DIRECTED PROTEIN EVOLUTION OF AGRICULTURAL TRAIT IN VITRO

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DIRECTED PROTEIN EVOLUTION OF AGRICULTURALLY IMPORTANT TRAITS IN VITRO:

1 DIRECTED PROTEIN EVOLUTION OF AGRICULTURALLY IMPORTANT TRAITS IN VITRO

CONTENT:

2 CONTENT INTRODUCTION STRATEGIES OF PROTEIN EVOLUTION IN VITRO METHODS OF PROTEIN EVOLUTION IN VITRO APPLICATIONS OF DIRECTED EVOLUTION AGRICULTURALLY IMPORTANT TRAITS CONCLUSION FUTURE THRUST

INTRODUCTION:

3 INTRODUCTION Describe various techniques for generation of protein mutants and selection of desirable functions. Campbell et al . (1973) and Hall et al . (1973 & 1974) systematically documented events of directed evolution of proteins. Campbell et al . (1973) evolved EbgA protein as a Beta galactosidase from Escherichia coli that sufficient to replace the lac Z gene function under controlled mutagenesis and selection. Jenson (1976) studied ability to recruit new protein functions. Hall, (1981, 2002 & 2003) developed EbgA enzyme variants with newly acquired function contain only one to three mutations. He also predicted evolutionary pathway in TEM-1 beta-lactamase six amino acid substitution. Emerged as a powerful technology in protein engineering. Chemical, pharmaceutical and agricultural sciences. Gradual accumulations of beneficial mutation and recombination.

STRATEGIES OF PROTEIN EVOLUTION IN VITRO:

4 STRATEGIES OF PROTEIN EVOLUTION IN VITRO Libraries of variants are searched experimentally for clones possessing the desired properties. Rational design modify proteins based on an understanding of structural and mechanistic consequences of a particular change or set of changes. DNA mutagenesis and recombination Rational design to facilitate development of proteins with new and improved properties .

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5 METHODS OF PROTEIN EVOLUTION IN VITRO

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6 DNA Shuffling Whole Genome Shuffling Heteroduplex Random Chimeragenesis on Transient Templates (RACHITT) Assembly of Designed Oligonucleotides (ADO) Mutagenic and Unidirectional Reassembly (MURA) Exon Shuffling Y-Ligation Based Block Shuffling (YLBS) Non- Homologous Recombination (ITCHY) Combining Rational Design with Directed Evolution

Table : 1. SELECTED METHODOLOGIES FOR DIRECTED EVOLUTION:

7 Table : 1. SELECTED METHODOLOGIES FOR DIRECTED EVOLUTION Method for Mutagenesis & Recombination Target Genes Selection Reference(s) Single gene DNA shuffling Beta Lactamase Cefotaxime degradation Stemmer (1994) Gene family shuffling Cephalosporinase Moxalactam degradation Crameri et al . (1998) Staggered extention process Subtilisin E Protease thermostability Zhao et al . (1998) In vitro-in vivo DNA recombination GFP Restore flurescence Volkov et al . (1999) Error- prone PCR Geranyl diphosphate synthase Lycopene production Wang et al . (2000) Combinatorial libraries enhanced by recombination in yeast Cytochrome P450 Aromatic hydrocarbon oxidation Abecassis et al . (2000) Compartmentalized self replication DNA polymerase Thermostability, heparin resistance Ghadessy et al . (2001) Random chimeragenesis on transient templates Monooxygenase Reaction rate ,altered substrate specificity Coco et al . (2001) Incremental truncation for the creation of hybrid enzymes N terminus of E.coli PurnN and C terminus of human hGART PurnN-Hgart functional hybrid enzymes Lutz et al . (2001) ; Ostermeier et al .(1999) Sequence homology-independent protein recombination Cytochrome P450 Deethylation of 7-ethoxyresorufin Sieber et al . (2001) Microbiol. Mol. Biol. Rev.69(3): 373 - 392 Yuan et al . (2005)

Table : 1 CONTD.:

