RNA Silencing of Lactate Dehydrogenase Gene in Rhizopus Oryzae

RNA Silencing of Lactate Dehydrogenase Gene in Rhizopus oryzae

Neda Haghayegh Jahromi Ali Hashemi Gheinani

This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science with a Major in Resource Recovery – Industrial Biotechnology, 120 ECTS credits No. 9/2010

Neda Haghayegh Jahromi, [email protected] Ali Hashemi Gheinani, [email protected]

Master thesis

Subject Category: Technology

University College of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640

Examiner: Dr.Ilona Sarvari Horvath

Supervisor, name: Dr.Elisabeth Feuk-Lagerstedt

Supervisor, address: University of Borås ,School of Engineering

SE-501 90 BORÅS

Date: 10.12.03

Keywords: RNA interference, Knockdown, Rhizopus oryzae, siRNA, Lactate dehydrogenase, Lactic acid, Ethanol

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Abstract

RNA silencing with direct delivery of siRNA has been used to suppress ldhA gene expression in filamentous fungus Rhizopus oryzae. Here, for the first time we show that, introducing small interfering RNA which consequently forms silencing complexes can alter the gene expression and we report a significant reduction of lactic acid production for isolates containing short (25 nt) synthetic siRNA. In all samples lactic acid production was reduced comparing with wild types. The average concentration of lactic acid production by Rhizopus oryzae during batch fermentation process where glucose has been used as a sole carbon source, diminished from 2.06 g/l in wild types to 0.36 g/l in knockdown samples which signify 5.7 times decrease. Interestingly, the average concentration of ethanol production was increased from 0.38 g/l in wild types to 0.45 g/l in knockdown samples. In some samples we were able to report even a 10 fold decrease in lactic acid production. Since R.oryzae is capable to assimilate a wide range of carbohydrates hydrolysed from lignocellulosic material in order to produce many economically valuable bulk material such as ethanol, these results suggest that RNA silencing is a useful method for industrial biotechnology to be applied in fungus Rhizopus oryzae in order to trigger the metabolism and gene expression toward a desired product.

Keywords: RNA interference, Knockdown, Rhizopus oryzae, siRNA, Lactate dehydrogenase, Lactic acid, Ethanol

List of Figures

FIGURE 1.RHIZOPUS ORYZAE ...... 1 FIGURE 2. A SIMPLIFIED PATHWAY FROM CARBON SOURCE TO PRODUCTS ...... 2 FIGURE 3.VARIOUS GROWTH FORMS OF FUNGI. (A) ASEPTATE HYPHA OF MUCORMUCEDO (ZYGOMYCOTA).THE HYPHA BRANCHES TO FORM A MYCELIUM. (B) SEPTATE BRANCHED HYPHA OF TRICHODERMA VIRIDE (ASCOMYCOTA). SEPTA ARE INDICATED BY ARROWS. (C) YEAST CELLS OF SCHIZOSACCHAROMYCES POMBE (ASCOMYCOTA) DIVIDING BY BINARY FISSION. (D) YEAST CELLS OF DIOSZEGIA TAKASHIMAE (BASIDIOMYCOTA) DIVIDING BY BUDDING. (E) PSEUDOHYPHA OF CANDIDA PARAPSILOSIS (ASCOMYCOTA), WHICH IS REGARDED AS AN INTERMEDIATE STAGE BETWEEN YEAST CELLS AND TRUE HYPHAE. (F) THALLUS OF RHIZOPHLYCTIS ROSEA (CHYTRIDIOMYCOTA) FROM WHICH A SYSTEM OF BRANCHING RHIZOIDS EXTENDS INTO THE SUBSTRATE. (G) PLASMODIA OF PLASMODIOPHORA BRASSICAE (PLASMODIOPHOROMYCOTA) INSIDE CABBAGE ROOT CELLS. SCALE BAR ¼ 20 MM (A,B,F,G) OR 10 MM (C_E).(WEBSTER AND WEBER, 2007) ...... 6 FIGURE 4.R.ORYZAE ON AGAR ...... 6 FIGURE 5.R.ORYZAE HYPHAE ...... 6 FIGURE 6.R.ORYZAE MYCELIA ...... 6 FIGURE 7.THE HIERARCHY OF BIOLOGICAL CLASSIFICATION'S EIGHT MAJOR TAXONOMIC RANKS FOR R.ORYZAE (INTERMEDIATE MINOR RANKINGS ARE NOT SHOWN.) ...... 7 FIGURE 8.A REPRESENTATION OF THE UNIVERSAL PHYLOGENETIC TREE, BASED ON COMPARISONS OF THE GENES ENCODING SMALL SUBUNIT (16S OR 18S) RIBOSOMAL RNA. THE LENGTHS OF THE LINES LINKING ORGANISMS TO THEIR NEAREST BRANCH POINT REPRESENT INFERRED EVOLUTIONARY DISTANCES (RRNA GENE SEQUENCE DIVERGENCE)(DEACON, 2006)...... 7 FIGURE 9.NJ TREE OF RHIZOPUS STRAINS INFERRED FROM 622 NUCLEOTIDES FROM LSU RDNA D1 AND D2 REGION. NUMBERS GIVEN ON BRANCHES INDICATE THE CONFIDENCE LEVEL FROM 1K REPLICATES BS SAMPLING [44] ...... 8 FIGURE 10.MICROGRAPH SHOWING MUCORALES(KENDRICK, 2000) ...... 9 FIGURE 11.MATURE SPORANGIUM OF MUCOR SP. FUNGUS(KENDRICK, 2000) ...... 9 FIGURE 12.SCHEMATIC DIAGRAM OF RHIZOPUS SP. (KENDRICK, 2000) ...... 10 FIGURE 13.DIAGRAMMATIC REPRESENTATION OF A FUNGAL HYPHA(DEACON, 2006) ...... 11 FIGURE 14.R.ORYZAE AFTER 48 HOURS BATCH FERMENTATION ...... 12 FIGURE 15.R.ORYZAE AFTER 48 HOURS BATCH FERMENTATION ...... 12 FIGURE 16.DIAGRAM OF A HYPHAL MODULAR UNIT(MAHESHWARI, 2005) ...... 12 FIGURE 17.ERGOSTEROL(HANSON, 2008) ...... 13 FIGURE 18.N-ACETYLGLUCOSAMINE(HANSON, 2008) ...... 14 FIGURE 19.TRANSMISSION ELECTRON MICROSCOPY OF ULTRATHIN SECTIONS OF FUNGAL CELL SHOWING DIFFERENT LAYERS OF CELL WALL AND PLASMA MEMBRANE (MICHAEL J. CARLILE, 2001)...... 15 FIGURE 20.STRUCTURAL FORMULAE OF THE PRINCIPAL POLYMERS IN R.ORYZAE ...... 15 FIGURE 21.FLUORESCENCE MICROSCOPIC IMAGES (100×) OF RHODAMINE B BY (A) R. ORYZAE MYCELIA, (B) INTACT CELL WALL, (C) CHITIN, (S) CHITOSAN, (E) PHOSPHOMANNAN AND (F) GLUCAN(DAS ET AL., 2008) ...... 17 FIGURE 22.THE CYTOSKELETON IN FUNGI(WEBSTER AND WEBER, 2007) ...... 19 FIGURE 23.THE ROLE OF CONVENTIONAL KINA AND KIP2 FAMILY A COMPARISON OF A WILD TYPE WITH A CONVENTIONAL KINESIN DELETION MUTANT (TAKEN FROM REQUENA ET AL. 2001). B SCHEME OF AMTWITH THEMTPLUS END COMPLEX. THIS PROTEIN COMPLEX CONSISTS OF SEVERAL PROTEINS, E.G. KIPA OR LIS1, CONVENTIONAL KINESIN TRANSPORTS VESICLES AND COMPONENTS OF THE PLUS END COMPLEX, FOR INSTANCE DYNEIN (ZHANG ET AL. 2003). A DIRECT INTERACTION BETWEEN KINA AND DYNEIN OR DYNACTIN HAS NOT YET BEEN VERIFIED. MODIFIED AFTER HESTERMANN ET AL. (2002) C, D WHEN KIPA, WHICH IS SUGGESTED TO BE INVOLVED IN THE DELIVERY OF CELL END MARKERS, IS MISSING, HYPHAE LOSE DIRECTIONALITY. IMAGES TAKEN FROM (FISCHER, 2007)...... 19 FIGURE 24.THE ROLE OF CLAMP CONNECTIONS IN MAINTAINING A REGULAR DIKARYON(DEACON, 2006)...... 21 FIGURE 25.ELECTRON MICROGRAPHS OF YEAST NUCLEUS SHOWING NUCLEOLAR SUBCOMPARTMENTALIZATION. A–B—FROM S. CEREVISIAE SHOWING FC, GC, AND DFC (A) AND RDNA LOCALIZATION ONLY IN THE FC (B); C–D—THE NUCLEOLUS OF S. POMBE SHOWING NDB FROM GAR-1 MUTANT CELLS, THE TRUNCATED GAR2P IS ACCUMULATED IN THE DB (C). THE BAR ¼ 1MM REPRODUCED FROM CHROMOSOMA 1997, 105:542–552 AND CHROMOSOMA 1999, 108:103–113, BY COPYRIGHT PERMISSION OF SPRINGER VERLAG ...... 21 FIGURE 26.IMAGES OF GFP-TUBA (A TUBULIN) IN A. NIDULANS SHOWING CYTOPLASMIC MICROTUBULES (A) SPINDLE MICROTUBULES (B) AND ASTRAL MICROTUBULES EMANATED FROM THE POLES OF AN ELONGATING SPINDLE (B, C)(XIANG AND FISCHER, 2004)...... 23

FIGURE 27.RIBBON-LIKE AGGREGATES OF CHITIN MICROFIBRILS (R) PRODUCED IN VITRO FROM CHITOSOMES (C) ISOLATED FROM MUCOR ROUXII, WHEN INCUBATED WITH SUBSTRATE (N-ACETYLGLUCOSAMINE) AND A PROTEOLYTIC ACTIVATOR. (COURTESY OF C.E. BRACKER; FROM BARTNICKI-GARCIA ET AL(DEACON, 2006)...... 25 FIGURE 28.TWO POSSIBLE PATHWAYS TO CONVERT C:G TO A:T (IRELAN AND SELKER, 1996) ...... 28 FIGURE 29.SCHEMATIC ILLUSTRATION OF RIP AND MIP(IRELAN AND SELKER, 1996).A:BOTH COPIES OF DUPLICATION ARE RETAINED THROUGH THE SEXUAL CYCLE. MOST OFTEN BOTH ARE FOUND TO HAVE SUFFERED FROM RIP OR MIP (FILLED BOXES).B: DUPLICATION HAPPED BUT RIP AND MIP WERE NOT HAPPENED. C: DUPLICATED SEQUENCE IS DELETED. D: FIRST RIP HAPPED AND THEN DUPLICATION WAS DELETED...... 29 FIGURE 30.FLOWER PHENOTYPE OF THE CHS SENSE AND DFR SENSE TRANSFORMANTS (VAN DER KROL ET AL., 1990)...... 33 FIGURE 31.THE PROPOSED MECHANISM FOR ANTISENSE RNA BY NELLEN, W. AND C. LICHTENSTEIN IN 1993(NELLEN AND LICHTENSTEIN, 1993A) ...... 33 FIGURE 32. PETUNIAS CONTAINING WILD TYPE (SEPARATED PICTURE IN THE LEFT ) AND 4 TRANSGENE COPIES(VAN DER KROL ET AL., 1990) ...... 34 FIGURE 33.PETUNIAS CONTAINING WILD TYPE (SEPARATED PICTURE IN THE LEFT ) AND 4 TRANSGENE COPIES(NAPOLI ET AL., 1990) ...... 34 FIGURE 34.INTERFERENCE CONTRAST MICROGRAPHS SHOW IN SITU HYBRIDIZATION IN EMBRYOS OF C.ELEGANS. A, NEGATIVE CONTROL SHOWING LACK OF STAINING IN THE ABSENCE OF THE HYBRIDIZATION PROBE. B, EMBRYO FROM UNINJECTED PARENT (SHOWING NORMAL PATTERN OF ENDOGENOUS MEX-3 RNA). C, EMBRYO FROM A PARENT INJECTED WITH PURIFIED MEX-3B ANTISENSE RNA. THESE EMBRYOS (AND THE PARENT ANIMALS) RETAIN THE MEX-3 MRNA, ALTHOUGH LEVELS MAY BE SOMEWHAT LESS THAN WILD TYPE. D, EMBRYO FROM A PARENT INJECTED WITH DSRNA CORRESPONDING TO MEX-3B; NO MEX-3 RNA IS DETECTED(FIRE ET AL., 1998) ...... 35 FIGURE 35.HIGHLY SIMPLIFIED SCHEMATIC MECHANISM OF RNA INTERFERENCE ...... 37 FIGURE 36.RNAI STRATEGIES: (A) CONVENTIONAL HPRNAI, (B) PSILENT1 (HETEROGENEOUS NUCLEAR RNA EXPRESSING VECTOR SYSTEM), (C) PSILENT-DUAL1 SYSTEM (OPPOSING DUAL PROMOTER SYSTEM (BHADAURIA ET AL., 2009)...... 38 FIGURE 37.CRYSTAL STRUCTURE OF GIARDIA DICER. (A) FRONT AND SIDE VIEW RIBBON SYMBOLIZE OF DICER SHOWING THE N-TERMINAL PLATFORM DOMAIN (BLUE), THE PAZ DOMAIN (ORANGE), THE CONNECTOR HELIX (RED), THE RNASE IIIA DOMAIN (YELLOW), THE RNASE IIIBDOMAIN (GREEN) AND THE RNASE- BRIDGING DOMAIN (GRAY). DISORDERED LOOPS ARE DRAWN AS DOTTED LINES. (B) CLOSE-UP VIEW OF THE DICER CATALYTIC SITES; CONSERVED ACIDIC RESIDUES (STICKS); ERBIUM METAL IONS (PURPLE); AND ERBIUM ANOMALOUS DIFFERENCE ELECTRON DENSITY MAP, CONTOURED AT 20S (BLUE WIRE MESH) (MACRAE ET AL., 2006)...... 39 FIGURE 38.DICER RNASE III DOMAINS (MACRAE ET AL., 2006) ...... 39 FIGURE 39.A MODEL FOR DSRNA PROCESSING BY DICER SHOWING GIARDIA DICER WITH MODELED DSRNA (MACRAE ET AL., 2006)...... 39 FIGURE 40.DICER AND RISC (RNA-INDUCED SILENCING COMPLEX ...... 41 FIGURE 41.ROLES OF THE ARGONAUTE COMPLEX IN MIRNA AND RNAI PATHWAYS(HUTVAGNER AND SIMARD, 2008) ...... 41 © FIGURE 42.STRUCTURAL FEATURES OF ARGONAUTE PROTEINS(HUTVAGNER AND SIMARD, 2008) TAKEN FROM (2004) AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE...... 42 FIGURE 43.POTATO DEXTROSE AGAR SLANT CULTURE OF R.ORYZAE USED FOR THIS WORK ...... 46 FIGURE 44.7.1.2 CULTURING R.ORYZAE ON AGAR PLATE ...... 47 FIGURE 45.SUBCULTURING OF R.ORYZAE IN PDA PLATES ...... 47 FIGURE 47.PDA CULTURED PLATES OF R. ORYZAE IN INCUBATOR (IN 30 °C) ...... 48 FIGURE 46.R.ORYZAE AFTER 4 DAYS INCUBATION IN 30 °C ...... 48 FIGURE 48.PLOT OF LDHA VS. LDHB ...... 50 FIGURE 49.THE PELLET OF LABELED SIRNA ...... 53 FIGURE 50.R.ORYZAE CELLS UNDER THE MICROSCOPE ...... 54 FIGURE 51.HARVESTED MYCELIA USED FOR PROTOPLAST PREPARATION ...... 55 FIGURE 52.TRANSFORMED R.ORYZAE ...... 55 FIGURE 53.PLIERS, CORK BORER AND OTHER EQUIPMENT NEEDED TO SET A FERMENTOR ...... 57 FIGURE 54.ASSEMBLED CAP ...... 57 FIGURE 55.SAMPLING HOSE WITH NEEDLE ...... 57 FIGURE 56.A GLASS FERMENTATION LOCK ...... 57 FIGURE 57.A SET OF CORK BORER ...... 57 FIGURE 58.COMPLETLY ASSEMBLED FERMENTORS ...... 57 vi

FIGURE 59.INCUBATION OF R.ORYZAE IN SUBMERGED BATCH CULTURE ...... 58 FIGURE 60.HPLC ALLIANCE/WATERS 2695 ...... 59 FIGURE 61.BRIGHT FIELD OF R.ORYZAE AFTER 7 H IN THE BATCH CULTURE WITH 10X MAGNIFICATION ...... 61 FIGURE 62.BRIGHT FIELD OF R.ORYZAE AFTER 9 H IN THE BATCH CULTURE WITH 10X MAGNIFICATION ...... 61 FIGURE 63.BRIGHT FIELD OF R.ORYZAE AFTER 10 H IN THE BATCH CULTURE WITH 10X MAGNIFICATION ...... 61 FIGURE 64.PRODUCTION OF MAJOR FERMENTATION PRODUCTS BY R.ORYZAE. ENZYMATIC ACTIVATION SHOWN AS: A LACTATE DEHYDROGENASE; B PYRUVATE DECARBOXYLASE; C ALCOHOL DEHYDROGENASE; D PYRUVATE CARBOXYLASE; E MALATE DEHYDROGENASE; F FUMARASE ...... 67

