium inoculation.:

53 Table 18 : Nodulation response of Nod - chickpea genotypes and their F 1 , F 2 , and backcross progenies to rhizobium inoculation. Generation Parent or cross Plants Expected ratio  2 <P< Nod + Nod - Annigeri NN  P319-1 NN Parent (P 1 ) Annigeri NN 0 107 All Nod - Parent (P 2 ) P319-1 NN 0 117 All Nod - F 1 P 1  P 2 0 24 All Nod - F 2 P 1  P 2 0 467 All Nod - BC 1 F 1  P 2 0 92 All Nod - BC 1 F 1  P 2 0 97 All Nod - Annigeri NN  Rabat NN Parent (P 1 ) Annigeri NN 0 107 Parent (P 2 ) Rabat NN 0 86 F 1 P 1  P 2 31 0 All Nod + F 2 P 1  P 2 262 224 9 : 7 1.082 0.30-0.25 BC 1 F 1  P 2 53 47 1 : 1 0.360 0.70-0.50 BC 1 F 1  P 2 46 53 1 : 1 0.495 050-0.40 Continue….

Continue….:

54 Continue…. Generation Parent or cross Plants Expected ratio  2 <P< Nod + Nod - Rabat NN  P319-1 Parent (P 1 ) Rabat NN 0 86 Parent (P 2 ) PM233 0 46 F 1 P 1  P 2 27 0 All Nod + F 2 P 1  P 2 274 205 9 : 7 0.177 0.30-0.25 BC 1 F 1  P 2 55 44 1 : 1 1.222 0.70-0.50 BC 1 F 1  P 2 54 45 1 : 1 0.495 0.50-0.40 Rabat NN  PM233 Parent (P 1 ) Rabat NN 0 86 Parent (P 2 ) PM233 0 46 F 1 P 1  P 2 29 0 All Nod + F 2 P 1  P 2 244 194 9 : 7 0.052 0.90-0.80 BC 1 F 1  P 2 45 50 1 : 1 0.263 0.70-0.50 BC 1 F 1  P 2 51 34 1 : 1 3.400 0.10-0.05 ICRISAT,(Patancheru, A.P) Singh and Rupela (1998)

Biotechnological Approaches to Study Nodulin Genes in Legumes :

55 Biotechnological Approaches to Study Nodulin Genes in Legumes Identification of plant genes necessary for root nodule formation and function Genome mapping. Identification of DNA markers linked to nodulation genes RAPD (Random Amplified Polymorphic DNA) RFLP (Restriction Fragment Length Polymorphism) AFLP (Amplified Fragment Length Polymorphism) SSR (Simple Sequence Repeat) Genome sequencing. Medicago truncatula - 155,000 sequenced BAC clones Lotus japonicus - 162 Mb sequenced

METHODS TO IDENTIFY NODULIN GENES:

56 METHODS TO IDENTIFY NODULIN GENES Differential display Differential hybridization Subtractive hybridization Expressed sequence tags (ESTs) Insertional Mutagenesis Promoter Trapping cDNA array Real time PCR

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57 Expressed sequence tags (ESTs) Sequence obtained from the 5 ’ or 3 ’ ends of isolated cDNA clones 218,000 ESTs from Soybean 137,000 ESTs from Medicago truncatula 032,000 ESTs from Lotus japonicus cDNA array An array of 2,304 cDNA clones derived from Lotus japonicus was produced. Expression of 36 genes was detected only in nodules but not in roots.(Leghemoglobin, Enod 16, early nodulin 12A etc.) Transcript of 83 genes were abundant in nodules.(ammonia assimilation, asparagine synthesis etc ) More than 50 genes have never been identified as nodule induced in any species. (transport, hormone metabolism, and signal transduction etc) Colebatch et.al.,( 2002)

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58 Germany, Colebatch et.al.,( 2002) Scatter plots of gene activity ( relative transcript level) in L japonicus nodules and roots.

Identification of Nodule-Specific Transcripts in the Model Legume Medicago truncatula1 :

59 Identification of Nodule-Specific Transcripts in the Model Legume Medicago truncatula 1 Maryland Fedorova et.al., (2002)

Structure of the NIN protein and a working model for the role in early phases of root nodule initiation. :

60 Structure of the NIN protein and a working model for the role in early phases of root nodule initiation. Denmark Stougaard Jens (2002)

LIN (lumpy infection ) gene in Medicago truncatula maintain Rhizobial infection and differentiation of nodule from nodule primordia :

61 LIN (lumpy infection ) gene in Medicago truncatula maintain Rhizobial infection and differentiation of nodule from nodule primordia Symbiotic phenotype of Wild type and lin mutant roots California Kuppusamy et.al., (2004)

Table 19 : Comparison of nodulation phenotype between A17and lin in medicago truncatula:

62 Table 19 : Comparison of nodulation phenotype between A17and lin in medicago truncatula Phenotype A17* Lin* No of infection per root at 3 dpi 116 ± 20.0 25.0 ± 6.5 No of primordia at 18 dpi 0.0 ± 0.0 18.0 ± 2.0 No of nodules at 18 dpi 8.0 ± 2.0 0.0 ± 0.0 Nitrogenase activity at 18dpi (C 2 H 4 nmol/plant/h) 8.00 ± 2.00 0.01 ± 0.00 Parental lines F 2 progeny analysed No of plants observed Nod + Nod -  2 value P values lin × A17 122 99 23 2.459 >0.100 lin × A20 125 93 0.024 >0.25 >0.25 California Kuppusamy et al., (2004) * values represent mean ± SD Table 20 : Genetic analysis of the M.truncatula nodulation lin

Genetic mapping of the Nod- trait in the tetraploid alfalfa :

63 Genetic mapping of the Nod - trait in the tetraploid alfalfa Hungary Kalo et al., (2002)

Limitations to Study Legume Plants Role in Symbiosis:

64 Limitations to Study Legume Plants Role in Symbiosis Important grain and forage legumes large genome size and polyploidy These plants are difficult to transform and regenerate.

Conclusion :

65 Conclusion Rhizobium -Legume symbiosis is the result of interaction between both symbionts. Biotechnological tools speed up the process of identification of different genes involved in the nodulation and nitrogen fixation from Rhizobium as well as legumes Genetic modification in the Rhizobium leads to improve the efficiency of N 2 fixation Little is known about the plant genes involved in nodulation.

Future thrusts :

66 Future thrusts To develop GM Rhizobium strains with wide range host-specificity and high compatibility. To produce genetically engineered nitrogenase which is insensitive to O 2 Transfer of Nif gene from prokaryotes to green plants and to develop techniques to maintain nitrogenase in active form in plant and retain their expression Development of staple food crops i.e. Wheat and Rice cultivars as genetically improved nitrogen fixing plants capable of fixing their own nitrogen from atmosphere “Legume plants acquired the ability to form symbiotic nitrogen fixing nodules by recruiting genes that have common functions in all plants.”

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67 Thank you

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