8 Table : 1 CONTD. Sequence independent site directed chimeragenesis Beta Lactamases TEM-1and PSE-4 Functional hybrid enzymes Hiraga et al .(2003) Nonhomologous random recombination Chorismate mutase and fumarase Chrismate mutase activity Bittker et al .(2004) Synthetic shuffling Subtilisin Protease thermostability at pH 10 Ness et al .(2002) Mutagenic and unidirectional reassembly Phospholipase A1 Altered substrate specificity Song et al .(2002) Y-ligation-based block shuffling GFP Fluorescence Kitamura et al .(2002) Assembly of designed oligonucleotide Lipases Enantioselectivity Zha et al . (2003) Microbiol. Mol. Biol. Rev.69(3): 373 - 392 Yuan et al . (2005)

DNA SHUFFLING:

9 DNA SHUFFLING Stemmer et al . (1994) developed DNA shuffling technique to generate improved and efficient protein through repeated mutation and recombination. Library diversity created through mutagenesis and recombination * Libraries generated by random point mutagenesis (error prone PCR) or site directed mutagenesis of a starting sequence. * Libraries are screened and best variants selected for additional mutagenesis. * DNA shuffling overcomes limitations of point- mutation based approaches for protein improvements. ♦ Allowing directed recombination of beneficial mutation from multiple genes.

DNA SHUFFLING (contd.):

10 DNA SHUFFLING (contd.) Relationship between library diversity, library size and assay capability dictates evolution of phenotypes requiring larger steps (not take care in the Stemmer’s technique) through sequence space employ a more efficient search strategy. To overcome this problem, Crameri et al . (1998) suggested family shuffling method. * Naturally occurring homogenous genes as the source of starting diversity. * Allows block exchanges of sequences that are typically >60% identical.

Figure : 1. Phylogenetic tree of the four cephalosporinase genes:

11 Figure : 1. Phylogenetic tree of the four cephalosporinase genes Nature, 391, 288 – 291, California (USA) Crameri et al . (1998) Numbers on the vertical bars indicate the percentage of DNA sequence similarity.

Figure : 2. a, Comparison of single sequence shuffling versus sequence family shuffling. b, Sequence of chimaeric mutant A obtained by family shuffling. :

12 Figure : 2. a, Comparison of single sequence shuffling versus sequence family shuffling. b, Sequence of chimaeric mutant A obtained by family shuffling. Nature 391, 288 - 291 California (USA) Crameri et al . (1998)

Figure : 3.Computer model of evolved mutant A obtained by a single cycle of family shuffling.    :

13 Figure : 3.Computer model of evolved mutant A obtained by a single cycle of family shuffling.    Nature 391, 288 - 291 California (USA) Crameri et al . (1998)

Figure : 4. Searching sequence space by family shuffling versus by single sequence shuffling. :

14 Figure : 4. Searching sequence space by family shuffling versus by single sequence shuffling. Nature 391, 288 - 291 California (USA) Crameri et al . (1998)

DNA SHUFFLING (contd.):

15 DNA SHUFFLING (contd.) Much larger number of mutations are tolerated in a given sequence without introducing deleterious effects on the structure or function. Obtained increased sequence diversity of the chimeric libraries resulting in sparse sampling of much greater regions of sequence and function space. Ness et al . (2002) recommended synthetic shuffling to get greater control over the incorporation of sequence diversity * No physical starting genes required * A series of degenerated oligonucleotides that incorporate all desired diversity use to assemble a library of full length gene * Every amino acid from of a set of parents is allowed to recombine independently of every other amino acid. * Breaking the linkages between amino acids normally present in parental genes. * Access unique regions of sequence space.

Figure : 5. Results of sequencing genes from 10 randomly-selected unscreened clones from DNA shuffled library. :

16 Figure : 5. Results of sequencing genes from 10 randomly-selected unscreened clones from DNA shuffled library. Nucleic Acids Res. 25 :1307-1308 (USA) Zhao et al .(1997)

Table : 2 . Frequency of active clones obtained after DNA shuffling under different conditions a Clones exhibiting >10% wild-type subtilisin E activity. :

17 Table : 2 . Frequency of active clones obtained after DNA shuffling under different conditions a Clones exhibiting >10% wild-type subtilisin E activity. Nucleic Acids Res. 25 :1307-1308 (USA) Zhao et al .(1997)