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List of Tables

TABLE 1.DETECTED METABOLITES DURING CULTIVATION OF R.ORYZAE IN SHAKE FLASK(TAHERZADEH ET AL., 2003) ...... 1 TABLE 2.RNA SILENCING EFFORTS IN SOME FUNGI ...... 4 TABLE 3. MAIN CHARACTERISTICS OF FUNGI (WEBSTER AND WEBER, 2007) ...... 5 TABLE 4.THE CELL WALLS COMPOSITION OF SELECTED STRAINS OF FUNGI (PERCENT OF TOTAL DW CELL WALL) ADAPTED FROM (GRIFFIN, 1994) , (RUIZ-HERRERA, 1992) AND (WEBSTER AND WEBER, 2007) ...... 15 TABLE 5.SUGAR CONTENTS IN ENZYMIC HYDROLYSATES OF RHIZOPUS CELL WALL (TOMINAGA AND TSUJISAKA, 1981) ...... 16 TABLE 6.AMOUNT OF CHITOSAN PRODUCED BY DIFFERENT FUNGI(POCHANAVANICH AND SUNTORNSUK, 2002) ...... 17 TABLE 7.ADSORPTION CAPACITY OF RHODAMINE B BY DIFFERENT CELLULAR COMPONENTS OF R.ORYZAE ...... 17 TABLE 8.GENOME STATISTICS OF R.ORYZAE © 2010 BROAD INSTITUTE ...... 21 TABLE 9.SEQUENCES PRODUCING SIGNIFICANT ALIGNMENTS ...... 49 TABLE 10.SIRNA USED IN THIS WORK ...... 52 TABLE 11.TRANSFORMATION METHODS IN VARIOUS FUNGI ...... 52 TABLE 12.VARIATION IN SAMPLE PREPARATION ...... 56 TABLE 13.LACTIC ACID PRODUCTION IN R.ORYZAE ...... 62 TABLE 14.ETHANOL PRODUCTION IN R.ORYZAE ...... 63 TABLE 15.COMPARISION ON DIFFERENT SAMPLE PREPARATION WITH LACTIC ACID AND ETHANOL PRODUCTION ...... 68

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List of Charts

CHART 1.MYCELIAL GROWTH (■) AND CHITOSAN PRODUCTION (●) OF RHIZOPUS ORYZAE TISTR3189 (POCHANAVANICH AND SUNTORNSUK, 2002) ...... 16 CHART 2.DRY WEIGHT OF MYCELIA, ALKALINE INSOLUBLE MATERIALS AND EXTRACTABLE CHITOSAN OF RHIZOPUS ORYZAE USDB 0602 (TAN ET AL., 1996) ...... 16 CHART 3.LACTIC ACID PRODUCTION IN KNOCKDOWN AND WILD TYPES DETECTED BY UV IN HPLC ...... 62 CHART 4.ETHANOL PRODUCTION IN KNOCKDOWN AND WILD TYPES DETECTED BY RI IN HPLC ...... 63 CHART 5.GRAPHICAL REPRESENTATION OF GROWTH CURVE OF R.ORYZAE IN SEMI SYNTHETIC GROWTH MEDIA IN FERMENTOR #1 ACCORDING CHANGES OD OVER TIME ...... 64 CHART 6.GRAPHICAL REPRESENTATION OF GROWTH CURVE OF R.ORYZAE IN SEMI SYNTHETIC GROWTH MEDIA IN FERMENTOR #2 ACCORDING CHANGES OD OVER TIME ...... 64 CHART 7.STATISTICAL PLOTS FOR LACTIC ACID PRODUCTION IN SILENCED SAMPLES (X1) AND WILD TYPE SAMPLES (X2) TAKEN FROM R ...... 69 CHART 8.STATISTICAL PLOTS FOR ETHANOL PRODUCTION IN SILENCED SAMPLES (X3) AND WILD TYPE SAMPLES (X4) TAKEN FROM R . 71

DEDICATION

We would like to dedicate this thesis work to our loved parents for their love, encouragement and support specially to one who rest in peace

Acknowledgments

We are deeply indebted to Dr.Elisabeth Feuk-Lagerstedt for supervising the study and providing us a fantastic time during this project. Without her guidance, support and good nature, we would never have been able to pursue experiments. We never forget her patience and trust.

We would like to gratefully and sincerely thank Prof.Mohammad Taherzadeh whose idea builds up this project.

Special thanks to Patrik Lennartsson for supporting us in theoretical issues and his lessons in fermentations and analytical methods.

We are very grateful to Jonas Hanson for his great help in practical problems and providing us with materials and facilities during this work.

We also appreciate Dr.Peter Threning in engineering department of Borås University for supporting us in our experiments by providing access to lab during holidays, weekends and until late at the night.

Abreviations

BAC bacterial artificial chromosome NF B nuclear factor B nt nucleotide bDNA branched DNA OAS1 2 ,5 -oligoadenylate synthase CCT2 T-complex protein 1, -subunit ORF open reading frame cDNA complementary DNA PCR polymerase chain reaction COPAS Complex Object Parametric Analyzer and Sorter PEG polyethylene glycol cPPT central polypurine tract PKR protein kinase R Dicer intracellular endonuclease complex PMSG pregnant mare‘s serum gonadotropin DLBCL diffuse large B-cell lymphoma pri-miRNA primary miRNA dNTP Deoxyribonucleotide triphosphate PTGS post-transcriptional gene silencing DPC days postcoitum PVP polyvinylpyrrolidone dsRNA double-stranded RNA QD quantum dots DTT dithiothreitol qde quelling-deficient ELISA enzyme-linked immunosorbent assay qRT-PCR quantitative RT-PCR ER endoplasmic reticulum RDA rhodamine ES embryonic stem RdRP RNA-dependent RNA polymerase EXT extinction RISC RNA-induced silencing complex FACS fluorescence-activated cell sorting RISC* activated RISC FLU1 green fluorescence emission RNA ribonucleic acid FSH follicle-stimulating hormone RNA pol RNA polymerase FYCO1 FYVE and coiled coil containing protein 1 RNAi RNA interference GFP green fluorescent protein RNase ribonuclease hCG human chorionic gonadotropin RPA RNA protection assay HDAC4 histone deacetylase 4 RSK4 ribosomal S6 kinase HTA-TIP histone acetyl transferase TIP60 RT reverse transcriptase IL-8 interleukin-8 SAHS S-adenosyl-L-homocysteine hydrolase IRF-3 interferon regulatory factor-3 shRNA short hairpin RNA JAK-STAT Janus kinase–signal transducers and activators of siRNA small interfering RNA transcription LATS2 large tumor suppressor homologue 2 SV40 simian virus 40 LC-MS liquid chromatography-mass spectrometry TBDMS tertiary-butyldimethylsilyl LH luteinizing hormone TERT telomerase catalytic subunit LTR long terminal repeat TGCT testicular germ cell tumor LV lentivirus vector THN trihydroxynaphthalene reductase MALDI-TOF matrix-assisted laser desorption ionization–time of TNF- tumor necrosis factor- flight MBT mid-blastula transition TOF time of flight MCS multiple cloning site TOM triisopropylsiloxymethyl miRNA micro-RNA tsLT temperature-sensitive allele of SV40 large T antigen MMR Marc‘s Modified Ringer UPR unfolded protein response MPP membrane-permeant peptides UTR untranslated regions mRNA messenger RNA UV ultraviolet MSCV murine stem cell virus VSV vesicular stomatitis virus MSUD meiotic silencing by unpaired DNA

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Table of Contents Abstract ...... iv List of Figures ...... v List of Tables ...... viii List of Charts ...... ix DEDICATION ...... x Acknowledgments ...... xi Abreviations ...... xii 1. Chapter 1 - Introduction ...... 1 1.1 Research Purpose ...... 1 1.2 Literature Review ...... 3 2. Chapter 2 – Introduction to Fungus Rhizopus oryzae ...... 5 2.1 Taxonomy and Classification of R.oryzae ...... 7 2.2 Morphology ...... 11 2.2.1 The Plasma Membrane ...... 13 2.2.2 Cell Wall ...... 14 2.2.2.1. Covalently Linked Cell Wall Proteins ...... 18 2.2.2.2. Synthesis of the Cell Wall ...... 18 2.2.3 The Cytoskeleton ...... 18 2.2.4 The Nucleus ...... 20 2.2.4.1. The Nucleolus ...... 21 2.2.5 Mitochondria and Mitochondrial DNA ...... 24 2.2.6 Vacuoles ...... 25 2.2.7 Endoplasmic Reticulum ...... 26 2.2.8 Golgi apparatus ...... 26 2.2.9 Exocytosis/Secretion...... 26 3. Chapter 3 – RNA Interference a Gene Silencing Tool ...... 27 3.1 Background ...... 27 3.1.1 Gene Inactivation in DNA Sequence Level...... 27 3.1.2 Gene Inactivation in Transcriptional Level ...... 28 3.1.2.1. Repeat-Induced Point Mutations (RIP) ...... 28 3.1.2.1. MIP: Methylation Induced Premeiotically ...... 29 3.1.3 Gene Inactivation in Post-Transcriptional Level (PTGS) ...... 29 3.1.3.1. Antisense Oligodeoxynucleotide (ODN) ...... 30 3.1.3.1.1. Phosphorothioates (S-DNA) ...... 30 3.1.3.1.2. LNA ...... 30 3.1.3.1.3. Morpholino ...... 30 3.1.3.2. Ribozyme ...... 30 3.1.3.3. RNA Interference ...... 31 3.2 History and Discovery of RNA Silencing ...... 31 3.3 Mechanism ...... 37 3.3.1 Dicer ...... 38 3.3.2 RISC ...... 40 3.3.2.1. Argonaute ...... 41 3.4 Biological Function ...... 44 3.4.1 Immunity...... 44 3.4.2 Downregulation of Genes ...... 45 3.4.3 Upregulation of Genes ...... 45 xiii

3.4.4 Offtarget Effects ...... 45 4. Chapter 4 - Methods and Materials ...... 46 4.1 Fungal Strain and Growth Media Preparation ...... 46 4.1.1 Strain ...... 46 4.1.2 Culturing R. oryzae on Agar Plate (Subculturing) ...... 46 4.1.3 Preparation of Inoculum ...... 47 4.1.4 Culture Conditions ...... 48 4.2 Designing the siRNA ...... 48 4.2.1 Target Lactate Dehydrogenase mRNA ...... 48 4.2.2 Direct Comparison of Two Sequences ...... 49 4.2.2.1. Dot Matrix View ...... 50 4.2.2.2. Alignment ...... 50 4.2.3 siRNA Design ...... 51 4.3 Fungal Transformation ...... 52 4.3.1 Labeling siRNA ...... 53 4.3.2 Inducing siRNA in Rhizopus oryzae ...... 54 4.3.3 Protoplast Preparation ...... 54 4.3.4 Transformation of R.oryzae by the (CaCl2/ PEG) method ...... 55 4.4 Fermentation Test...... 56 4.4.1 Equipment: ...... 56 4.4.2 Preparation of Media ...... 58 4.4.3 Inoculation: ...... 58 4.4.4 The incubation ...... 58 4.5 Analytical method - HPLC ...... 59 4.6 Growth Curve ...... 60 5. Chapter 5 – Results ...... 62 5.1 Lactic acid Production...... 62 5.2 Ethanol Production ...... 63 5.3 Growth Curves ...... 63 6. Chapter 6 –Discussion ...... 65 6.1 Labelling of siRNA ...... 65 6.2 Defining Growth Pattern of R.oryzae...... 66 6.3 Reduction of Lactic Acid Production ...... 66 6.4 Increasing Ethanol Production ...... 67 6.5 Statistical Analysis of Lactic Acid Production Results...... 68 6.5.1 Commands: ...... 69 6.5.2 Graphs: ...... 69 6.5.3 Statistical test: ...... 70 6.6 Statistical Analysis of Ethanol Production Results ...... 70 6.6.1 Commands: ...... 70 6.6.2 Graphs: ...... 71 6.6.3 Statistical test: ...... 72 7. Chapter 7 – Conclusions ...... 73 7.1 Conclusions ...... 73 7.2 Limitations of the Study ...... 73 7.3 Further Works ...... 73 7.3.1 RT-PCR analysis ...... 73 7.3.2 RNA extraction and Northern blot analysis...... 73 7.3.3 Protein extraction and determination of LDH activity ...... 73 xiv

7.3.4 Construction of silencing vectors ...... 73 8. References ...... 74

Appendix A: Protocols

Appendix B: Information about ldhA gene from ncbi

Appendix C: Information about ldhB gene from ncbi

1. Chapter 1 - Introduction

1.1 Research Purpose Lignocellulosic biomass is regarded nowadays to be an economically eye-catching carbohydrate feedstock for industrial fermentation of chemicals. There are many microorganisms that can utilize lignocellulosic biomass and convert them to numerous valuable material but among them R. oryzae is a towering one. R. oryzae is a filamentous fungus (Figure 1) and is capable to uptake a wide range of sugars such as mannose, xylose, glucose and galactose (Edebo, 2000) as its substrate.

R. oryzae has been used as a host microorganism to Figure 1.Rhizopus oryzae produce mainly lactic acid (Skory, 2004a), but it can also produce a variety of other valuable materials, such as gallic acid (Misro et al., 1997), lipase (Salah et al., 1994), protease (Tunga et al., 1999) , cellulolytic enzymes (Amadioha, 1993) and ethanol (Sorahi Abedinifar, 2009). Also in some countries like Indonesia, China and Japan R. oryzae is consumed as a food (Buyukkieci, 2007). In case of ethanol production and specially when sustainable processes are intended to be applied R. oryzae is a good choice to be served as a host microorganism because of its tolerance to inhibitors in lignocellulose acid hydrolyzates(Karimi et al., 2006) , valuable material contents in its biomass (Taherzadeh et al., 2003) and its ability to grow at higher temperatures (rather than Baker‘s yeast ) which results in lower risks of contamination (Millati et al., 2005) .

When the aim of process is production of ethanol it is better to minimize other products (by-products) to increase the ethanol yield. In 2003 Taherzadeh and co-workers in their work showed (as it is depicted in Table 1) one of the by products which are produced in a notable amount along with ethanol is lactic acid (Taherzadeh et al., 2003). The hypothesis is if the gene involving in producing lactic acid is silenced a portion of the carbon source which had been supposed to convert to lactic acid will convert to ethanol and contribute to higher yield (see Figure 2).

Table 1.Detected metabolites during cultivation of R. oryzae in shake flask (Taherzadeh et al., 2003)

Metabolite Minimum yield (mg/g) Maximum yield (mg/g) Ethanol 200 374 Lactic acid 68 298 Glycerol 48 86 Pyruvic Acid 2.6 14 Succinic Acid 1.6 3.8 Acetic Acid 0.0 2.0

The main purpose of this project is to decrease the amount of lactic acid production by silencing the corresponding gene.

Thus, in this project the goal is prevention of lactic acid production in fermentation process by genetic engineering tools and investigation of lactic acid reduction effect on yield of ethanol production. To prevent the production of lactic acid it was decided to use an epigenetic method called RNA interference which alters post-transcriptional system of the fungus.

Lactic Acid Lactate dehydrogenase Glucose → Glucose-6-P→Fructose-6-P→ Pyruvate

Ethanol

Figure 2. A simplified pathway from carbon source to products

1.2 Literature Review

In industrial biotechnology field when researchers were supposed to have R. oryzae as a host microorganism the goal of the researchers have been production of lactic acid and a few of them aimed to produce ethanol (Yang et al., 2010, Thongchul et al., 2010). So it goes without saying that there should be a shortage in literatures talking about genetically triggering R.oryzae in the direction of ethanol production.

In the other hand ,most functional genomics applications of RNAi have been made on Caenorhabditis elegans, a nematode that is frequently used as a model organism in genetics research (Fire et al., 1998, Fraser et al., 2000) and on fruit fly (Drosophila melanogaster) (Elbashir et al., 2001c). In 2006, Andrew Fire and Craig C. Mello jointly were awarded the Nobel Prize for their work on RNAi on the nematode worm C. elegans, which they published in 1998 (Fire et al., 1998, Daneholt, 2010). Although RNA interference also have been used to silence many genes in fungi (see Table 2) but only few of them were tried on R.oryzae . Among those work in 2004 Skory put some efforts to silence the ldhA gene with translational fusion construct using the phosphoglycerate kinase promoter. Skory in his work tried several types of gene expression systems for Rhizopus to produce altered transcript that can form dsRNA of the lactate dehydrogenase gene, ldhA. But Skory was not able to achieve any significant reduction of lactic acid for isolates containing short (20-25 nt) synthetic ldhA RNAi in the expression plasmids. However, expression of a 430 nt inverted repeat of ldhA resulted in a 25% decrease in lactic acid production. The most decline of acid production was with a translational fusion construct using the phosphoglycerate kinase promoter where isolates had drastically lower LDH enzymatic activity and up to 95% less lactic acid. He found out that the products of fermentation are altered mainly to ethanol and fumaric acid, although growth was moderately unchanged (Skory, 2004b).

As it is depicted in Table 2 there are many evidences in using RNAi in fungi. The first evidences of silencing in fungi can be find in a work which Romano and Machino in 1992 transformed a wild-type strain of Neurospora crassa (a red bread mold of the phylum Ascomycota) with different pieces of the carotenogenic albino-3 (al-3) and albino-1 (al-1) genes and they observed that up to 36% of Neurospora crassa transformants showing an albino phenotype. It was done by random integration in ectopic locations resulting in a severe destruction in the expression of the endogenous al-1 or al-3 genes (Romano and Macino, 1992). Romano and Machino named this phenomenon, ‗quelling‘, they found it to be spontaneously and progressively reversible, leading to wild-type or intermediate phenotypes. Since then till know there have been many researches comprising application of RNA interference technique.

Table 2.RNA silencing efforts in some fungi

Item Microorganism Target Gene Year Authors Ref Aspergillus nidulans Khatri,M. et (Khatri and 1 ornithine decarboxylase (ODC) 1992 al. Rajam, 2007) Cladosporium fulvum Hamada, W. (Hamada and 2 hydrophobin gene HCf-1 1998 et. al Spanu, 1998) Phytophthora infestans van West, P., (van West et 3 inf1 1999 et al., al., 1999) Cryptococcus neoformans (Liu et al., 4 CAP59 and ADE2 2002 Liu, H., et al., 2002) Dictyostelium discoideum Martens, H., et (Martens et al., 5 RrpA, RrpB, and DosA 2002 al. 2002) Mucor circinelloides (Nicolas et al., 6 carotenogenic gene carB 2003 Nicolas et al. 2003) Magnaporthe oryzae Kadotani, N., (Kadotani et 7 eGFP 2003 et al al., 2003a) Rhizopus oryzae 8 2004 Skory (Skory, 2004b) lactate dehydrogenase gene (ldhA) Aspergillus oryzae Yamada, O., et (Yamada et al., 9 brlA gene 2004 al., 2007) Venturia inaequalis Fitzgerald, A (Fitzgerald et 10 GFP, trihydroxynaphthalene reductase (THN) 2004 et al. al., 2004) Histoplasma capsulatum Rappleye, (Rappleye et 11 AGS1 (encoding alpha-(1,3)-glucan synthase 2004 C.A., et al. al., 2004) Fusarium verticillioides McDonald, T., (McDonald et 12 Alfatoxin regulatory gene aflR 2005 et al al., 2005) Aspergillus parasiticus McDonald, T., (McDonald et 13 Alfatoxin regulatory gene aflR 2005 et al al., 2005) Aspergillus flavus McDonald, T., (McDonald et 14 Alfatoxin regulatory gene aflR 2005 et al al., 2005) Coprinus cinereus Namekawa, (Namekawa et 15 LIM15/DMC1 2005 S.H., et al., al., 2005) Neotyphodium uncinatum Spiering, M.J., (Spiering et 16 loline-alkaloid production(LOL-1 and LOL-2) 2005 et al., al., 2005) Mortierella alpina Takeno, S., et (Takeno et al., 17 Δ12-desaturase gene 2005 al. 2005) Phytophthora infestans Latijnhouwers, (Latijnhouwers 18 Pigpa1 2005 M., et al et al., 2004) Schizophyllum commune de Jong, J.F., (de Jong et al., 19 SC15 2006 et al 2006) Ophiostoma floccosum Tanguay, P., et (Tanguay et 20 polyketide synthase (PKS1) gene 2006 al. al., 2006) Ophiostoma piceae Tanguay, P., et (Tanguay et 21 polyketide synthase (PKS1) gene 2006 al. al., 2006) Aspergillus fumigatus Mouyna, I., et (Mouyna et al., 22 ALB1/PKSP and FKS1 2007 al., 2004) Neurospora crassa Romano,N. et. (Romano and 23 carotenogenic albino-3 (al-3) and albino-1 (al-1) genes 2007 al Macino, 1992) Aspergillus niger (Barnes et al., 24 uidA gene, encoding beta-glucuronidase (GUS) 2008 Barnes, S. 2008) Phanerochaete chrysosporium Matityahu, A., (Matityahu et 25 manganese-containing superoxide dismutase gene (MnSOD1) 2008 et al al., 2008)

2. Chapter 2 – Introduction to Fungus Rhizopus oryzae

In terms of biodiversity, it is believed that at least 1.5 million different species of fungi is existing , but no more than about 75,000 species (5% of the total) have been described up to now (Deacon, 2006) and R. oryzae is one of those species. Fungi have conquered an amazingly wide range of habitats in the course of evolution, carrying out important roles in diverse ecosystems (Dix and Webster, 1995). The conquest of new environment, they produce numerous small spores (see Figure 4).

Table 3. Main Characteristics of fungi (Webster and Weber, 2007)

Characteristic Description

Nourishment Mostly Heterotrophic, rather than ingestion their feeding is by absorption.

Life cycle Their life cycle can be simple or, more typically, complex.

Cell wall Typically they have cell wall, usually comprising glucans and chitin, in some cases glucans and cellulose (Oomycota).some of them have chitosane too.

Nuclear status Eukaryotic, uni- or multinucleate, heterokaryotic, haploid, dikaryotic or diploid

Habitat in terrestrial and freshwater habitats they are ubiquitous

Reproduction. sexual , parasexual , asexual

Propagules Microscopic spores.

Sporocarps Microscopic, macroscopic

Ecology saprotrophs, mutualistic symbionts, parasites, or hyperparasites.

Distribution. Cosmopolitan

Vegetative on or in the substratum, as a non-motile mycelium of hyphae

Figure 3.Various growth forms of fungi. (a) Aseptate hypha of Mucormucedo (Zygomycota). The hypha branches to form a mycelium. (b) Septate branched hypha of Trichoderma viride (Ascomycota). Septa are indicated by arrows. (c) Yeast cells of Schizosaccharomyces pombe (Ascomycota) dividing by binary fission. (d) Yeast cells of Dioszegia takashimae (Basidiomycota) dividing by budding. (e) Pseudohypha of Candida parapsilosis (Ascomycota), which is regarded as an intermediate stage between yeast cells and true hyphae. (f) Thallus of Rhizophlyctis rosea (Chytridiomycota) from which a system of branching rhizoids extends into the substrate. (g) Plasmodia of Plasmodiophora brassicae (Plasmodiophoromycota) inside cabbage root cells. Scale bar ¼ 20 mm (a,b,f,g) or 10 mm (c_e).(Webster and Weber, 2007)

Figure 4.R. oryzae on Agar Figure 5.R. oryzae Hyphae Figure 6.R. oryzae Mycelia

2.1 Taxonomy and Classification of R.oryzae

Taxonomic thought on the fungi is in such a state of flux that it is difficult to Kingdom: Fungi find a solid classification for fungi .But Phylum: Zygomycota for R. oryzae the situation is not that Class: Zygomycetes bad and most of the authors believe in a Subclass: Incertae sedis same classification. Order: Mucorales The most agreed scientific classification Family: Mucoraceae declares that species Rhizopus oryzae is among eukaryotes in universal Genus: Rhizopus phylogenetic tree (Figure 8) and is Species: R. oryzae classified under the genus Rhizopus, family Mucoraceae in the order Figure 7.The hierarchy of biological classification's eight major taxonomic ranks for R. oryzae (intermediate minor Mucorales of the phylum Zygomycota. rankings are not shown.)

In 1985 a method based on Electrophoretic patterns of sporangiospore proteins were chosen as taxonomic characters for a range of isolates of the genus Rhizopus. Outcomes, showed as dendrograms obtained after cluster analyses of simple matching coefficients, question the currently accepted classifications, and give support for a reduction in the number of species (Seviour et al., 1985). In 2005 Oda Yuji and Sone Teruo proposed a new classification for R. oryzae on the basis of their taxonomical properties that have been found through studies on their ability to produce lactic acid(ODA YUJI, 2005). Later in 2007 Abe et al. divided R. oryzae strains into two groups, LA (lactic acid producer) and FMA (fumaric-malic acid producers). They carried out an rDNA ITS analysis and revealed that lactate dehydrogenase B, actin, translation elongation factor-1α and genome-wide AFLP resolved the same two exclusive clusters so they proposed reclassification of strains in the FMA group as R. Delemar. So based on their findings the groupings were confirmed as phylogenetically distinct, corresponding with the organic acid grouping (Abe et al., 2007).

Figure 8.A representation of the Universal Phylogenetic Tree, based on comparisons of the genes encoding small subunit (16S or 18S) ribosomal RNA. The lengths of the lines linking organisms to their nearest branch point represent inferred evolutionary distances (rRNA gene sequence divergence)(Deacon, 2006). Also Liou et al. in 2007 using a polyphasic approach showed that the R. oryzae group are found to include species of the genus Amylomyces. Phylogenetic analysis of Rhizopus strains was done based 7

on the D1/D2 region of LSU rDNA sequences yielded a phylogram (see Figure 9) with four well- supported clades ((Liou et al., 2007)).

Figure 9. NJ tree of Rhizopus strains inferred from 622 nucleotides from LSU rDNA D1 and D2 region. Numbers given on branches indicate the confidence level from 1K replicates BS sampling [44]

Phylum Zygomycota:

The phylum Zygomycota consists of two classes, the Zygomycetes and the Trichomycetes. Within these two classes the sexual process consists of the fusion of two gametangia to give a resting spore, the zygospore (Michael J. Carlile, 2001) . Since R. oryzae in classified under phylum Zygomycota the five major features serve to characterize the phylum Zygomycota is reviewed: I. The cell wall is composed of a combination of chitosan , chitin and polyglucuronic acid II. Hyphae that classically is deficient in cross walls, therefore all the nuclei are contained within a shared cytoplasm (a coenocytic mycelium); III. Thick-walled resting spore – the zygospore – that if produced by a sexual process IV. The production of asexual spores by cytoplasmic cleavage within a sporangium; V. A haploid genome. Zygomycota as pathogens of humans Many common members of the Mucorales, particularly Absidia corymbifera, Rhizopus arrhizus, and Rhizomucor pusillus (a thermophilic species) can cause serious, life-threatening infections of humans. Collectively, these diseases are termed zygomycosis (infections that are caused by fungi members of the Zygomycota). Rhizopus oryzae is also the greatest communal cause of zygomycosis that usually occurs in immunocompromised patients. Interactions between R. oryzae and vascular endothelial cells are likely of central importance to the organism's pathogenetic strategy. R. oryzae spores and germ tubes can adhere to human umbilical vein endothelial cells (HUVECs), while only spores adhere to subendothelial matrix proteins. (Ibrahim et al., 2005). Patients with zygomycosis in India, Pakistan, New Guinea, Taiwan, Central and South America , Africa, Iraq, Somalia, Egypt, Libya, Israel, Turkey, Spain, Italy, Hungary, Czechoslovakia, Germany, Ukraine, the British Isles, and the United States (Domsch et al., 1995) have been mostly attacked by Rhizopus oryzae (William E. Dismukes, 2003, Ribes et al., 2000) . Actually Rhizopus oryzae is a highly critical infection in immunocompromised hosts. The typical treatment for invasive zygomycosis comprises of reversal of the predisposing causes, common surgical debridement, and aggressive antifungal drug. Unfortunately all treatments the general mortality of zygomycosis is >50% , and it slants 100% in patients with disease (Ibrahim et al., 2005).

Order Mucorales Mucorales sometimes called "Pin molds, is best studied order among Zygomycete fungi. Order Mucorales is consisting of a number of species of the genera Mucor, Rhizopus, Phycomyces and Thermomucor. All of them grow principally as saprotrophs in soil, on animal dung, on composts or on a range of other substrates such as over-ripe fruits. They can grow fast, often covering an agar plate in 24–36 hours, and they are among the commonest fungi found on soil dilution plates(Deacon, 2006).

Figure 11.Mature sporangium of Mucor sp. Figure 10.Micrograph showing fungus(Kendrick, 2000) mucorales(Kendrick, 2000) 9

Genus Rhizopus

Rhizopus is a genus of that includes filamentous fungi types. Rhizopus species produce both asexual and sexual spores. Inside the sporangium, a pinhead-like structure, the asexual sporangiospores are produced (Figure 12) which is genetically identical to their parent. In Rhizopus, the sporangia are supported by a large apophysate columella, and the sporangiophores arise among rhizoids.

Some species are plant pathogens and many are used in food industry like Rhizopus oligosporus, which is used in the production of tempeh, a fermented food derived from soybeans and, oncom; Rhizopus oryzae used to produce pito , a Nigerian fermented beverage, produced from maize, sorghum or a mixture of both (Ekundayo, 1969).

The synonyms of R. oryzae are: R. tritici, R. thermosus, R. tamarii, R. suinus, R. peka, R. hangchow, R. formosaensis, R. formasaensis var. chylamydosporus, R. delemar, R. chiuniang, R. arrhizus, R. liquefaciens, R. javanicus Y. Takeda, R. Pseudochinensis.

Figure 12.Schematic diagram of Rhizopus sp. (Kendrick, 2000)

2.2 Morphology Fungal morphology is often regarded as important parameters in industrial biotechnology hence the desired morphology for the production of fungal metabolite products varies from one product to another. Morphology of fungi during the biotechnological process can be affected by many parameters like of agitation, specific growth rate, dissolved oxygen and number of spores or conidia per liter of fermentation broth. When higher yield is desired in the process those parameters have to be considered. And therefore it is worth to understand the mechanism underlying the morphology of the cell, its growth and product formation by filamentous fungi (Pazouki and Panda, 2000). Cellular growth and its morphology are affected by many factors which include medium shear , level and medium constituents, type of inoculum and the pH (Atkinson and Daoud, 1976). Nutrient consumption and oxygen uptake rate in submerged culture can be changed by changes in morphology (Schügerl et al.). Filamentous growth increases the viscosity of the medium, thereby requiring a higher power input to maintain adequate mixing and aeration.

Filaments fungi and among them R. oryzae are composed of microscopic, tube-like structures called hyphae (singular hypha). Rather than a few large propagules, they grow this system of branching tubes to achieve the food efficiently (see Figure 5). The hyphae together make up a mass called the mycelium (see Figure 3 and Figure 6); the mass of hyphae is sometimes called shiro.

Figure 13.Diagrammatic representation of a fungal hypha (Deacon, 2006)

Figure 13 is showing progressive aging and vacuolation behind the hyphal tip. In the oldest regions, the walls may break down by autolysis or the mycelial nutrients may accumulate in chlamydospores (thick-walled resting spores that serve in dormant survival). Aut = autolysis; AVC = apical vesicle cluster; Chlam = chlamydospore; ER = endoplasmic reticulum; G = Golgi/Golgi equivalent; Gl = glycogen; L = lipid; M = mitochondria; MT = microtubules; MW = melanized wall; N = nucleus; P = plasmalemma; R = ribosomes; S = septum; SP = septal plug; V = vacuole; W = wall; Wo = Woronin body.

In different taxonomic groups of fungi hyphae are pretty uniform. The presence or absence of cross- walls or septa is among those few features of distinction (see Figure 16). The Zygomycota and Oomycota normally have aseptate hyphae wherein the nuclei are in a cytoplasm. Such a condition is called coenocytic. When the nuclei are genetically alike the mycelium is said to be homokaryotic, but if a cell or mycelium hold nuclei of dissimilar genotype it is said to be heterokaryotic, for example because of anastomosis of hyphae which are genetically different.

Figure 14.R. oryzae after 48 hours batch fermentation Figure 15.R. oryzae after 48 hours batch fermentation

Figure 16.Diagram of a hyphal modular unit(Maheshwari, 2005)

Influence of engineering variables upon the morphology (van Suijdam and Metz, 1981):

I. No influence of pH upon morphology could be shown. While others observed the influence of pH. The difference between observations must probably be accounted for by the presence of precursors like phenylacetic acid in the growth medium used. II. No influence of oxygen tension upon morphology could be shown for oxygen tensions in the range of 12-300 mm Hg. 12

III. The hyphal growth unit was constant at varying growth rates. This means that the hyphal extension rate must be proportional to the growth rate for the strain used. IV. Effective hyphal length and total hyphal length increased with increasing growth rates, which was in agreement with the qualitative V. The influence of the shear stress upon the hyphal length was very limited VI. Changes in the tensile strength of the cell wall could have influenced the experimental results very well, both in the experiments to investigate the effect of growth rate as well as in those where the energy input was varied. A decrease of tensile strength with increasing growth rate and with increasing hyphal length could account for differences of the experimental data with the model where no fatigue is assumed to play a role in breakup of hyphae VII. The shear stresses in the fermentor, expressed as the energy dissipation per unit mass, influence the hyphal morphology

Measuring the Morphology

Characterization of mycelial morphology is important for design and operation of industrial production. Image analysis has been developed as a fast and accurate method for this measuring the morphology. Recently some fully automatic system has been developed, in which speed is gained, but with loss of accuracy in some cases. The method has been tested on Streptomyces clavuliegrus and Penicillium chrysogenum batch fermentation and a fed-batch Penicillium chrysogenum fermentation, in which the medium contains solid ingredients (Packer and Thomas, 1990, Treskatis et al., 1997).

Measuring Viability

In industrial biotechnology it is important to maintain a uniform inoculum for consistency of fungal fermentations, because once a mycelia structure has formed, it remains a discrete entity for long periods, extending and growing older (Paul et al., 1993). Swelling of spores and germination processes constitute a major part of the lag phase. The subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. In 2004 Paul and co- workers developed an image analysis method for assessing the viability and germination characteristics of fungal spores in submerged culture. Paul and co-workers ´s method has a number of advantages over photomicroscopy or colony counting. It is rapid, more accurate and consistent which can discriminate between non-germinated and just germinated spores. In particular, this can be used on spores germinating in actual submerged fermentation medium. Structural variation during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distribution of spore volume and of germ tube length and volume (Paul et al., 1993).

2.2.1 The Plasma Membrane All cells are bounded by a plasma membrane (plasmalemma). The fungal plasma membrane consists of a phospholipid bilayer with associated transmembrane proteins, many of which are involved directly or indirectly in nutrient uptake. One important difference between plasma membrane of fungi from plasma membrane of plants and animals is that ergosterol is the major sterol of the Figure 17.Ergosterol(Hanson, 2008)

plasma membranes of fungi, in contrast to cholesterol in membranes of animal and phytosterols plants (Michael J. Carlile, 2001, Kavanagh, 2005). In filamentous fungi, the cell membrane and wall may be intimately bound, as hyphae are often resistant to plasmolysis, so they lack the periplasm. The membrane also can anchor some enzymes. In fact, the main wall-synthetic enzyme, chitin synthase integral membrane proteins; they become anchored in the membrane in such a way that they produce polysaccharide chains from the outer membrane face. The plasma membrane has a third important role, in relaying signals from the external environment to the cell interior – the process termed signal transduction (Deacon, 2006). The plasma membrane of fungi, especially during the vegetative growth of saprophytic species, contains vast numbers of permeases and transporters to facilitate the uptake of macromolecules required to maintain what can often be prolific growth rates (Esser and Lemke, 1994).

2.2.2 Cell Wall

The fungal cell wall is an essential organelle that accounts for 15–30% of the cellular dry weight and therefore represents a substantial metabolic investment for the cell (Klis et al., 2007). Cell wall helps fungi to: I. Withstand turgor pressures varying from about half a megapascal in normal cells up to eight megapascals in specialized cells (Howard et al. 1991). II. Helps to maintain stable osmotic conditions inside the cell. III. It maintains cell shape. IV. Allows morphogenesis. V. Protects the cell against physical damage. VI. Adhesiveness (Ibrahim et al., 2005). VII. Protection against desiccation. The last two are related to the frequent presence of an external protein coat surrounding the skeletal part of the wall.

The chemistry of the fungal cell wall can be used as a taxonomic marker. The fungal cell wall is different from structural components both in bacterial cell wall and mammalian cell membranes. While among and inside different groups of fungi the chemical composition of cell walls is said to be different (Table 4), the basic design has many components in common. Figure 18.N- This wall comprises of a crosslinked structural scaffold of fibres, acetylglucosamine(Hanson, 2008) and a gel-like matrix or crystalline material. Cell wall in fungi is a complex of chitin [a polymer of N-acetylglucosamine] (see Figure 18), various mannoproteins together with a- and b-linked 1, 3-D-glucans. Ergosterol (Figure 17) is major sterol rather than cholesterol which is found in mammalian systems (Hanson, 2008).