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18 WHOLE GENOME SHUFFLING

Figure: 6 . Asexual versus sexual evolution in bacteria (Streptomyces fradiae ) :

19 Figure: 6 . Asexual versus sexual evolution in bacteria ( Streptomyces fradiae ) Nature 415, 644-646 (USA) Zhang et al . (2002)

Figure : 7 . Genome shuffling versus classical strain improvement in Streptomyces fradiae :

20 Figure : 7 . Genome shuffling versus classical strain improvement in Streptomyces fradiae Nature 415, 644-646 (USA) Zhang et al . (2002)

Figure : 8. Principle of the library construction. :

21 Figure : 8. Principle of the library construction. Nucleic Acids Research,Vol. 28, No. 20 e88 (France) Valérie et al . (2000)

Figure : 9. Construction of enhanced crossover SCRATCHY library. :

22 Figure : 9. Construction of enhanced crossover SCRATCHY library. Nucleic Acids Research, Vol. 31, No. 21 e126(USA) Kawarasaki et al . (2003)

Mutational Bias Affects Protein Evolution in Flowering Plants :

23 Mutational Bias Affects Protein Evolution in Flowering Plants Wang et al.(2004) reported that those rice genes that display increased divergence in their nucleotide composition (specifically, increased G+C content) showed a corresponding, predictable change in the amino acid compositions of the encoded proteins relative to their Arabidopsis homologs. This trend was not seen in a "control" set of rice genes that had nucleotide contents closer to their Arabidopsis homologs. Investigated the biased patterns of amino acid substitution since the divergence of these two species. Results concluded that the amino acid exchange matrix was highly asymmetric when comparing the High G+C rice genes with their Arabidopsis homologs.

Mutational Bias Affects Protein Evolution in Flowering Plants (contd.):

24 Mutational Bias Affects Protein Evolution in Flowering Plants (contd.) Very strong negative correlation between the level of nucleotide bias and the length of the coding sequences within the rice genome. Mutational bias can be a major determinant of the patterns of protein evolution in eukaryotes. The rice genome does not, however, have a uniformly elevated G+C content among its coding sequences. The result of this heterogeneity in the nucleotide content among the coding sequences is reflected in the very different amino acid compositions among the encoded proteins.

Table : 3.AVERAGE NUCLEOTIDE CONTENTS OF HOMOLOGOUS GENES IN RICE AND Arabidopsis:

25 Table : 3.AVERAGE NUCLEOTIDE CONTENTS OF HOMOLOGOUS GENES IN RICE AND Arabidopsis Codon Position(%) First Second Third Average All homologous pairs (n= 4,447) Rice 58.1 44.7 66.4 56.4 Arabidopsis 51.5 41.0 43.0 45.5 High G+C Genes a (n= 1,000) Rice 65.4 51.6 91.8 69.6 Arabidopsis 51.7 43.5 46.9 47.3 Low G+C Genes a (n= 1,000) Rice 51.6 39.1 43.3 44.7 Arabidopsis 50.8 38.9 41.0 43.5 Mol. Biol. Evol. 21(1):90-96. 2004 (Canada) Wang et al .(2004)

Table : 4. EXON-INTRON STRUCTURE OF RICE GENES AND THEIR Arabidopsis HOMOLOGS:

26 Table : 4. EXON-INTRON STRUCTURE OF RICE GENES AND THEIR Arabidopsis HOMOLOGS Number of Exons 1 2 3 4 High G+C genes a (n =1000 ) Rice 434 294 165 107 Arabidopsis 308 224 182 286 Low G+C genes a (n =1000 ) Rice 260 72 165 503 Arabidopsis 159 89 57 695 Mol. Biol. Evol. 21(1):90-96. 2004 (Canada) Wang et al .(2004)

Figure: 10 .Distribution of G+C contents among rice and Arabidopsis genes. Homologous gene pairs only were used for this analysis. This data set included 8,894 genes—4,447 rice genes and 4,447 homologs from Arabidopsis :

27 Figure: 10 .Distribution of G+C contents among rice and Arabidopsis genes. Homologous gene pairs only were used for this analysis. This data set included 8,894 genes—4,447 rice genes and 4,447 homologs from Arabidopsis Mol. Biol. Evol . 21(1):90-96. 2004 (Canada) Wang et al .(2004)