In 1981 Yoshio Tominaga and Yoshio Tsujisaka used purified lytic enzymes, protease and chitosanase from Bacillus R-4 and chitinase II from Streptomyces orientalis to degrade the cell wall of R. oryzae. When these enzymes were used individually they could only partially lysed the cell wall, but when they allowed the cell wall to react with all of the enzymes, a complete lysis was achieved by cooperative action so they concluded that wall structure of R. oryzae might be composed mainly of

chitin fibers cemented by chitosan and protein or peptides scattered in a mosaic manner (TOMINAGA and TSUJISAKA, 1981).

As shown in Table 5, chitosanase alone or the mixed solution of protease and chitosanase were subjected to react on the Rhizopus cell wall, and approximately 60% to 70% of total hexosamine in the

Figure 19.Transmission electron microscopy of ultrathin sections of fungal cell showing different layers of cell wall and plasma membrane (Michael J. Carlile, 2001). cell wall were contained in their solubilizing fractions. Even in the fraction solubilized by protease, 16% of total hexosamine contained in the cell wall were found. The total sugar determined by the method of phenol-sulphuric acid was very small in all fractions.

Table 4.The cell walls composition of selected strains of fungi (percent of total DW cell wall) adapted from (Griffin, 1994) , (Ruiz-Herrera, 1992) and (Webster and Weber, 2007) Group Example Chitin Cellulose Glucans Protein Lipid Oomycota Phytophthora 0 25 65 4 2 Chytridiomycota Allomyces 58 0 16 10 ? Zygomycota Mucor 9 0 44 6 8 Saccharomyces 1 0 60 13 8 Ascomycota Fusarium 39 0 29 7 6 Schizophyllum 5 0 81 2 ? Basidiomycota Coprinus 33 0 50 10 ?

The main polysaccharide component in Rhizopus cell wall is chitosan and chitin. Furthermore, no glucose derived from glucan was detected in the acid hydrolysate of Rhizopus cell wall. Therefore, in the Rhizopus cell wall, chitosan probably plays an important role in maintaining the structure of the cell wall instead of glucan (TOMINAGA and TSUJISAKA, 1981). Yoshio Tominaga and Yoshio Tsujisaka also proposed an indistinct Layer structure in the Rhizopus cell wall. They stated that each protein, chitosan and chitin is partially exposed on the surface of the cell wall, (since were directly degraded by the lytic enzyme). The plasticity (extensibility) of the wall is determined Figure 20.Structural formulae of the principal polymers in R.oryzae 15

according to the degree of cross-linking, whilst permeability or the size of pore is the wall matrix property. The scaffold forms the inner layer of the wall and the matrix is found predominantly in the outer layer (Webster and Weber, 2007) . Table 5.Sugar Contents in Enzymic Hydrolysates of Rhizopus Cell Wall (TOMINAGA and TSUJISAKA, 1981)

Treated cell wall (mg/l0mg cell wall) Protease Untreated cell wall Protease Chitosanase +chitosan Sol. Res. Sol. Res. Sol. Res. Total sugar * 0.18 0.03 0.16 0.15 0.02 0.16 0.03 Reducing Sugar 4.07 1.00 3.17 2.48 1.39 2.83 1-26 Total Hexamine 3.59 0.60 2.07 2.23 1.28 2. 40 1.26 Sol., solubilized fraction; Res., residue. * Sugars determined by the method of phenol-sulfuric acid.

P. Pochanavanich and W. Suntornsuk in 2002 show that the chitosan accumulation of each strain of fungi varies in different stage of cell growth, (Chart 1 ) where chitosan yield in Rhizopus oryzae TISTR3245 was shown to give a maximal yield of chitosan at 138 mg g–1 dry weight (14% chitosan) (see Table 6) (Pochanavanich and Suntornsuk, 2002). In addition, previously in 1996 it has been showed that the most extractable chitosan is produced at the late exponential phase (Tan et al., 1996) .

Chart 1.Mycelial growth (■) and chitosan Chart 2.Dry weight of mycelia, alkaline production (●) of Rhizopus oryzae insoluble materials and extractable chitosan of TISTR3189 (Pochanavanich and Rhizopus oryzae USDB 0602 (Tan et al., 1996) Suntornsuk, 2002)

In the Zygomycota, after chitin fibres are synthesis they will get modified by complete or partial deacetylation to construct poly-b-(1,4)- glucosamine (chitosan) (see Figure 20) (Calvo-Mendez and Ruiz-Herrera, 1987, Yoshihara et al., 2003, Chatterjee et al., 2008, Zamani et al., 2007, Peter, 2005). This Chitosan fibres will be cross-linked by various neutral sugars and glucuronic acid (Datema et al., 1977). The cell wall matrix is consisting of proteins and glucans, just like other fungal groups. Also in the cell wall of the fungi specially R.oryzae there are glucosamine (GlcN) and N-acetyl glucosamine 16

(GlcNAc) (Zamani et al., 2008). In 2010 Kleekayai and co-workers could obtain the maximum chitosan yield which was 10.8 g/kg substrate. In their work properties of fungal chitosan showed 86– 90% degree of deacetylation, 80–128 kDa for molecular weight of and finally viscosity was 3.1–6.1 mPa·s. consequently, as a low cost feed stock for chitosan production from R. oryzae potato peel could be used (Kleekayai and Suntornsuk, 2010).Comparing with the Hang in 1990 who successfully produced Chitosan as a second valuable product from Rhizopus oryzae mycelia and the highest yield of extractable chitosan was 700 mg/L (Hang, 1990) the work done by Kleekayai is notable. Fungal chitosan molecular weight would vary upon substrate, medium supplementation and fermentation mode. Chatterjee et al. (2008) reported that the molecular weight of chitosan obtained from R.oryzae mycelia under submerged fermentation was 120 kDa with a low polydispersity index of 0.22 and a uniform chain length of chitosan molecules. After the supplementation with a plant growth hormone, chitosan molecular weight of R. oryzae was about two fold larger. Table 6.Amount of chitosan produced by different fungi(Pochanavanich and Suntornsuk, 2002)

Species Chitosan produced(mg g-1) Chitosan content (%) Aspergillus niger TISTR3245 107 11 107 11 Rhizopus oryzae TISTR3189 138 14 138 14 Lentinus edodes no. 1 33 3.3 Pleurotus sajo-caju no. 2 12 1.2 Zygosaccharomyces rouxii TISTR5058 36 3.6 Candida albicans TISTR5239 44 4.4

In 1981 Yoshio Tominaga and Yoshio Tsujisaka in a report Table 7.Adsorption capacity of stated that there is no evidence of glucans but in 2008 Das rhodamine B by different cellular and co-workers published a report describing extracting of components of R.oryzae mannan and glucan from the lyophilized R. oryzae cell walls Type of cell wall Component Uptake following the methods described by Northcote and Horne component (mg/g) (Northcote and Horne, 1952) where they used Fourier transformed infrared spectroscopic (FTIR) study to Cell wall 7.6 ± 0.19 characterize the modification of the functional groups due to Mannan 11.25 ± 0.31 chemical treatments. They described the role of different Chitosan 9.61 ± 0.28 functional groups (i.e. amino, carboxyl, hydroxyl as well as Chitin 2.86 ± 0.09 phosphate) and cell wall components (such as chitin, Glucan 1.725 ± 0.04 chitosan, glucan and phosphomannan) of Rhizopus oryzae on adsorption of rhodamine B (Das et al., 2008).

Figure 21.Fluorescence microscopic images (100×) of rhodamine B by (a) R. oryzae mycelia, (b) intact cell wall, (c) chitin, (s) chitosan, (e) phosphomannan and (f) glucan(Das et al., 2008)

Adsorption capacity of rhodamine B by different cellular components Yoshio Tominaga and Yoshio Tsujisaka isolated the Cell wall components (chitin, chitosan, glucan and phosphomannan) from R. oryzae in order to understand their relative participation in the adsorption of rhodamine B. 17

The adsorptive capacity of the different cell wall components for rhodamine B was reported (see Table 7). Fluorescence microscopic images done by Day et al. also supported the differential adsorption capacity of the various cell wall components

2.2.2.1. Covalently Linked Cell Wall Proteins

I. GPI-Dependent and Non-GPI Cell Wall Proteins

Glycosylphosphatidylinositol (GPI anchor) is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification. As in other eukaryotic organisms, fungal GPI proteins follow the secretory pathway. GPI anchor addition takes place in the lumen of the ER, a step that is mediated by a transamidase complex that exchanges the C-terminal region of these proteins for a pre- formed glycosylphosphatidylinositol lipid membrane anchor (Fraering et al. 2001; Fontaine et al. 2004). II. Glycosylation

Most fungal cell wall proteins are N- and O-glycosylated, and their carbohydrate side-chains often account for the bulk of their molecular mass (Dean 1999; Strahl-Bolsinger et al. 1999). N- Glycosylation is also initiated in the endoplasmic reticulum and starts with the stepwise assembly of a lipid-linked 14-residue oligosaccharide and transfer of the sugar chain by the oligo saccharyltransferase complex to selected asparagines residues of nascent polypeptide chains (Knauer and Lehle 1999)

2.2.2.2. Synthesis of the Cell Wall The chitin synthesis is done by particular organelles named chitosomes (Bartnicki- Garcia et al., 1979; Sentandreu et al., 1994) where inactive chitin synthases are transported to the apical plasma membrane and turn out to be activated as it contacts with the lipid bilayer membrane (Montgomery & Gooday, 1985). The number of chitin synthase genes varies according to the fungal species, from one gene in the ancestral fungus Encephalitozon cuniculi to > 20 genes in Rhizopus oryzae. BLAST analysis of amino acid sequences identifies six families of fungal chitin synthases. Three are specific for filamentous fungi (class III, V and VI) (Latgé, 2007). The composition and structure of the cell wall can diverge immensely during the growth and development of the fungal colony (Arora et al., 2004).

2.2.3 The Cytoskeleton Filamentous fungi have both main elements of the cytoskeleton, i.e. microtubules and actin (Figure 22) filaments (Heath, 1994, Heath, 1995). Long-distance transportation of organelles for instance secretory vesicles (or nuclei) and in the positioning of mitochondria, vacuoles or nuclei are mediated by Microtubules. Microtubules and actin filaments, two main essentials of the cytoskeleton, are plentiful in filamentous fungi (Heath, 1994, Heath, 1995). Intermediate filaments, that play skeletal roles in animal cells, are most likely of lesser significance in fungi. Microtubules are naturally positioned longitudinally comparing to the hypha involving in transportation of organelles such as secretory vesicles or nuclei to long-distance (Seiler et al., 1997), and also they are responsible for positioning of nuclei or vacuoles and mitochondria (Steinberg et al., 1998). They preserve the polarized distribution of lots of organelles in the hyphal tip consequently.

Figure 22.The cytoskeleton in fungi(Webster and Weber, 2007)

Kinesins: The presence of members of the kinesin superfamily in fungi is well documented. Lee and Plamann (2001) have described the structural and functional attributes of fungal (Lee et al., 2001). The role of conventional and kinesin-like proteins in vesicle transport towards the hyphal tip as well as mitochondrial and nuclear movement have been demonstrated, yet their function in different filamentous fungi may vary (Steinberg et al., 1998).

Figure 23.The role of conventional KinA and Kip2 family a Comparison of a wild type with a conventional kinesin deletion mutant (taken from Requena et al. 2001). b Scheme of aMTwith theMTplus end complex. This protein complex consists of several proteins, e.g. KipA or LIS1, conventional kinesin transports vesicles and components of the plus end complex, for instance dynein (Zhang et al. 2003). A direct interaction between KinA and dynein or dynactin has not yet been verified. Modified after Hestermann et al. (2002) c, d When KipA, which is suggested to be involved in the delivery of cell end markers, is missing, hyphae lose directionality. Images taken from (Fischer, 2007). Dynein: Dynein and its accompanying multisubunit component, known as dynactin have been implicated to be involved in vesicle trafficking and mitosis. The multisubunit dynein/dynactin motor is

complex and all indications are that their structure is highly similar to those present in higher eukaryotes (Arora et al., 2004). The complex is involved in: I. Retrograde transport II. Vesicle movement associated with the endocytic pathway III. Formation of the endoplasmic reticulum network IV. Organization of the Golgi apparatus V. Formation of the mitotic spindle (Karki and Holzbaur, 1999)

Regulation of Elongation / Branching The formation of the common cylindrical cell extension/branches by the polarized synthesis of new membrane and cell wall material must be both temporally and spatially regulated. Filamentous fungi may have additional or alternate regulatory pathways and are most likely to have more downstream elements governed by the regulatory systems involved rather than yeast. Morphology governed by the regulatory systems involved. A case illustrating a significant difference in a key regulatory pathway involved in polar growth of yeasts and filamentous fungi is described later. It is believed that the increase in hyphal tips per unit of biomass does not necessarily result in a parallel increase in secretion of extracellular enzymes (Arora et al., 2004).

2.2.4 The Nucleus The hyphal compartments of fungi may contain one or many nuclei. Some fungi can have about 50 nuclei. The nuclei of the vegetative phase of most filamentous fungi are haploid. The nuclei of most fungi are small (ca 1-2 pm in diameter) compared with those of animals and plants and they have comparably small chromosomes (Michael J. Carlile, 2001). They are surrounded by a double nuclear membrane with pores, as in all eukaryotes. I. However, fungi are notable for several peculiar features of their nuclei and nuclear division (Heath, 1978). Primary, during most stages of mitosis the nuclear membrane and the nucleolus stay intact, while in most other organisms the nuclear membrane breaks down at an early stage during nuclear division (it helps to prevent dispersion of the nuclear contents in hyphal compartments that contain several nuclei and rapidly streaming protoplasm). II. Second, in fungi there is no clear metaphase plate; instead the chromosomes seem to be randomly dispersed and at anaphase the daughter chromatids pull apart along two tracks, on spindle fibres of different lengths. III. A third point of difference is that fungi have various types of spindle-pole bodies (also called microtubule-organizing centres). They are responsible for microtubule assembly during nuclear division. All fungi need a microtubule-organizing center to ensure that the chromosomes separate correctly during nuclear division. The vast majority of fungi are haploid with chromosome numbers ranging from about 6 to 20 and a few fungi are naturally diploid. Some fungi can alternate between haploid and diploid generations – e.g. S. cerevisiae, and Allomyces spp. (Chytridiomycota) – or exist as polyploid series (Allomyces and several Phytophthora spp.). R. oryzae occurs as a haploid has 15 chromosomes. Although R. oryzae has a defined sexual cycle involving mating between the positive and the negative strains, classical genetic approaches have rarely been applied to study this organism. That is because germination of zygospores takes months and the rate of germination is erratic. The following are the data from Broadinstitute:

Summary Size Chrs %GC Genes tRNAs rRNAs R. oryzae RA 99-880 46.09 Mb -- 35.60 17,459 239 7 R. oryzae RA 99-880 mito 61.76 Kb -- 26.36 23 18 2

Table header definitions: Size length of complete genome sequence, calculated by adding lengths of all scaffolds together Chrs number of chromosomes, %GC GC content of scaffolds, Genes number of predicted protein-coding genes in genome, tRNAs number of predicted tRNA genes in genome, rRNAs number of predicted rRNA genes in genome

Figure 24.The role of clamp connections in maintaining a regular dikaryon (Deacon, 2006).

2.2.4.1. The Nucleolus The largest and most clear body in the nucleus is nucleolus which can be visualized under a light microscope by the help of a simple basic stain, such as acetocarmine or propiono-hematoxylin. All fungi appear to have only one nucleolar organizer (nor) located in a specific chromosome, which is known as the nucleolar chromosome. Under the squashed preparation, the nor is often seen as a highly condensed body associated with the nucleolus (Arora et al., 2004). The nucleolus is known to be the site of rRNA transcription and processing, i.e., the 35s rRNA was first transcribed from rDNA and then processed into 18s, 25s, and 5.8s rRNA. These are combined with 5s rRNA and ribosomal proteins to form preribosomal particles for export to the cytoplasm (Shaw et al., 1995). It is possible that the nucleolus has other important functions that are not commonly known. Some function of nucleolus is as following: I. The nucleolus may be a depot for mRNA exports from nucleus to cytoplasm II. The nucleolus is the site of signal recognition particle (srp) assembly and export Figure 25.Electron micrographs of yeast nucleus showing nucleolar III. The nucleolus may hold keys to cell Subcompartmentalization. A–B—from S. cerevisiae showing FC, GC, and DFC (A) and rDNA localization only in the FC (B); C–D— cycle and check point controls the nucleolus of S. pombe showing NDB from gar-1 mutant cells, the truncated Gar2p is accumulated in the DB (C). The bar ¼ 1mm Reproduced21 from Chromosoma 1997, 105:542–552 and Chromosoma 1999, 108:103–113, by copyright permission of Springer Verlag

IV. Nucleolar Silencing and Its Control of Aging

Nucleolar Structure

With the help of electron microscopy, the nucleolus is seen to have substructures—the fibrillar component, the granular component (GC), the DNA containing component, and sometimes nucleolar vacuole I. Nucleolar Subcompartmentalization

The presence of nucleolonema has recently been confirmed by using cryofixation and cryosubstitution techniques in yeast, S. cerevisiae and S. pombe (Léger-Silvestre et al., 1999, Léger-Silvestre et al., 1997). An example of which is shown in Figure 25. These authors demonstrated three major compartments of the nucleolus: the dense fibrillar component (DFC), the fibrillar centre (FC) and GC.

II. The Nucleolar Dense Body (NDB)

The NDB or simply nucleolar body (NB) or dense body (DB) is another prominent fibrillar compartment It was initially noted only in meiotic nuclei, in the wild-type C. cinereus (Lu 1984; 1996). It was never observed in mitotic nuclei. These observations raise the question of whether or not the organization of this NDB compartment is related to some specific function(s), maybe in meiotic or in mitotic nucleolus (Lu, 1996).

Centromere

The centromere sequence of S. cerevisiae is the simplest among fungi. It contains a 220–250-bp sequence, which is divided into three functionally distinct sequences, namely CDEI, CDEII, and CDEIII (Bloom and Carbon, 1982, Clarke and Carbon, 1980). The CEDII is an A T rich central region of 78–86 bp and A deletion of all or part of it leads to increased nondisjunction of chromosomes in mitosis and premature separation of sister chromatids in meiosis (Gaudet and Fitzgerald-Hayes, 1989). CDEIII is the most critical one. A point mutation of the central C to T in the inverted repeat will eradicate the centromere function and structure (Kenna et al., 1988).