Figure : 11. Amino acid content of homologous rice and Arabidopsis protein sequences. :

28 Figure : 11. Amino acid content of homologous rice and Arabidopsis protein sequences. Mol. Biol. Evol. 21(1):90-96. 2004 (Canada) Wang et al .(2004) The content of G, A, R, and P and F, Y, M, I, N, and K amino acids (expressed as percentages) for high G+C rice genes and their Arabidopsis homologs (1,000 genes each). These data are based on variant sites only in the aligned homologous sequences Proportions of individual amino acids at variant sites (expressed as numbers per 10,000 variant sites) plotted for the high G+C rice genes and their homologs from Arabidopsis .

AGRICULTURALLY IMPORTANT TRAITS:

29 AGRICULTURALLY IMPORTANT TRAITS Improvement of crop yields through * resistance to insect pests and diseases * tolerance to environmental stresses ( cold and drought) * post-harvest characteristics ( ripening control and prevention of potato sweetening However, genes have not provided sufficient efficacy to produce commercially viable genetically modified products. Laboratory directed evolution of protein technology may overcome the difficulties to improve effectively existing traits * Glyphosate resistance and Bacillus thuringiensis toxin expression in the crops * In addition to this, it can be applied to develop desirable gene functions from gene targets that have low or no activity.

GLYPHOSATE TOLERANCE (Herbicide):

30 GLYPHOSATE TOLERANCE (Herbicide) Existing herbicide resistance traits based on expression of a microbial enopyruvylshikimate-3 phosphate synthase(EPSPS) in corn, cotton and soybean . He et al . (2001) bred E.coli and Salmonella enterica serovar t yphimurium EPSPS to develop variants with superior properties. Several gene variants from a single round of DNA shuffling resulted in enzymes simultaneously improved over the best parent in * a two fold improve specific activity * a five fold improved K m for phosphoenolpyruvate * a five fold decrease in sensitivity to glyphosate Castle et al . ( 2004) discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM -1 min -1 to 8320 mM -1 min -1 . From the fifth iteration and beyond GAT enzymes conferred increasing glyphosate tolerance to E . coli , Arabidopsis , tobacco and maize.

Figure : 11. Catalyzing Reaction of Herbicide Round-Up ( glyphosate ) works by inhibiting 5-enolpyruvylshikimate 3-phosphate synthase, (EPSPS).:

31 Figure : 11. Catalyzing Reaction of Herbicide Round-Up ( glyphosate ) works by inhibiting 5-enolpyruvylshikimate 3-phosphate synthase, (EPSPS).

Figure : 12. Steps taken to bring glyphosate N-acetyltransferase trait from discovery to proof of concept in the field... :

32 Figure : 12. Steps taken to bring glyphosate N-acetyltransferase trait from discovery to proof of concept in the field... Microbial colonies from a diversity collection screening plate Model of glyphosate acetylation reaction carried out by GAT DNA shuffling process * Colored bars represent different starting genes for shuffling. In each iteration, genes are fragmented and reassembled. * Recovered genes contain recombined portions in the same linear order from each of the starting genes. * After screening for desired activity, best molecules are identified. * Entire process is repeated using these selected progeny as parents for the next shuffling iteration Maize plants expressing gat are tolerant to glyphosate spray under standard field conditions ISB News Report ,USA. Castle et al (2004)

Figure : 13. Sequence variation affects enzyme properties ( Glyphosate N – acetyltransferase ):

33 Figure : 13. Sequence variation affects enzyme properties ( Glyphosate N – acetyltransferase ) PNAS ,Vol. 102 : 25 , 8887-8892 (U.S.A.) Keenan  et al .(2005) SDS/PAGE comparing the expression and solubility of randomly selected, shuffled GAT variants with those of the three WT enzymes. Pair wise amino acid identity expressed as the number of differences for 11 enzymes that were selected for crystallization trials. Side-by-side comparison of the behavior of the three WT and eight variant enzymes in the HT crystal screen condition yielding the best unoptimized crystals for variant 5.