The Kinetochore

Although the centromere is simple, the kinetochore is complex with a large number of centromere binding proteins and associated proteins. It is made up of three distinctive compartments: the inner, the central, and the outer kinetochore. In the inner kinetochore, the most important one of the centromere binding proteins is the CBF3 complex (Ndc10p, Cep3p, Ctf13p, and Skp1p), which binds to CDEIII; without it, the kinetochore function is abolished. Other inner kinetochore proteins are Cbf1p/CENP-B and Mif2p/CENP-C (Cheeseman et al., 2002b, Cheeseman et al., 2002a). The function of the kinetochore is the organization of spindle microtubules during nuclear division and this is the function of the outer kinetochore where microtubules are attached by the help of microtubule-associated proteins (MAPs)(Arora et al., 2004).

Figure 26.Images of GFP-TUBA (a tubulin) in A. nidulans showing cytoplasmic microtubules (A) spindle microtubules (B) and astral microtubules emanated from the poles of an elongating spindle (B, C)(Xiang and Fischer, 2004).

The Telomeres

Eukaryotic chromosomes are capped with a very specialized chromatin structure known as telomere. The telomere has two important functions: I. to protect the integrity of the chromosome end II. to ensure complete replication of the chromosomal DNA

The telomere sequence was first identified in Tetrahymena as (30CCCCAA50)n/(50TTGGGG30)n (Shampay et al., 1984). In fungi, S. pombe has 50-TTACAGG-30, N. crassa has a hexanucleotide repeat of 50 TTAGGG30, a sequence it shares with human and other animals (Schechtman, 1990). Telomeres play an important role in homologous chromosome pairing during meiosis (Arora et al., 2004).

How Do Nuclei Move in Hyphae?

There is strong experimental evidence that microtubules and the microtubule-dependent motor protein dynein are key players for nuclear migration. While the exact mechanism of dynein-mediated nuclear positioning remains unclear, evidence suggests that the dynamic status of microtubules is important for nuclear positioning in filamentous fungi (Xiang and Fischer, 2004). In filamentous fungi the situation is more complicated because the active growing apical compartment contains multiple nuclei. Mitosis occurs in hyphae and this separates the two daughter nuclei from each other.

Plasmogamy Plasmogamy is a stage in the sexual reproduction of fungi. In this stage, the cytoplasm of two parents mycelia fuse together without the fusion of nuclei, as occurs in higher terrestrial fungi. After plasmogamy occurs, the secondary mycelium forms. The secondary mycelium consists of dikaryotic cells, one nucleus from each of the parent mycelia.

The Dikaryon The dikaryon is a nuclear feature which is unique to some fungi, in which after plasmogamy the two compatible nuclei of two cells pair off and cohabit without karyogamy within the cells of the hyphae, synchronously dividing so that pairs are maintained in the older cells while newer cells or hyphal tips are also binucleate.

2.2.5 Mitochondria and Mitochondrial DNA Mitochondria are: I. The site of vital biosynthetic and degradative metabolic pathways II. Responsible for generating most of the energy required for function and survival of eukaryotic cells.

Studies of mitochondrial function and biogenesis are involved bioenergetic studies of metabolism and oxidative phosphorylation, genetic analysis of inheritance and mitochondrial DNA (mtDNA) mutations, and phylogenetic comparison of mitochondrial genomes.

An mtDNA molecule normally consists of a circle of double-stranded DNA, but in a few organisms, including the yeast Hansenula mrakii, the molecule is linear. The mtDNA constitutes the mitochondrial genome, and contains the genes for the transfer RNA (tRNA) and ribosomal RNA (rRNA) of the mitochondria and for some of the enzymes involved in oxidative phosphorylation. There are differences between organisms with respect to which enzymes or enzyme subunits are specified by mtDNA and which by the cell nucleus. A thus in human subunit 9 of ATPase is specified by a nuclear gene, in S. cerevisiae by a mitochondrial gene, and in N. crassa the gene is present in both nucleus and mitochondrion. The mitochondrial genome of fungi differs in size between species.

Plasmids

Plasmids are pieces of DNA that are capable of replication independently of the replication of the chromosomes or mitochondrial DNA. Although plasmids are very common in bacteria, they are infrequent in fungi and other eukaryotes (Michael J. Carlile, 2001).

Chitosomes

Most of the vesicles seen in hyphal tips have not been chemically characterized, but some of the smaller ones (microvesicles) resemble particles that have been purified in vitro and are termed chitosomes small spheroidal bodies, 40–70 nm diameter, each surrounded by a ―shell‖ about 7 nm thick (Figure 27) (Deacon, 2006). 24

Figure 27.Ribbon-like aggregates of chitin microfibrils (R) produced in vitro from chitosomes (C) isolated from Mucor rouxii, when incubated with substrate (N-acetylglucosamine) and a proteolytic activator. (Courtesy of C.E. Bracker; from Bartnicki-Garcia et al(Deacon, 2006).

2.2.6 Vacuoles The vacuolar system of fungi has several functions: I. The storage and recycling of cellular metabolites. For example the vacuoles of several fungi, including mycorrhizal species, accumulate phosphates in the form of polyphosphate. II. They are the major sites for storage of calcium which can be released into the cytoplasm as part of the intracellular signalling system. III. They are containing proteases for breaking down cellular proteins and recycling of the amino acids. IV. Have a role in the regulation of cellular pH. V. The potential role in cell expansion and (possibly) in driving the protoplasm forwards as hyphae elongate at the tips.

Secretion and Membrane Traffic

2.2.7 Endoplasmic Reticulum In both lipid and protein pathways the ER has an essential role. Regardless of the ultimate destination of cellular membranes and transmembrane proteins endoplasmic reticulum is the site of biosynthesis of nearly all cellular membranes and transmembrane proteins (Esser and Lemke, 1994). Luminal proteins destined for the Golgi apparatus and vacuoles are initially co-translationally delivered into the ER lumen. Also, the ER lumen provides space to store calcium ions to be released into the cytosol upon induction via appropriate signalling (Martin et al., 2005). Though the ER is thought to represent a single interconnected membrane system (Voeltz et al., 2002) it can be subdivided into a collection of numerous functional domains (Staehelin and Driouich, 1997, Staehelin, 1997).

2.2.8 Golgi apparatus Golgi apparatus is the site where secretory products and plasma membrane resident proteins are packaged for delivery to the cell surface and other materials packaged in transit to vacuolar compartments. It can also be the endpoint for materials that enter the endocytic pathway. The structure of the Golgi apparatus in fungi is strikingly different from those found in higher plants and animals. The most salient difference is that stacks of flattened cisternae, or dictyosomes, are lacking in true fungi (Grove and Bracker, 1970, Satiat-Jeunemaitre et al., 1996).

2.2.9 Exocytosis/Secretion Exocytosis is the vesicle-mediated delivery of materials to the outside of the cell and transmembrane proteins to the plasma membrane, especially glycosylated proteins that necessarily pass through the Golgi apparatus.

3. Chapter 3 – RNA Interference a Gene Silencing Tool

3.1 Background

Approaches to make a specific gene inoperative can be categorised in different levels:

I. DNA Sequence level II. Transcriptional level III. Post-transcriptional Level

3.1.1 Gene Inactivation in DNA Sequence Level Gene inactivation in DNA level is known as knockout which results in creating organisms called knockouts of KO. They are used to investigate about the function of a sequenced gene. The difference between the KO and WT researchers draw inferences about the function of the desired gene. Knockout can be done via homologues recombination, transposons, etc.

Homologous Recombination In this method one allele will be replaced with an engineered construct but without affecting any other locus in the genome. Knowing the sequence of the gene is essential to perform homologous recombination. In fungi, some meiotic recombination seems to root from a double-strand break repair. During this repair, by conversion caused by the process of double-strand gap filling, recombination occurs where homologous molecules interact by complementary base pairing, and by crossing-over which is probably an occasional byproduct of the repair process(Hastings, 1992). Recently many methods have been described rapidly creating knockout mutants in fungi for high-throughput gene disruption at an efficiency >90% based on homologous recombination (Colot et al., 2006, Tonukari et al., 2000, Pöggeler and Kück, 2006).

PCR-Based Gene Disruption In disruption through homologous recombination intermediate one need to carry out subcloning of a target gene into a suitable plasmid and digestion with restriction enzymes to insert a selectable marker cassette. It is sometimes difficult to subclone fungi genome sequences, because of their genomic DNA has highly biased AT-rich content as well as repetitive sequences and is thus sometimes unstable in Escherichia coli. Furthermore, the low number of restriction enzyme sites prevents researchers from designing a gene disruption construct with a flexible configuration. To conquer these issues, a method that does not require subcloning had to be established (Kuwayama et al., 2002).

So, PCR-based construction of a gene disruption construct has been developed which involves the synthesis of a marker cassette flanked by short homology regions (Baudin et al., 1993). In 1995 Amberg et al. adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae (Amberg et al., 1995). In 1998 Nikawa et al. developed a novel method for synthesizing marker disrupted alleles of yeast genes. This method needs only short oligonucleotides as primers and PCR ligation-mediated methods (Nikawa and Kawabata, 1998). 27

Transposons Inactivation of a gene at DNA Sequence level mainly comprises creating a mutant. Generating mutants via a procedure which is called ―knockout‖ will results in a cell which its DNA sequence for a specific gene is different from its wild type. Hamer et al. in 2001 developed transposon-arrayed gene knockouts (TAGKO) in order to investigation about an amino acid oxidation pathway in M. grisea strain Guy11 and M. graminicola strain PG. Their results showed that insertions of transposons can create alterations in gene expression (Hamer et al., 2001a, Hamer et al., 2001b). Transposons or ―jumping genes" catalyzed by the enzyme transponase cause mutations that disrupt genes in a nontargeted way but an alternative is ―Homing‖ which transposons can be inserted into specific DNA target sites with high frequency. This method takes advantage of group II introns which are a large class of self-catalytic ribozymes. In a process Group II introns are released in a lariat form that can integrate into intronless alleles of the same gene.

3.1.2 Gene Inactivation in Transcriptional Level

Providing a situation that the heterochromatin around a gene becomes inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.) by imposing modifications in histone (Kass et al., 1997) is transcriptional gene inactivation which mainly alters the regulation of transcription process. For example In some articles DNA methylation, methyl-recruiting proteins (El-Osta, 2002).

3.1.2.1. Repeat-Induced Point Mutations (RIP) It has been shown that duplicated sequences during sexual cycle are mutagenized via multiple G:C to A:T transitions (Cogoni, 2001, Watters et al., 1999, Irelan and Selker, 1996). Since RIP occurs in a specific period of the sexual cycle in the premeiotic phase The RIP is highly regulated (Selker, 1990), and this happens when the two nuclei of opposite mating types share a common cytoplasm in dikaryotic cells formed before karyogamy. RIP has the capability to detect any duplication with a minimal size of 400 bp (Watters et al., 1999). This size look like the minimum length required for recombination, so one can infer that a recombination-like mechanism that can form a DNA-DNA pairing structure could be the substrate for RIP (Rossignol and Faugeron, 1994, Selker, 1990). Mutations by RIP are usually adequate to fully inactivate affected genes, and in general, G:C to A:T transitions result in an assortment of silent, missense, and nonsense mutations (Cogoni, 2001). Since RIP involves local mutagenesis, gene inactivation is (Barry et al., 1993).

Figure 28.Two possible pathways to convert C:G to A:T (Irelan and Selker, 1996)

3.1.2.1. MIP: Methylation Induced Premeiotically Considering the timing and the sequence requirements MIP and RIP seem closely related processes (see Figure 29) but notably differ in their consequences (Rossignol and Faugeron, 1995). MIP happens in the sexual phase, and a minimum required size of homology is 400 bp (Goyon et al., 1996). MIP is irreversible because involves only DNA methylation and this Methylation has been found to be coextensive with the region of homology and blocks the transcription elongation (Barry et al., 1993).

Figure 29.Schematic illustration of RIP and MIP(Irelan and Selker, 1996).A: Both copies of duplication are retained through the sexual cycle. Most often both are found to have suffered from RIP or MIP (filled boxes).B: Duplication happed but RIP and MIP were not happened. C: Duplicated sequence is deleted. D: First RIP happed and then duplication was deleted.

3.1.3 Gene Inactivation in Post-Transcriptional Level (PTGS) Post-transcriptional level gene inactivation generally refers to methods which destroy or block the mRNA of a particular gene. Post-transcriptional gene silencing (PTGS) phenomenon was termed ‗quelling‘ when it refers to gene silencing in Neurospora crassa (Romano and Macino, 1992) and related to co-suppression in plants and RNA interference (RNAi) in animals. 29

3.1.3.1. Antisense Oligodeoxynucleotide (ODN) Antisense oligodeoxynucleotide (ODN) is a nucleotide sequence that is complementary to a sequence of messenger RNA (mRNA). When antisense DNA or RNA is added to a cell, it attaches to a specific mRNA molecule and make it inoperative by physically blocking the ability of ribosomes to move along the messenger RNA or by simply accelerating its degradation (Yamamoto et al., 1997).

3.1.3.1.1. Phosphorothioates (S-DNA) First generation of antisense oligonucleotides are Phosphorothioates. They are a variant of normal DNA in which one of the no bridging oxygens is replaced by sulphur. These oligomers have resistance to nucleases and the ability to activate RNase H. The relatively low stability of duplexes with complementary RNA and also the toxic effects which is possibly related to non-specific protein–S-ODN interactions are the main shortcomings of S-ODNs (Yoo et al., 2004).

3.1.3.1.2. LNA Locked Nucleic Acid is a novel class of nucleotide analogue in which the conformational freedom of the ribose moiety is restricted. These oligonucleotides contain one or more nucleotide building blocks in which an extra methylene bridge fixes the ribose moiety either in the C3′-endo (β-D- LNA) or C2′-endo (α-L-LNA) conformation. The β-D-LNA modification results in significant increases in melting temperature of up to several degrees per LNA residue. LNA has improved mismatch discrimination and comparing with S-ODN lower toxicity, and increased metabolic stability (Grünweller and Hartmann, 2007).

3.1.3.1.3. Morpholino Morpholino is a molecule used to modify gene expression and to block access of other molecules to specific sequences within nucleic acid. Morpholinos block small (~25 base) regions of the base- pairing surfaces of RNA (Heasman, 2002, Paessler et al., 2008, Kanzler et al., 2003).

3.1.3.2. Ribozyme An RNA molecule that can catalyze a chemical reaction is called ―ribozymes‖ or RNA enzyme. They can catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Their advantage over antisense oligonucleotides (ODNs) is that ribozyme genes can be delivered to cells with plasmid or viral vectors, and ribozyme expression can be controlled with promoter-based expression.

3.1.3.3. RNA Interference RNA interference or RNAi is a technique which is used to silence a specific gene. While Gene silencing is a general term which describes epigenetic processes of gene regulation it can also be used to explain the "switching off" of a gene by a mechanism other than genetic modification. RNAi procedure is named ―co-suppression‖ when this is applied in plants, ―quelling‖ when is used to silence gene in fungi (Shiu et al., 2001), and ―RNA interference‖ (RNAi) in nematodes. In RNA interference dsRNA facilities the homologous mRNA degradation, in that way causes the diminishing or abolishing gene expression (Nakayashiki and Nguyen, 2008a, Fire et al., 1998). Despite numerous reports on RNA silencing in a variety of organisms, only few species among filamentous fungi have been proven to have this mechanism. One is N. crassa and the other is the human pathogenic fungus Cryptococcus neoformans (Cogoni and Macino, 1999). Also the operation of RNA silencing in Magnaporthe oryzae the rice blast fungus, using enhanced green fluorescence protein (eGFP) as a model was described by Naoki Kadotani and his colleagues in 2003 (Kadotani et al., 2003a). In 2004 Skory worked on R. oryzae to investigate the effect of lactate dehydrogenase gene of Rhizopus oryzae (Skory, 2004b).

3.2 History and Discovery of RNA Silencing

The breakthrough of RNAi was heralded primary by observations of expression inhibition in bacteria, transcriptional inhibition by antisense RNA expressed in transgenic plants. In 1981 Janice Light and Søren Molin investigated how genes involved in replication of R1 are expressed in vivo using gene fusion techniques in Escherichia coli. They described that a plasmid- specific cytoplasmic factor, which act as inhibitor of replication is a small, unstable RNA molecule (copA gene product) (Light and Molin, 1981). Their effort followed by a work at the same period of time (1981) done by R. M. Lacatena & G. Cesareni describing base pairing of RNA I with its complementary sequence in the primer precursor inhibits ColE1 replication (Lacatena and Cesareni, 1981). In 1983, J Light and S Molin shown that the CopA RNA interacts with its target (CopT) only when the region is transcribed to form RepA mRNA (Light and Molin, 1983). In 1984 , Izant, J. & Weintraub, H described Inhibition of thymidine kinase gene expression by antisense RNA which was a molecular approach to genetic analysis (Izant and Weintraub, 1984).

At the same year Coleman J, Green PJ and Inouye M constructed a plasmid that produces a complementary RNA to the E. coli lpp mRNA (mic[Ipp] RNA). Induction of the mic(Ipp) gene efficiently blocked lipoprotein production and reduced the amount of lpp mRNA. They suggest that micRNAs complementary to regions of the mRNA likely to come in contact with ribosomes were most effective (Coleman et al., 1984). In 1984 Mizuno et al. proposed that the micRNA inhibits the translation of the mRNA by hybridizing with it (Mizuno et al., 1984). In 1985 Coleman J et al proposed that the micRNA immune system provides an effective means of preventing viral infection as well as the expression of harmful genes in both prokaryotes and eukaryotes (Coleman et al., 1985). Later but in the same year Rosenberg, U.B., et al., investigated the Production of phenocopies by Krüppel antisense RNA injection into Drosophila embryos (Rosenberg et al., 1985). At the same year Melton reported blocking of translation in frog oocyte cytoplasm and suggested that the 5' region of the mRNA must be covered in order to block translation (Melton, 1985). Ecker, J.R. and R.W. Davis,

in 1986 stated that expression of antisense RNA Inhibit the gene expression in plant cells (Ecker and Davis, 1986). In 1988 van der Krol, A.R., et al., reported that in transgenic petunia and tobacco plants the constitutive expression of so called 'anti-sense' chalcone synthase gene altered pigmentation flower and they concluded that this happened because of decrease in amount of both the enzyme itself and the meRNA for the enzyme (van der Krol et al., 1988).

Attempts to alter flower colours in petunias, continued by Napoli, Lemieux, Jorgensen in 1990. They tryed to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene and surprisingly, the introduced gene created a block in anthocyanin biosynthesis. They found out that the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans was unclear for them, but the erratic and reversible nature of this phenomenon persuaded tham to suggest the involvement of methylation (Napoli et al., 1990). At the same year Van der Krol et al.transfored petunia with additional dihydroflavonol- 4-reductase (DFR) or chalcone synthase (CHS) genes in order to evaluate the effect of increased expression of genes involved in flower pigmentation and they noticed that In most transformants, the Increased expression had no measurable effect on floral pigmentation. They surprised with a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression. They inferred that the similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits (van der Krol et al., 1990).

Van der Krol et. al showed in Figure 30 (A) that Shown from left to right flowers on petunia VR plants transformed with the constitutive CHS gene initially did not exhibit an altered flower phenotype. However, when two of the transformants were grown, flowers with white sectors developed after 2 weeks (not observed for the wild-type petunia VR). Plants containing the CHS genomic clones showed flowers with either wild-type pigmentation or an even reduction in pigment synthesis. In section B of Figure 30 Flowers on petunia VR plants transformed with the DFR sense gene construct showed either unaffected flower pigmentation (top left) or a reduction in pigment synthesis.

In 1991 the antisense RNA investigations continued by Fire, A., et al., in a work named ―Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle”, used an antisense strategy to disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. They examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus was present at normal levels and that RNA was properly spliced in the region homologous to the antisense RNA. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. They conclude that the observed phenotypes resulted from interference with a late step in gene expression, such as transport into the cytoplasm or translation(Fire et al., 1991). Soon after, in 1992 a similar event termed quelling by Romano et.al but this time the blocking of expression was experienced in the fungus Neurospora crassa (Romano and Macino, 1992).

Figure 30.Flower Phenotype of the CHS Sense and DFR Sense Transformants (van der Krol et al., 1990).

In 1993, Nellen, W. & Lichtenstein, C proposed a mechanism for antisense RNA. They described that when sense and antisense transcripts are synthesized in a cell (a), hybridization will be facilitated by hybrid-promoting proteins such as RNP protein A1 (b) or by the 'transwindase' activity of RNA helicases which can eliminate intramolecular secondary structures in favour of more extensive intermolecular hybridization (e).

Figure 31.The proposed mechanism for Antisense RNA by Nellen, W. and C. Lichtenstein in 1993 (Nellen and Lichtenstein, 1993a)

Furthermore they suggested RNA helicases to disrupt hybrids and make them unavailable for further processing (d). They also described that when hybrids are formed (e), they can be digested by dsRNA- specific nucleases (t). They believed that the regulation of these mechanisms can be done by differentially activating and inactivating the respective enzymes. An example was given for a dsRNA- specific nuclease which was inactivated (h) by a protein phosphatase (g) and activated by a protein kinase such as DDPK which induced by dsRNA (I) (Nellen and Lichtenstein, 1993b, Nellen and Lichtenstein, 1993a).

Investigation of the gene inactivation phenomenon in plants carried out by Van Blokland, R., et al. In 1994 indicated that the reduction in steady-state chs mRNA was not the result of a transcriptional inactivation event. Van Blokland, R., et al. declared that transcription of suppressed genes was similar to that of non-suppressed genes. This designated that co-suppression occurs post-transcription ally. They named this suppression as ―co-suppression of gene expression” (Van Blokland et al., 1994).

Figure 32. Petunias Containing Wild type (Separated picture in the left ) and 4 Transgene copies(van der Krol et al., 1990)

Figure 33.Petunias Containing Wild type (Separated picture in the left ) and 4 Transgene copies(Napoli et al., 1990)

In 1995 Guo et al noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm (Guo and Kemphues, 1995). Between 1995 and 1998 experimental introduction of RNA into cells in certain biological systems to interfere with the function of an endogenous gene was quite usual and such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. Also using RNA interference in the nematode C. elegans to manipulate gene expression was usual (Fire et al., 1991, Guo and Kemphues, 1995). In 1998 Fire, A., et al. investigates the requirements for structure and delivery of the interfering RNA. To their surprise, they found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-

stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny (Fire et al., 1998).

Figure 34.Interference contrast micrographs show in situ hybridization in embryos of C. elegans. a, Negative control showing lack of staining in the absence of the hybridization probe. b, Embryo from uninjected parent (showing normal pattern of endogenous mex- 3 RNA). c, Embryo from a parent injected with purified mex-3B antisense RNA. These embryos (and the parent animals) retain the mex-3 mRNA, although levels may be somewhat less than wild type. d, Embryo from a parent injected with dsRNA corresponding to mex-3B; no mex-3 RNA is detected (Fire et al., 1998)

Finding of Fire and Mello was mainly notable since it signified the first identification of the reason for the phenomenon. Mello and Fire were awarded the Nobel Prize in 2006 (Daneholt, 2010, Biesaga, 2007, Pichlmeier, 2006b, Pichlmeier, 2006a).

Knowing the degradation of mRNA through a process known as RNA interference (RNAi) directed by Double-stranded RNA (dsRNA), in 2000 Zamore et al. examined the molecular mechanism underlying RNAi. They discovered that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21- 23 nucleotides in length. They also Reported processing of long dsRNA by RNase III (Dicer) into shorter fragments of 21-23nt intervals in Drosophila (Zamore et al., 2000). In 2001 Bernstein et al. identified an enzyme, Dicer, which can produce putative guide RNAs. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi (Bernstein et al., 2001b) .

In 2001 Tuschl and colleagues showed that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney and HeLa cells. This promise a brilliant future of therapeutics (Elbashir et al., 2001a). And later in 2003 in mammalian cells, shRNAs was used to carry out the sequence-specific silencing (Paddison et al., 2002, Sui et al., 2002, Paul et al., 2002). In 2003 Song et al. reported that siRNAs can be used therapeutically in whole animals (Song et al., 2003).

Kadotani and colleagues in 2003 (Kadotani et al., 2003b) used expression of the enhanced green fluorescence protein (eGFP) gene as a reporter M. oryzae. Their results indicated that RNA silencing operated in M. oryzae, which provided a new tool for genome-wide functional analysis of this fungus.

In 2004 Morris et al. some aspect of siRNA gene silencing at transcriptional level possibly through directing de novo DNA methylation emerged (Morris et al., 2004, Kawasaki and Taira, 2004) and in 2004 Acuity Pharmaceuticals first phase I clinical trial of siRNA drug for age-related macular degeneration (AMD). The importance of RNA interference became more highlighted when Andrew 35

Fire and Craig Mello Won Noble Prize in Physiology or Medicine for discovering RNAi mechanism in 2006 (Daneholt, 2010, Biesaga, 2007, Pichlmeier, 2006b, Pichlmeier, 2006a).

In 2006 Li et al first reported that small dsRNA induces gene expression activation, a phenomenon termed as RNAa (Li et al., 2006a) during 24th Annual Meeting of the American Society of Retina Specialists in Cannes, France in 2006 Acuity Pharmaceuticals, a clinical stage ophthalmic pharmaceutical company, announced positive results from its Phase II C.A.R.E(TM) trial for bevasiranib sodium (formerly Cand5). Bevasiranib is a first-in-class small interfering RNA (siRNA) therapeutic designed to silence the genes that produce vascular endothelial growth factor (VEGF), believed to be largely responsible for this leading cause of adult blindness. In 2006 Richard J Weld et al. discussed recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches (Weld et al., 2006)

Moriwaki and colleagues in 2007 (Moriwaki et al., 2007) demonstrated the applicability of targeted gene silencing as a useful reverse-genetics approach in Bipolaris oryzae, causal agent of brown leaf spot disease in rice. More recently and in 2008, Nguyen and colleagues (Nguyen et al., 2008) described an approach for gene function analysis using RNAi, which provides an alternative to the gene knock-out by homologous recombination in the rice-blast fungus, Magnaporthe oryzae.

Another promising application of RNA interference revealed when Quark Pharmaceuticals Announces Presentations of its Two siRNA Drug Candidates at Glaucoma & Retinopathies 2010 (Quark, 2010).

3.3 Mechanism

RNA Interference or RNAi (in some articles categorized in Knockdown methods) is an exciting strategy for reverse genetics (Bhadauria et al., 2009). In RNAi a double-stranded RNA (dsRNA) which operate as templates for a protein called ―Dicer ribonuclease III ― (which produce short interfering RNAs or siRNAs ) (Bernstein et al., 2001a) slices the long dsRNA molecules to fragments of ~20 nt. The produced siRNAs by dicer become separated into two ssRNA, called the guide strand the passenger strand. The guide strands are integrated into a protein complex called ― RNA-inducing silencing complex (RISC)‖ which serve as sequence-specific guides that target homologous mRNA molecules for destruction and the passenger strand will be degraded (Hammond et al., 2001, Hannon, 2002). Post-transcriptional gene silencing is the outcome of this event, which happened when the guide strand coupled with its complementary mRNA and induces splicing by argonaute which is the catalytic component of the RISC complex.

Figure 35.Highly simplified schematic mechanism of RNA interference

In 2009 Vijai Bhadauria et al. proposed three RNAi strategies (Bhadauria et al., 2009) (see Figure 36):

I. Conventional hpRNAi: transcripts with complementary or near-complementary 20- to 50-base pair inverted repeats form double-stranded RNA (dsRNA) hairpins are processed into micro- RNAs that mediate mRNA degradation. II. pSilent1 (heterogeneous nuclear RNA expressing vector system): the vector carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with multiple cloning sites and a spacer of an intron sequence.

III. pSilent-Dual1 system (opposing dual promoter system): contains trpC and gpdA promoters which are cloned in a convergent manner separated by multicloning site.

Figure 36.RNAi strategies: (a) conventional hpRNAi, (b) pSilent1 (heterogeneous nuclear RNA expressing vector system), (c) pSilent-Dual1 system (opposing dual promoter system (Bhadauria et al., 2009).

3.3.1 Dicer In 2001 Emily Bernstein et al. identified an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Emily Bernstein et al. described that the enzyme includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi (Bernstein et al., 2001a). Their results indicated that the process of RNAi can be divided into at least two distinct steps: I. Initiation of PTGS would occur on processing of a dsRNA by Dicer into, 22-nucleotide guide sequences, although they could not formally eliminate the possibility that another Dicer-associated nuclease that may participate in this process. II. Incorporation of these guide RNAs into a distinct nuclease complex (RISC) that targets single- stranded mRNAs for degradation. They infer this model from their observation, that guide sequences were themselves derived directly from the dsRNA that triggers the response. In accord with this model, they showed that 32P-labelled, exogenous dsRNAs that have been introduced into S2 cells by transfection were incorporated into the RISC enzyme as 22- nucleotide sequences (Bernstein et al., 2001a).

Dicer is categorized in endoribonucleases and in the RNase III family. Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA) into short double-stranded RNA fragments (small interfering RNA or siRNA). SiRNAs have about 20-25 nucleotides long, usually with a two-base overhang on the 3' end and carry a 5' phosphate group. (Sohail, 2005). As it is depicted in Figure 37 dicer contains two RNase III domains and one PAZ domain; the distance between these two regions of the molecule is determined by the length and angle of the connector helix and determines the length of the siRNAs it produces(Macrae et al., 2006). Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC).

Figure 37.Crystal structure of Giardia Dicer. (A) Front and side view ribbon symbolize of Dicer showing the N-terminal platform domain (blue), the PAZ domain (orange), the connector helix (red), the RNase IIIa domain (yellow), the RNase IIIb domain (green) and the RNase-bridging domain (gray). Disordered loops are drawn as dotted lines. (B) Close-up view of the Dicer catalytic sites; conserved acidic residues (sticks); erbium metal ions (purple); and erbium anomalous difference electron density map, contoured at 20s (blue wire mesh) (Macrae et al., 2006).

The two adjacent RNase III domains of Dicer form an internal heterodimer by sitting next to each other in the blade region and that is similar to the homodimeric structure of bacterial RNase III (Figure 38). Dicer RNase III domains are depicted in Figure 38 showing the Figure 38.Dicer RNase III superposition of the C-α atoms from M. tuberculosis RNase III (white) domains (Macrae et al., 2006) and the RNase IIIa (yellow) and IIIb (green) domains of Giardia Dicer. Asterisks denote regions implicated in sequence discrimination in bacterial RNase III. N and C denote N-terminal and C-terminal ends of each domain.

Figure 39.A model for dsRNA processing by Dicer showing Giardia Dicer with modeled dsRNA (Macrae et al., 2006).

A model for dsRNA processing by Dicer is presented in Figure 39. Front and side views of a surface representation of red and blue represent acidic and basic protein surface charge, correspondingly. Electrostatic surface potentials do not include contributions from bound metal ions. Putative catalytic metal ions are shown as green spheres. Also white arrows represent scissile phosphates. Asterisk denotes PAZ domain 3 overhang-binding pocket (Macrae et al., 2006).

Fungus Neurospora has two partially redundant Dicer proteins: first one is Dicer-like-1 (DCL-1) and the second one is DCL-2. Both of them can process dsRNA into about 25-nt small RNAs in an ATP- dependent manner. The double mutant of dcl-1 and dcl-2 completely abolished quelling and disrupted the processing of dsRNA into siRNA in vivo and in vitro, but the single dcl mutants had comparable quelling frequencies to the wild type. These results suggest that the two Dicers are functionally redundant, explaining why they escaped the earlier screening for quelling defective mutation. However, the accumulation of siRNA was significantly reduced in the dcl-2 mutant compared to that of the wild-type, indicating that the DCL-2 is the major dsRNA processing enzyme (Li et al., 2010).

3.3.2 RISC RISC contains short RNAs (22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown (Elbashir et al., 2001b). In 2001 Hammond et al. reported the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction was containing ribonucleoprotein complex of 500 kilodaltons. Protein microsequencing revealed that one constituent of this complex was a member of the Argonaute family of proteins, which are essential for gene silencing in C. elegans, Neurospora, and Arabidopsis too. This observation began the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that was emerging from Drosophila in vitro systems (Hammond et al., 2001). In 2009 Kawamata explained that unwinding of miRNA-miRNA duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12–15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Their findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins (Kawamata et al., 2009, Haley and Zamore, 2004).

Evidence obtained from studies of RNAi in Drosophila cells show that at least one protein, called R2D2, is involved in the transfer of siRNAs as they are generated by Dicer onto proteins that from part of the RISC complex(Liu et al., 2003). R2D2 may influence the polarity (sense/antisense orientation) with which the siRNA interacts with components of RISC. Several protein components of RISC have been isolated. The first to be identified, Argonaute 2 (Ago2) appears to form the core of RSIC(Hammond et al., 2001). Ago2 and Dicer are both members of the PAZ (PIWI, Argonaute, and Zwille/Pinhead) domain-containing family of proteins (Cerutti et al., 2000) Recent structural analysis of the Drosophila Ago2 protein has shown that the PAZ domain can form a nucleic acid-binding fold.40,41 The affinity of the interaction of this domain and RNA is relatively weak, but the presence of a 3' single stranded RNA overhang appears to be critical to the interactions (Rand et al., 2005, Liu, 2004, Hammond et al., 2001, Kim et al., 2006, Adams et al., 2009, Sofola et al., 2007).

RNA, single- and double-stranded, can also bind, though in the case of duplex DNA the binding is independent of a 3' overhang. Other proteins associated with RISC include in Drosophila, the Vasa

intronic gene product (a p68 RNA helicase homolog) and the fragile X mental retardation protein, and in human cells the homolog of Ago2–E2FC and Gemin3 and Gemin4 (Sohail, 2005). The antisense single-stranded siRNA component of RISC leads to localization of the RISC complex through perfect sequence alignment of the two RNA molecules. Cleavage of the target occurs at a position ~10 nts from the first nucleotide that represented the first based pair from the 5' end of the original siRNA (Timmons and Fire, 1998).

Figure 40.Dicer and RISC (RNA-induced silencing complex

3.3.2.1. Argonaute

Argonaute proteins are named after the argonaute (AGO) phenotype of Arabidopsis thaliana mutants, which itself has some similarity with argonauts (Bohmert et al., 1998). Argonaute proteins are part of the RNA-induced silencing complex (RISC) which have catalytic activity. They bind siRNA fragments and have endonuclease activity directed against messenger RNA (mRNA) strands that are complementary to their bound siRNA fragment. It is believed that argonaute proteins are also partially responsible for selection of the guide Figure 41.Roles of the Argonaute complex in miRNA and RNAi pathways(Hutvagner and Simard, 2008) 41

strand and destruction of the passenger strand of the siRNA substrate (Rand et al., 2005).

The structural basis for binding of RNA to the argonaute proteins was shown by X-ray crystallography and is comprising the entrance of phosphorylated 5' side of the RNA into a very conserved basic surface which creates interactions throughout a divalent cation for instance magnesium and by aromatic stacking between the 5' nt in the siRNA and a conserved tyrosine residue (Ma et al., 2005). In eukaryotes, argonaute proteins have been identified in high concentrations in cytoplasmic bodies, to which mRNA decay is also localized (Sen and Blau, 2005). Based on comparative genomics studies, the argonaute family is thought to have evolved from components of the translation initiation system (Anantharaman et al., 2002).

Figure 42.Structural features of Argonaute proteins(Hutvagner and Simard, 2008) taken from ©(2004) American Association for the Advancement of Science.

Role or argonaute in RNA interference (RNAi) pathway is depicted in Figure 41. Argonaute associated with small interfering RNA forms the active RNA-induced silencing complex (RISC), which can induce endonucleolytic cleavage of targeted mRNA (Hutvagner and Simard, 2008).

What RNA Species Is the Best Trigger for RNA Silencing in Filamentous Fungi?