Figure : 14 . Crystallographic analysis of Glyphosate N - acetyltransferase:

34 Figure : 14 . Crystallographic analysis of Glyphosate N - acetyltransferase PNAS ,Vol. 102 : 25 , 8887-8892 (U.S.A.) Keenan  et al .(2005) Crystals of selenomethionine-containing variant 5 GAT after optimization diffract beyond 1.6-Å resolution. Ribbon representation of GAT bound to oxidized CoA (gray), colored from the N terminus (blue) to C terminus (red)..

Figure: 15. Generalization of the crystallization strategy (glyphosate N-acetyltransferase):

35 Figure: 15. Generalization of the crystallization strategy (glyphosate N- acetyltransferase) PNAS ,Vol. 102 : 25 , 8887-8892 (USA) Keenan  et al .(2005)

Bacillus thuringiensis toxin ( Insect pest Tolerance):

36 Bacillus thuringiensis toxin ( Insect pest Tolerance) Genes govern B. thuringiensis toxin expressing in the plants – second most widely grown transgenic crops ( corn and cotton). Limitation: * Spectrum of insects controlled by any given by B. thuringiensis Cry protein is relatively narrow. * Bt. Cry proteins with broadened specificity have the potential to further reduce the use of synthetic pesticides is commercial agriculture. * Relatively difficult to express Bt. Cry proteins in transgenic plants at sufficiently high levels to control many insect pests. Lassner et al . (2002) successfully used directed evolution to address both of these limitations . Concluded that Bt. Cry proteins exhibiting increased specific activity against current insect targets could reduce the effort required to generate a commercially useful level of insect resistance.

Figure: 16 . Relatedness Dendogram of Bt crystal proteins on the basis of alignments of the full-length protein sequences.:

37 Figure: 16 . Relatedness Dendogram of Bt crystal proteins on the basis of alignments of the full-length protein sequences. Madan Babu & Geetha(2005) Chennai (India)

Figure: 17. Highly conserved regions of the amino acid sequence for the cry proteins among different subspecies ( The genes are Cry1B, Cry1A, Cry1D and Cry4B in order, all proteins weigh approximately 130kd and are composed of approximately 1170 amino acids):

38 Figure: 17. Highly conserved regions of the amino acid sequence for the cry proteins among different subspecies ( The genes are Cry1B, Cry1A, Cry1D and Cry4B in order, all proteins weigh approximately 130kd and are composed of approximately 1170 amino acids) Chennai ( India ) Madan Babu & Geetha(2005)

Figure: 18 . Searching sequence space by family shuffling versus by single sequence shuffling :

39 Figure: 18 . Searching sequence space by family shuffling versus by single sequence shuffling Chennai ( India ) Madan Babu & Geetha(2005)

Figure : 19. DNA shuffling methodology :

40 Figure : 19. DNA shuffling methodology Chennai ( India ) Madan Babu & Geetha(2005)

GOLDEN RICE:

41 GOLDEN RICE Beyer et al .(2002) define a rice variety developed to express elevated levels of Beta-carotene (precursor of vitamin A) in the grain. Enhanced nutritional qualities to save the lives of millions of people. Nestle (2001) who developed genetically engineered Golden rice enough amount of vitamin-A precursor reported. Directed evolution could be of great benefit in an improvement of the metabolic pathway engineered into golden rice requires the coordinated expression of multiple transgenes. Potential to significantly boost the amount of Beta-carotene in next generation of golden rice varieties.

Figure : 20. Provitamin A biosynthetic pathway :

42 Figure : 20. Provitamin A biosynthetic pathway J.Nutr. 132:506S-510S (Germany & Switzerland ) Beyer et al .(2002) CrtI denotes a bacterial carotene desaturase capable in performing all necessary desaturation reactions for which two enzymes are required in plants. Arrows indicate the prenyllipid biosynthetic capacity of wild-type rice endosperm and the necessary reaction sequence to be completed to yield provitamin A.