A variety of species of RNA, such as sense, antisense, double strand, and hairpin, are all known to have the capability to trigger RNA silencing in plants and animals. In filamentous fungi, quelling (RNA silencing in N. crassa) occurred in transformants with multiple transgenes having no promoter(Romano and Macino, 1992). Another example of RNA silencing in fungi performed in pathogenic fungus C. neoformans by expressing the hairpin RNA of the cryptococcal genes (Liu et al., 2002). The philosophy behind the selection of RNA should be based on a systemic analysis in order to determine what RNA species is the best trigger for RNA silencing in filamentous fungi.

The Length of siRNA

In the fungus N. crassa, siRNAs have been demonstrated to be involved in the silencing pathway with a single molecular size reported at 25 nt (Catalanotto et al., 2002).

Delivery of siRNA to the Fungal Cells siRNAs it selves do not cross the membrane of the cell for the reason that they are negatively charged and also their size is bigger than they could pass the pores (Aagaard and Rossi, 2007). To date, direct delivery of siRNA have been very rarely reported in fungi (Nakayashiki and Nguyen, 2008b).the first demonstration of up-take of siRNA by germinating fungal spores from culture medium was published in 2007 by Madhu Khatri and M. V. Rajam (Khatri and Rajam, 2007). DNA-based (vector) expression cassettes could be constructed that are able to express the short hairpins (shRNA) or separate antisense and sense 21 mers (Aagaard and Rossi, 2007).

Direct Delivery

Direct delivery of siRNA/dsRNA into fungal cells have been very rarely reported in fungi (Nakayashiki and Nguyen, 2008b). Silencing a polyamine biosynthesis gene in Aspergillus nidulans by treating germinating spores with synthetic 23 nt siRNA duplex done with 2 nt overhangs at the 30- terminus (Khatri and Rajam, 2007). The results showed that in the culture medium siRNA could be taken up by germinating fungal spores, which is a fast and handy method to induce RNAi in fungal cells. In addition, it has been previously showed in Phytophthora infestans that direct delivery of longer dsRNA into protoplasts is possible (WHISSON et al., 2005).

Lipofection: a Highly Efficient, Lipid-mediated Transfection Procedure

Lipofection is reported to be more efficient from 5- to greater than 100-fold than either the DEAE- dextran transfection or calcium phosphate technique (Felgner et al., 1987). in vitro researches of the application of cationic liposome reagents has advanced DNA and mRNA transfection and show their utility for in vivo gene transfer (Felgner et al., 1994). Also Magnetic force combined with magnetic nanoparticles has shown possibilities for enhancing nucleic acid delivery. There are some reports describing Magnetofection as a reliable and gentle alternative method with low cytotoxicity for siRNA delivery into difficult to transfect cells such as mammalian fibroblasts (Ensenauer et al., 2010).

3.4 Biological Function

3.4.1 Immunity Not only the RNAi technology scores over many approaches with its ease, cost-effectiveness and rapidity in order to be used in research (Gadkari, 2005) but also RNA interference is now regarded to be an evolutionarily conserved existing in the entire eukaryotic kingdom and its original function is serving as a defence mechanism against viruses and foreign nucleic acids (Stram and Kuzntzova, 2006). Likewise to the silencing of genes by RNAi, viral functions can be silenced by the same mechanism as well. Knowing this, virus expression vectors were developed to be used as vehicles with which to deliver siRNAs into cells.

RNAi in Immune System of Fungi Studies shows that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism (Segers et al., 2006). Zhang, X., et al. reported the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus infected wild-type and dicer gene dcl-1 mutant C. parasitica strains. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing (Zhang et al., 2008).

RNAi in Immune System of Plants Even earlier than the deciphering of RNAi pathway in 1997, scientists were aware of the fact that in plants induced gene silencing possibly will spread all the way through the plant, and via grafting could be conveyed from stock to scion plants (Palauqui et al., 1997). Later it was found that plants utilize DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing to regulate gene expression and mediate defence against viruses (Blevins et al., 2006) and the silencing process may also prevent self-propagation by transposons (Stram and Kuzntzova, 2006, Blevins et al., 2006). Continuing investigation in plants In 2006 Katiyar-Agarwal et al. reported the discovery of an endogenous siRNA, nat-siRNAATGB2, which is specifically induced by the bacterial pathogen Pseudomonas syringae carrying effector avrRpt2 (Katiyar-Agarwal et al., 2006).

RNAi in Immune System of Drosophila An RNA interference system defends adult flies from infection by two evolutionarily diverse viruses. Studies describe a molecular framework for the viral immunity, where a viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, correspondingly. These findings establish a Drosophila model for studying the innate immunity against viruses in animals (Wang et al., 2006).

RNAi in Immune system of Caenorhabditis elegans It is also not known if small RNA-guided RNAi provides protection against viruses in single-Dicer C. elegans and mammals as it has been proven in plants (Katiyar-Agarwal et al., 2006) and insects (Wang et al., 2006). Studies demonstrated a complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. Investigations showed that FHV replication triggers potent antiviral silencing in C. elegans that requires RDE-1, a worm Argonaute protein (Hammond et al., 2001, Tabara et al., 1999) some studies establishes C. elegans model for genetic studies of animal 44

virus-host interactions and indicates an antiviral potential for RNAi in mammals via the siRNA pathway (Lu et al., 2005).

RNAi in HeLa cells Nowak M. et al. reported a new method for inhibition of RNA viruses induced by dsDNA. They demonstrated that both long dsDNA molecules and short interfering DNA with a sequence complementary to that of viral RNA inhibited tobacco mosaic virus expression and prevented virus spread. Also, the expression of the HIV-1 gp41 gene in HeLa cells was inhibited by complementary short interfering DNA. They showed that Dicer processed dsDNA, which suggests activation of the cellular machinery involved in silencing of RNA. For the silencing of viral RNA affected with dsDNA, they coined the term DNA interference technology (Nowak et al., 2009).

3.4.2 Downregulation of Genes

Importance of miRNAs which are endogenously expressed, as well as both intergenic and intronic miRNAs in repression of translation was revealed by (Ambros et al., 2003, Sullivan and Ganem, 2005, Saumet and Lecellier, 2006). Roles of miRNA also have been reported in the regulation of development, mainly on morphogenesis timing and the maintenance of incompletely differentiated or undifferentiated cell types for example in stem cells (Carrington and Ambros, 2003).

Role of MiRNA in Downregulation of Gene in C. elegance In 1993 the application of endogenously expressed miRNA in downregulating gene expression was initially described in C. elegans where Lee et al. used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Their results suggested that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction (Lee et al., 1993).

Role of MiRNA in Downregulation of Gene in Plants Palatnik et al. in a genetic screen identified the JAW locus that produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter which in Arabidopsis this process was reported to be consist of regulation of some genes that their role is controlling the shape of the plant (Palatnik et al., 2003).

3.4.3 Upregulation of Genes Gene transcription can be enhanced by RNA sequences complementary to sequence of a promoter this RNA sequences are known as miRNA and siRNA. This can double the RNA activation. Dicer and argonaute are part of this mechanism also there is demethylation of histone involved in the event (Check, 2007, Li et al., 2006b).

3.4.4 Offtarget Effects Offtarget effects can be reduced by avoiding the incorporation of sense strand (siRNA) and promoting the incorporation of antisense strand (siRNA) of siRNA duplex into the RISC complex. 5´- phosphate group is essential for the siRNA strand to act as a guide strand. So, modification of the 5´-phosphate group of the sense strand to a 5´-O-methyl, can effectively avoid sense strand RISC (siRNA–RISC) formation (Chen et al., 2008) 45

4. Chapter 4 - Methods and Materials

4.1 Fungal Strain and Growth Media Preparation

4.1.1 Strain Fungus Rhizopus oryzae CCUG 289581 (Culture Collection University of Göteborg, Sweden) was used in all experiments. CCUG 28958 is the named after Culture Collection University of Göteborg for CBS 112.072 of CBS Fungal Collection and its name in NBRC is NBRC 5414. The strain was maintained at 4 °C on 2% (wt/vol) potato dextrose agar slants (Figure 43). The slants could be stored for one year at 4 °C.

Figure 43.potato dextrose agar slant culture of R. oryzae used for this work

4.1.2 Culturing R. oryzae on Agar Plate (Subculturing) A swap from slant was taken and spread on the PDA agar plate in order to propagate the fungi. The fungus grew and formed spores on potato dextrose agar plates prepared as described in Protocol No. 13. PDA (potato dextrose agar) was prepared with Potato extract 4 g/l, glucose 20 g/l, agar 15 g/l and distilled RNase free water to reach the final volume 1 litre.

1 To see more about the strand see the web page of CCUG: http://www.ccug.se/default.cfm?page=search_record.cfm&id=6397&db=mc 2 http://www.cbs.knaw.nl/fungi/BioloMICS.aspx?Link=T&DB=0&Table=0&Rec=41751&Fields=All&ExactMatch=T 46

Figure 44.7.1.2 Culturing R.oryzae on Agar plate

4.1.3 Preparation of Inoculum To prepare the inoculum, agar plates containing sporulated fungi were washed with 20 ml sterile water to obtain a spore suspension. And 3ml of that was transferred to the new potato dextrose agar plates.

Figure 45.Subculturing of R. oryzae in PDA plates

4.1.4 Culture Conditions The fungus grew and formed spores on potato dextrose agar plates at 30 °C. After 24 hours some signs of growth could be observe on the surface of agar plate and after 4 days incubation the fungi and its spore were spread on the surface of the PDA plates.

Figure 46.PDA cultured plates of R. oryzae in Figure 47.R. oryzae after 4 days incubation in 30 °C incubator (in 30 °C)

4.2 Designing the siRNA

Victorious gene silencing by RNA interference mechanism necessitates a powerful and specific depletion of the target mRNA. Choosing target candidates must be done in a way that their corresponding short interfering RNAs are likely to be effectual against that target and improbable to unintentionally silence other transcripts due to sequence resemblance (Snove et al., 2004).

4.2.1 Target Lactate Dehydrogenase mRNA It is known that there are two genes, ldhA and ldhB, which code for NAD+-dependent L-lactate dehydrogenases (LDH) (EC 1.1.1.27), in R. oryzae (Skory, 2000, OBAYASHIi et al., 1966, Ono et al., 1990, Pritchard, 1971, Yu and Hang, 1991). According to NCBI (NCBI, 2010), ldhA and ldhB genes are similar to each other and exhibit more than 89% nucleotide sequence identity (see Table 9 ) and they contain no introns (Skory, 2000). The first description of ldh genes in a fungus, and sequence comparisons revealed that ldhA and ldhB are distinct from previously isolated prokaryotic and eukaryotic ldh genes. In 2000 Studies on protein sequencing of the LDH isolated from R. oryzae during lactic acid production revealed that that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of ldhA was greatest when glucose is the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid (Skory, 2000, Bai et al., 2003). Also no

transcripts from ldhB were detected when glucose, xylose or trehalose were substrate for R. oryzae. But transcripts from ldhB were detected when R. oryzae was grown on glycerol, ethanol, and lactate (Skory, 2000). So, the mRNA of lhdA was the target of silencing. Sequence of ldhA of R. oryzae CCUG 28958 is shown below:

1 atggtattac actcaaaggt cgccatcgtt ggagctggtg cagtaggagc ctccactgct 61 tatgcactta tgtttaaaaa catttgtaca gaaatcatta ttgtggatgt taatcctgac 121 atcgttcaag ctcaagtcct tgaccttgca gatgctgcca gtataagtca cacgcccatc 181 cgagcaggta gcgcagagga ggcagggcag gcagatattg ttgtcatcac ggccggtgcg 241 aaacaaaggg aaggtgagcc tcggacaaag ctcattgaac gaaacttcag agtgttgcaa 301 agtatcattg gtggcatgca acccattcga ccagacgcag tcatcttggt ggtagcaaat 361 ccagtcgata tcttgacaca cattgcaaag accctctctg gactgcctcc aaaccaggtc 421 attggctccg gtacctacct tgacacgacc cgtcttcgcg tccatcttgg cgatgtcttt 481 gatgtcaatc ctcaatcggt ccatgctttt gtcttgggtg aacatgggga ttcccagatg 541 atcgcttggg aggctgcttc gattggtggg cagccgttga caagtttccc ggaattcgca 601 aagctggata aaacagcaat ttcaaaagcg atatcaggta aagcgatgga gatcattcgt 661 ttgaaaggag ccacgtttta tggaattggt gcctgtgcag cggatttagt gcacactatc 721 atgttgaata ggaaatcagt acatccagtt tctgtttatg ttgaaaagta tggagccact 781 ttttctatgc ctgctaaact tggatggaga ggtgttgaac agatctatga agtaccactg 841 acggaagaag aagaagcgct gcttgtaaaa tctgtagaag cactgaaatc agttgaatat 901 tcatctacaa aagtcccaga aaaaaaagtt catgctactt ccttttctaa aagtagctgt 961 tga

4.2.2 Direct Comparison of Two Sequences In order to design the siRNA it is important to know the part of gene with lowest homology with other genes (Hutvagner and Zamore, 2002, Jackson et al., 2003, Khvorova et al., 2000, Kretschmer-Kazemi Far and Sczakiel, 2003, Krol et al., 2004, Lai, 2002). So ldhA blasted against ldhB to know which part of sequence have to be avoided.

Description: Rhizopus oryzae ldhB gene for lactate dehydrogenase Molecule type: nucleic acid Program :BLASTN 2.2.24+(Altschul et al., 1997)

Table 9.Sequences producing significant alignments

Accession Description Max Total Query E Max score score coverage value ident AB281557.1 Rhizopus oryzae ldhB gene for lactate 1371 1371 100% 0.0 89% dehydrogenase, complete cds, strain: CBS 112.07

4.2.2.1. Dot Matrix View

Figure 48.Plot of ldhA vs. ldhB

4.2.2.2. Alignment

Query 1 ATGGTATTACACTCAAAGGTCGCCATCGTTGGAGCTGGTGCAGTAGGAGCCTCCACTGCT 60 |||||| |||| |||||||| |||||| |||||||||| |||||||| |||||||||||| Sbjct 22 ATGGTACTACATTCAAAGGTTGCCATCATTGGAGCTGGGGCAGTAGGGGCCTCCACTGCT 81

Query 61 TATGCACTTATGTTTAAAAACATTTGTACAGAAATCATTATTGTGGATGTTAATCCTGAC 120 |||||||||||||||||||||||||||||||||||||||||||| ||| ||||||| ||| Sbjct 82 TATGCACTTATGTTTAAAAACATTTGTACAGAAATCATTATTGTTGATATTAATCCCGAC 141

Query 121 ATCGTTCAAGCTCAAGTCCTTGACCTTGCAGATGCTGCCAGTATAAGTCACACGCCCATC 180 |||||||||||||||||||||||||||||||| || |||||| |||| ||||||||||| Sbjct 142 ATCGTTCAAGCTCAAGTCCTTGACCTTGCAGACGCAGCCAGTGTAAGCAACACGCCCATC 201

Query 181 CGAGCAGGTAGCGCAGAGGAGGCAGGGCAGGCAGATATTGTTGTCATCACGGCCGGTGCG 240 |||||||| || |||||||||||||||||| |||||||| |||||||||||||||||||| Sbjct 202 CGAGCAGGCAGTGCAGAGGAGGCAGGGCAGTCAGATATTATTGTCATCACGGCCGGTGCG 261

Query 241 AAACAAAGGGAAGGTGAGCCTCGGACAAAGCTCATTGAACGAAACTTCAGAGTGTTGCAA 300 ||||||| |||||||||||||||||||||||||||||||||||| | |||||||||| || Sbjct 262 AAACAAAAGGAAGGTGAGCCTCGGACAAAGCTCATTGAACGAAATTACAGAGTGTTGAAA 321

Query 301 AGTATCATTGGTGGCATGCAACCCATTCGACCAGACGCAGTCATCTTGGTGGTAGCAAAT 360 | ||||||||||||||||||||| |||||| |||||||| ||||||||||||||| |||| Sbjct 322 AATATCATTGGTGGCATGCAACCTATTCGATCAGACGCAATCATCTTGGTGGTAGTAAAT 381

Query 361 CCAGTCGATATCTTGACACACATTGCAAAGACCCTCTCTGGACTGCCTCCAAACCAGGTC 420 |||||||||||||||||||||||||| |||| |||||||||||| | ||||||||||| Sbjct 382 CCAGTCGATATCTTGACACACATTGCTCAGACACTCTCTGGACTGGCACCAAACCAGGTG 441

Query 421 ATTGGCTCCGGTACCTACCTTGACACGACCCGTCTTCGCGTCCATCTTGGCGATGTCTTT 480 |||||||||||||||||||||||||||||||| ||||| ||||||||||| ||| ||||| Sbjct 442 ATTGGCTCCGGTACCTACCTTGACACGACCCGCCTTCGTGTCCATCTTGGTGATATCTTT 501

Query 481 GATGTCAATCCTCAATCGGTCCATGCTTTTGTCTTGGGTGAACATGGGGATTCCCAGATG 540 |||||||||||||||||| |||||||||||||||||||||| || |||||||| |||||| Sbjct 502 GATGTCAATCCTCAATCGATCCATGCTTTTGTCTTGGGTGAGCACGGGGATTCACAGATG 561

Query 541 ATCGCTTGGGAGGCTGCTTCGATTGGTGGGCAGCCGTTGACAAGTTTCCCGGAATTCGCA 600 ||||||||||||||||||||||||||||||||||||||||||||||| || ||||||||| Sbjct 562 ATCGCTTGGGAGGCTGCTTCGATTGGTGGGCAGCCGTTGACAAGTTTTCCAGAATTCGCA 621

Query 601 AAGCTGGATAAAACAGCAATTTCAAAAGCGATATCAGGTAAAGCGATGGAGATCATTCGT 660 |||||||||||| |||||||||||||||||||||||| ||||||||||||||||||||| Sbjct 622 GAGCTGGATAAAAAAGCAATTTCAAAAGCGATATCAGGCAAAGCGATGGAGATCATTCGT 681

Query 661 TTGAAAGGAGCCACGTTTTATGGAATTGGTGCCTGTGCAGCGGATTTAGTGCACACTATC 720 ||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||| Sbjct 682 TTGAAAGGAGCCACGTTTTATGGAATTGGTGCCTGTGCAGCGGATTTAGTGCATACTATC 741

Query 721 ATGTTGAATAGGAAATCAGTACATCCAGTTTCTGTTTATGTTGAAAAGTATGGAGCCACT 780 ||||||||||||||||||||||||||||||||||||||||||||||| ||||||| |||| Sbjct 742 ATGTTGAATAGGAAATCAGTACATCCAGTTTCTGTTTATGTTGAAAAATATGGAGTCACT 801

Query 781 TTTTCTATGCCTGCTAAACTTGGATGGAGAGGTGTTGAACAGATCTATGAAGTACCACTG 840 ||||||||||| ||||||||||||||||||||||||||| |||| ||||| ||||||||| Sbjct 802 TTTTCTATGCCAGCTAAACTTGGATGGAGAGGTGTTGAAAAGATATATGAGGTACCACTG 861

Query 841 ACGGAAGAAGAAGAAGCGCTGCTTGTAAAATCTGTAGAAGCACTGAAATCAGTTGAAT-- 898 ||||||||||| |||||||||||| ||||||||||||| ||||||||| ||||||||| Sbjct 862 ACGGAAGAAGAGGAAGCGCTGCTTTTAAAATCTGTAGAGGCACTGAAAGCAGTTGAATAT 921

Query 899 ------ATTCATCTACAAAAGTCCCAGa 920 ||||||||| |||| ||||||| Sbjct 922 TTATCATAATTTATACTCATAAAAATAAAAATGGTGTCATTCATCTATAAAAATCCCAGA 981

Query 921 aaaaaaaGTTCATGCTACTTCCTTTTCTAAAAGTAGCTGTTGA 963 ||||||||| ||| ||||||||||||| ||||||| |||||| Sbjct 982 AAAAAAAGTCCATACTACTTCCTTTTCCAAAAGTAAATGTTGA 1024

4.2.3 siRNA Design To pick up an efficient design of siRNA molecules of, Shabalina et al. performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature(Shabalina et al., 2006). They used this training set to select siRNA features and optimize computational models and identified 18 parameters. A number of these parameters characterize only the siRNA sequence, whereas others are connected to the whole mRNA. Most significantly, they derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand (Khvorova et al., 2000, Shabalina et al., 2006). In the design of siRNA for this work the authors preferably used A (adenosine) at the 1st position of antisense strand and choosing G at positions 1–3 and C at the 1st position and at the positions 13–14th G. (Jackson et al., 2003), at The 17–19th positions A was avoided. C and U were preferred in the 17th and 18th positions and C and G in the 19th position as well (Ding et al., 2004, Krol et al., 2004, Matveeva et al., 2003).