Figure :21. DNA constructs used in single transformations and cotransformations. RB, right borders; LB, left borders; tp, transit peptide from pea ribulose bis-phosphate carboxylase; !, terminator; p, promoter; gt, glutelin; psy, phytoene synthase; crtI, bacterial carotene desaturase; lcy, lycopene ß-cyclase. DNA sequence coding for carotenoid biosynthetic enzymes are given in black. :

43 Figure :21. DNA constructs used in single transformations and cotransformations. RB, right borders; LB, left borders; tp , transit peptide from pea ribulose bis-phosphate carboxylase; !, terminator; p, promoter; gt, glutelin; psy , phytoene synthase; crtI , bacterial carotene desaturase; lcy , lycopene ß-cyclase. DNA sequence coding for carotenoid biosynthetic enzymes are given in black. J.Nutr. 132:506S-510S (Germany & Switzerland ) Beyer et al .(2002)

Figure : 22. Northern and Western blot analysis of wild-type (-) and of trans-lycopene-accumulating, CPTA-treated petals (+). :

44 Figure : 22. Northern and Western blot analysis of wild-type (-) and of trans -lycopene-accumulating, CPTA-treated petals (+). J.Nutr. 132:506S-510S (Germany & Switzerland) Beyer et al .(2002)

Figure :23. Examples of a new yellow rice line obtained using PMI as a selectable marker gene. :

45 Figure :23. Examples of a new yellow rice line obtained using PMI as a selectable marker gene. J.Nutr. 132:506S-510S (Germany & Switzerland ) Beyer et al .(2002)

NEXT-GENERATION TRAITS:

46 NEXT-GENERATION TRAITS Traits for which promising results were seen in the laboratories but which did not translate into commercially viable products * Chitinase for antifungal properties * Mycotoxin detoxification * Viral vectors

CHITINASE FOR ANTIFUNGAL PROPERTIES:

47 CHITINASE FOR ANTIFUNGAL PROPERTIES Jach et al .(1995) reported enhanced quantitative resistance against fungal diseases by combinational expression of different barley antifungal proteins in transgenic tobacco. No commercial crop plant products based on expression of chitinase. Through the application of directed evolution * Increase the activity of antifungal chitinase expressed in transgenic crop plants. * Potential of controlling fungal diseases.

MYCOTOXIN DETOXIFICATION:

48 MYCOTOXIN DETOXIFICATION Mycotoxin is a toxic side effect of fungal infection of crop plants. Blackwell et al . (1999) suggested transgenic approach to reducing fumonisin( Fusarium moniliforme produces mycotoxins that contaminate maize produce). Starting enzymes had no activity in extra cellular space where they were required to work. Five rounds of DNA shuffling and screening performed using surrogate hosts. Significant improvement generated in enzyme activity at the low pH environment and efficiency of protein secretion. Functional assays showed significant improvement of plant fumonisin detoxification.

VIRAL VECTORS:

49 VIRAL VECTORS Directed evolution reported by using random mutagenesis coupled with recombination to improve the performance of their vectors in plant. Mutagenized tobacco mosaic virus variants were subjected to gene shuffling and screened for faster movement around the plant as well as transgene expression. Toth et al . (2002) recorded variants that moved significantly faster throughout the plant.

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50 Figure:24. .  Schematic representation of 30B.GFPe genome organization with enlargement of the shuffled region showing position of nucleotide substitutions found in clones R1 (1), R2 (2) and R3 (3).MP, movement protein; GFP, green fluorescent protein; CP, coat protein. The Plant Journal 30 (5), 593-600. (USA) Toth et al .(2002)

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51 Figure :25. Comparison of fluorescent infection foci by half-leaf inoculations. N. tabacum cv. Xanthi nn were passaged with the progenitor, GFP-expressing vector (lower half-leaf) or a variant identified in the primary screen of the first round shuffled population (upper half-leaf). The Plant Journal 30 (5), 593-600(USA) Toth et al .(2002)

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52 Figure : 26. Systemic infections of N. tabacum cv. MD609 plants 10 days post-inoculation.Plants were inoculated with reassembled virus and maintained at 33°C prior to observation of green fluorescence under UV illumination. The Plant Journal 30 (5), 593-600(USA) Toth et al .(2002) R0 R1 R2 R3

Table : 5. FLUORESCENT INFECTION FOCI AREA MEASUREMENTS ON INOCULATED LEAVES OF N. tabacum cv.MD609 AND N. benthamiana :

53 Table : 5. FLUORESCENT INFECTION FOCI AREA MEASUREMENTS ON INOCULATED LEAVES OF N. tabacum cv.MD609 AND N. benthamiana Inoculum Mean Area of Fluorescent Infection Foci (mm2) N.tabacum cv. MD 609 N. banthamiana R0 0.219 0.172 R1 0.309 0.554 R2 0.816 0.660 R3 0.967 0.913 The Plant Journal , 30: (5), 593-600(USA) Toth et al .(2002)

Table : 6. TIMING OF SYSTEMIC GREEN FLUORESCENCE DEVELOPMENT(Inoculated with reassembled virus for each construct and maintained at 28oC ):

54 Table : 6. TIMING OF SYSTEMIC GREEN FLUORESCENCE DEVELOPMENT(Inoculated with reassembled virus for each construct and maintained at 28 o C ) Inoculum Days post-inoculation to systemic fluorescence N.tabacum cv. MD 609 N. banthamiana R0 > 19 5 R1 6-7 4 R2 5-6 4 R3 5-6 4 The Plant Journal 30 (5), 593-600(USA) Toth et al .(2002)

Table : 7. EFFECT OF INDIVIDUAL R 3 MUTATIONS ON CELL TO CELL MOVEMENT ON INOCULATED LEAVES OF N.tabacum cv. MD 609 . LSD value at 5% level – 0.3655:

55 Table : 7. EFFECT OF INDIVIDUAL R 3 MUTATIONS ON CELL TO CELL MOVEMENT ON INOCULATED LEAVES OF N.tabacum cv. MD 609 . LSD value at 5% level – 0.3655 Clone Mean area of fluorescent infection foci (mm 2) at 3 days post inoculation R 0 0.499 R 2 2.715 R 3 3.347 30B.GFP.L 72 V 2.093 30B.GFP.N 10 0.919 30B.GFP.T 104 1.642 30B.GFP.G184 0.305 30B.GFP.R 3SIL 1.399 The Plant Journal 30 (5), 593-600(USA) Toth et al .(2002)

Table : 8. EFFECT OF BASE CONTENT OF SUBSTITUTED CODONS ON CELL TO CELL MOVEMENT ON INOCULATED LEAVES OF N.tabacum cv.MD 609 LSD value at 5% level – 0.191 . Nucleotide differences from the wild type codons ( L72= TTG, T104= ACT) are underlined:

56 Table : 8. EFFECT OF BASE CONTENT OF SUBSTITUTED CODONS ON CELL TO CELL MOVEMENT ON INOCULATED LEAVES OF N.tabacum cv.MD 609 LSD value at 5% level – 0.191 . Nucleotide differences from the wild type codons ( L72= TTG, T104= ACT) are underlined Clone / codon variant Mean area of fluorescent infection foci (mm 2) at 3 days post inoculation R 3 1.417 V 72 VARIANT 1 ( G TG) 0.875 V 72 VARIANT 2 ( G T T ) 1.140 V 72 VARIANT 3 ( G T C ) 1.081 I 104 VARIANT 1(A T T) 1.000 I 104 VARIANT 2(A TA ) 1.271 I 104 VARIANT 3(A TC ) 1.309 The Plant Journal 30 (5), 593-600(USA) Toth et al .(2002)

CONCLUSION:

57 CONCLUSION A system that compares and utilizes the genetic information generated ( Predicting evolution). Mechanism to expand the genetic diversity in our search for novel functions. Generate a quality library and perform effective screening to find the desired properties. A process where progressive partial change built upon previous partial changes. Techniques to accelerate the improvement by performing multiple rounds of evolution. Mutants with small but measurable degrees of enhancement are identified by a limited number of assays. These mutants use as parents for the next round of evolution.

FUTURE THRUST:

58 FUTURE THRUST Intensive laboratory directed evolution of protein research is to be needed in agriculturally important traits. New proteins should be eco-friendly (Crop improvement). Nutritionally value addition basic research may be given more weightage. High skill, technical knowledge and well established laboratory to operate such finely sophisticated technology need to be developed.

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59 “Struggle for existence and survival of the fittest”

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60 THANK YOU

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