Length of siRNA It is believed that the immune system is capable of distinguishing between self- and nonself-nucleic acids, based on features that are not fully understood (Hajeri and Singh, 2009). Also studies demonstrated that dsRNAs of greater than 30 bases can activate the interferon response, leading to apoptosis, and smaller RNAs (>30 base pairs) can induce RNAi without activating the interferon response(Stevenson, 2003). Sledz et al. found that transfection of siRNAs cause the interferon (IFN)- mediated activation of the Jak-Stat pathway and global upregulation of IFN-stimulated genes. This

effect was mediated by the dsRNA-dependent protein kinase, PKR, which was activated by 21-base- pair (bp) siRNAs and required for upregulation of IFN-beta in response to siRNAs (Sledz et al., 2003).

Off-Targeting Off-target effects could be due to two factors: the binding of siRNAs to sequences other than the target, resulting in their suppression or the induction or enhancement of innate immune response by some motifs or patterns in the siRNAs. As Birmingham et al (Birmingham et al., 2006) reported, offtarget effects mostly take place as a result of 3´-UTR seed matches, rather than overall homology between the siRNAs and targets (Xuan et al., 2006, Kittler and Buchholz, 2005, Jackson et al., 2006). Finally considering the above mentioned criteria and minimum 55% GC content using BLOCK-iT™ RNAi Designer (Invitrogen) (Invitrogen, 2010) was used to design and order siRNA (see Table 10).

Table 10.siRNA used in this work

N0. Type Sequence Quantity

1 Sense GGAGGCAGGGCAGGCAGAUAUUGUU 80NMOL

2 Antisense AACAAUAUCUGCCUGCCCUGCCUCC 80NMOL

4.3 Fungal Transformation

The first DNA-mediated transformation of the fungi was performed at 1973 in the laboratory of E. L. Tatum in Rockefeller University. They used Neurospora crassa strain for transformation which was an inositol-requiring mutant (inl). For transformation process, the growing cultures with DNA isolated from the wild type (inl+) and calcium were prepared. After cultivation they selected inositol- independent strains from the conidiospores (conidia) formed on the cultures (Fincham, 1989, Mishra and Tatum, 1973). Different transformation systems have been developed for the fungi. Some of the methods that have been used in different species of the fungi were gathered in Table 11 :

Table 11.Transformation methods in various fungi

Transformation Methods Species References Calcium chloride /polyethylene Neurospora (Case et al., 1979) glycol (PEG) Aspergillus (Yelton et al., 1984) Lithium acetate Saccharomyces (Clancy et al., 1984) Coprinus (Binninger et al., 1987) Electroporation Neurospora (Chakraborty et al., 1991) Penicillium (Chakraborty et al., 1991) Microparticle bombardment Saccharomyces (Armaleo et al., 1990) Neurospora (Armaleo et al., 1990) Trichoderma (Lorito et al., 1993) Agrobacterium tumefaciens- Aspergillus (de Groot et al., 1998) mediated transformation (ATMT)

4.3.1 Labeling siRNA We used acridin orange dye solution for labeling siRNA to trace it in the transformed R. oryzae cells under the microscope. AO is a fluorescent dye which used to distinguish between DNA and RNA in the cell. AO interacts with RNA and its luminescence is in red wavelength with maximum emission at 640 nm. The acridin orange can also stain DNA with an excitation maximum at 502 nm and an emission maximum at 525 nm which is in green wavelength (Darzynkiewicz, 1990, Beltrame et al., 1981). Acridin orange was supplied as a dye solution in our experiments but if the powder of dye was available then dye solution could be prepared via the Protocol No. 1.

The siRNA that was supplied from Invitrogen dried in a buffer solution and we had to dilute it with RNase free water to reconstitute the buffer to10mM Tris-HCl; pH=8, 20mM NaCl, 1mM EDTA. The storage and resuspension of duplex siRNA was described in Protocol No. 24. In this experiment, 5 µg of 25-mer duplex siRNA at 20 µM was used for labeling reaction. Stock solution of siRNA was prepared and afterward by using Protocol No. 23. The volume of duplex siRNA was calculated to find the sufficient volume consist of 5 µg siRNA.

We used two forms of siRNA for labeling procedure:

1. siRNA from 20 µM working stock solution prepared according to Protocol No. 27. 2. siRNA which cleaned up before labeling (See Protocol No. 28)

The siRNA that was cleaned and labeled via Protocol No. 25 and Protocol No. 28 should be treated with ethanol precipitation step to remove unreacted dye solution; it was performed by using the Protocol No. 29.

Figure 49.The pellet of labeled siRNA

4.3.2 Inducing siRNA in Rhizopus oryzae After preparation of labeled siRNA, induce it to the fungal cells is the next step. In this study Calcium chloride /polyethylene glycol (PEG) was used to transform R. oryzae with siRNA by using the Protocol No. 30. As it was mentioned in the protocol the preparation of the protoplast is required for this method of transformation.

Figure 50.R.oryzae cells under the microscope

4.3.3 Protoplast Preparation Transformation in fungi consists of many limitations because of the different cell wall specifications in fungal species. Some of the transformation methods need protoplast preparations which can be difficult in some species because lytic enzymes cannot perform sufficiently and digest the cell properly (Latterich, 2008). As it was described in part 2.2.2, Yoshio Tominaga and Yoshio Tsujisaka in1981 noticed that the cell wall of R. oryzae might be composed mainly of chitin fibers cemented by chitosan and protein or peptides scattered in a mosaic manner, so they used purified lytic enzymes, protease chitosanase and chitinase for digestion of the cell wall (TOMINAGA and TSUJISAKA, 1981).

In this project we tried two different enzymes for degrading the cell wall of the R. oryzae. According to Protocol No. 15 and Protocol No. 16, we used Lysozyme and Yatalase to prepare two different digestion buffers.The enzymes were used in protoplast preparation supplied from: 1. Lysozyme from chicken egg white (dialyzed, lyophilized, powder, ~100000 units/mg) which was supplied from Sigma-Aldrich, Fluka Biochemika, 62970-5G-F, Lot # BCBB3704 (See Protocol No. 15). 2. Yatalase from culture supernatants of Corynebacterium sp. OZ-21(Lyophilized powder containing lactose) which was supplied from Takara Bio, T017 (See Protocol No. 16). Yatalase™ is used to lyse cell walls of filamentous fungi and it consists mainly of chitinase, chitobiase and β-1, 3-glucanase.

The above prepared digestion enzymes were used for protoplast preparation of R. oryzae according to the Protocol No. 19 and Protocol No. 20. .

Figure 51.Harvested mycelia used for protoplast preparation

4.3.4 Transformation of R. oryzae by the (CaCl2/ PEG) method PEG-mediated transformation of R. oryzae is a modified method that was originally used by vollmer and yanofsky in 1986 (Vollmer and Yanofsky, 1986). CaCl2/ PEG method is applicable for some species such as M. oryzae, Neurospora, Aspergillus and Fusarium (Nakayashiki et al., 2005). The method that we used for transformation has been described in Protocol No. 30, the labeled siRNA used to induce to the protoplast.

Figure 52.Transformed R. oryzae

Table 12 indicates the variety of knockdown samples used in the current work. This table clarifies the procedures used for preparation of each sample. For instance in knockdown sample #1 the siRNA used for transformation taken directly from siRNA stock solution (20 µM). The cleanup procedure was not performed and labeling step used according to Protocol No. 27. Since cleaning up step was not implemented so the whole procedure did not continue with the ethanol precipitation. The volume of sample #1 was 100 µl. Transformation with this labeled siRNA was occurred by Protocol No. 30 and

the transformed protoplasts with siRNA were spread on the surface of PDA plate then incubation was done.

Table 12.Variation in sample preparation

Cleaned Labelling Ethanol V Transform Cultured Mixed up siRNA siRNA precipitation of (μl) ation on agar with agar Labelled RNA Knockdown #1 N Y N 100 Y N Y Knockdown #2 N Y N 100 Y Y N Knockdown #3 N Y N 50 Y Y N Knockdown #4 Y Y Y 50 Y N Y Knockdown #5 Y Y Y 50 Y Y N Knockdown #6 Y Y Y 100 Y Y N

4.4 Fermentation Test

To examine the efficiency of knockdown a fermentation test was carried out with both wild type and knockdown R.oryzae.So, eight fermentation sets were set up. In the following there are details of preparation of fermentors:

4.4.1 Equipment: 500 ml Erlenmeyer flasks were used as fermentors because the volume of the medium was 150 ml and the 500 ml is proper volume considering the shaking required during fermentation. A silicon cap was used to close the Erlenmeyer flask. Silicon caps drilled and a glass fermentation lock was passed through each silicon cap to let the gases out the fermentor and also a sampling pipe was passed through the silicon cap for sampling without letting the air go inside the fermentor. A set of cork borer is needed to make a proper bore in the silicon cap. Using some drops of glycerol can make drilling easier also it is much safer to pass the glass fermentation lock and sampling pipe through the cap when the hole is lubricated with glycerol. Wash the cap to clean the remained glycerol. Attach the hose with a needle to the end of sampling pipe and clean them all with compressed air. The hose should be closed by the clamp while sampling is not performed from the fermentor (See Figure 58).

Figure 53.Pliers, cork borer and other equipment needed to set a fermentor

Figure 57.A set of cork borer Figure 56.A glass Figure 55.Sampling Figure 54.assembled cap fermentation lock hose with needle

Figure 58.Completly assembled Fermentors

4.4.2 Preparation of Media The media which was used in this work was same as the general Semi-Synthetic Growth Medium for Cultivation of Zygomycetes Fungi (see Protocol No. 12). In this media there are normally following components: Materials: Glucose Yeast extract

(NH4)2SO4 (See Protocol No. 9)

KH2PO4 (See Protocol No. 9)

CaCl2.2H2O (See Protocol No. 9)

MgSO4.7H2O (See Protocol No. 9) Vitamin solution (See Protocol No. 11) Trace metal solution (See Protocol No. 10) Inoculum (See Protocol No. 33) 2M NaOH The media was prepared according Protocol No. 12. So, The fermentation medium contained (per 150 ml): 4.5 g glucose, 3.0 g (NH4)2SO4, 0.75 g MgSO4,7H2O, 3.0 g KH2PO4, 0.75 g CaCl2.2H2O, 0.75 g yeast extract , 150 µl vitamin solution and 1.5 ml trace metal solution.

4.4.3 Inoculation: The PDA plates contain grown R. oryzae were washed with 20 ml of distilled RNase free water and 3 ml of that suspension transferred to each fermentor in order to start fermentation (see Protocol No. 33).

4.4.4 The incubation The incubation was carried out for 62 h in rotary shaker with an agitation rate of 140 rpm and 30 °C.

Figure 59.Incubation of R. oryzae in submerged batch culture

4.5 Analytical method - HPLC

The measurement of ethanol, glycerol, lactic acid, pyruvic acid, succinic acid, citric acid and glucose was performed by High-performance liquid chromatography (alliance/waters 2695 ) (see Figure 60). The separation system was consisted of:

I. A solvent delivery system (equipped with an autosampler II. A refractive index detector (410 differential refractometer, Waters) III. A dual λ absorbance detector (2487, Waters) IV. A computer software (Waters:Empower). V. There were two ion moderated partition chromatography columns: a. Aminex HPX-87P with De-ashing and Carbo-P micro-guard cartridges b. Aminex HPX 87H with Cation H micro-guard cartridge. For this study the Aminex HPX- 87H (Bio-Rad, USA) column was used and maintained at 65 °C,

the sugars, organic acids, and ethanol were eluted with 5 mM H2SO4 prepared using ultrapure water at a flow rate of 0.6 mL min-1. Samples were prepared according Protocol No. 34 and standard were made according Protocol No. 35. Peaks were detected by refractive index and UV absorption (210 nm) and were identified and quantified by comparison to retention times of authentic standards (ethanol, glycerol, lactic acid, pyruvic acid, succinic acid, citric acid and glucose) according Protocol No. 36.

Figure 60.HPLC alliance/waters 2695

4.6 Growth Curve

When fungal cells are inoculated into a medium and incubated under optimal growth conditions, then it is the time to analysis the growth curve of desired fungi. Typically the fungal growth curve consists of three phases: lag, exponential and stationary phases.

The lag phase represents a period that the inoculated cells require to adapt to the new growth culture and their population growth is zero. The exponential phase is a period which cells or mycelia biomasses in filamentous fungi grow with logarithmic rate The stationary phase shows a period that the accumulated biomass remains constant and the specific growth rate returns to zero which means that again the population growth of the fungus cells become zero. In this step the cells may die and autolysis (Kavanagh, 2005).

In this experiment two flask fermentors were run with the semi synthetic growth medium according to the Protocol No. 12 and then inoculated with spores of R. oryzae wild type strain according to the Protocol No. 33 and rapidly taken the sample from both fermentors with using the Protocol No. 34. These samples were the samples in time zero. Then continue with sampling once an hour for 15 hours.

We measured the OD600 of the cell suspensions of each sample exactly at the time of sampling. On the other hand we putted one drop of each sample on the Bürker counting chamber 0.100 mm and count the cells (hyphae). We decided to draw a growth curve for the wild type of R. oryzae by using the OD over time and also the number of cells over time but we could follow to count number of cells just 9 hours after running the fermentors not more because the mycelia was constructed and counting of them became so difficult. Figure 61 shows the cells under the microscope after 7 hours passed from running the fermentors. Figure 62 illustrate the mycelia cell after 9 hours passed from running the fermentors. Figure 63 shows the mycelia web after 10 hours passed from running the fermentors and display how it was difficult to distinguish between the cells and counting them.

Figure 61.Bright field of R. oryzae after 7 h in the batch culture with 10X magnification

Figure 62.Bright field of R. oryzae after 9 h in the batch culture with 10X magnification

Figure 63.Bright field of R. oryzae after 10 h in the batch culture with 10X magnification

5. Chapter 5 – Results

The results of cultivation of the filamentous fungi R. oryzae are summarized here in this chapter.

5.1 Lactic acid Production

After RNA silencing, as shown in Table 13 Table 13.Lactic acid production in R. oryzae in all 6 samples lactic acid production was reduced comparing with wild types. The Sample Concentration (g/l) Yield (g/g) average concentration of lactic acid production detected by UV detector was Knockdown #1 0.23 0.01 Knockdown #2 0.29 0.01 0.36 g/l in knockdown samples while the Knockdown #3 0.20 0.01 average of lactic acid production by wild Knockdown #4 0.84 0.03 types of Rhizopus oryzae was 2.06 g/l Knockdown #5 0.41 0.01 which represents 5.7 times decrease. Knockdown #6 0.20 0.01 Wild #1 2.13 0.07

Wild #2 2.00 0.07 These results suggest that this reduction in lactic acid production is due to the siRNA. The lactic acid concentration in fermentation media was also detected by RI detector via HPLC which its results were same as UV detector.

3,00

2,00

1,00 concentration (g/l) concentration

0,00

Chart 3. Lactic acid production in Knockdown and wild types of R. oryzae, detected by UV in HPLC

5.2 Ethanol Production

HPLC results showed that ethanol production Table 14.Ethanol production in R.oryzae have been increased after RNA silencing of lactate dehydrogenase gene, since the average Sample Concentration (g/l) Yield concentration of ethanol production has been (g/g) increased from 11.82 g/l in wild types to 13.37 Knockdown #1 13.51 0.45 g/l in knockdown samples (see Table 14 and Knockdown #2 13.41 0.45 Chart 4). The average yield of ethanol from Knockdown #3 13.57 0.45 glucose was increased from 0.39 g/g in wild Knockdown #4 13.37 0.45 types to 0.45 g/g in samples which their lactate Knockdown #5 13.12 0.44 Knockdown #6 13.25 0.44 dehydrogenase gene was silenced. Wild #1 12.34 0.41 Wild #2 11.30 0.38

14,00

13,50

13,00

12,50

12,00

Concentration (g/l) Concentration 11,50

11,00

Chart 4. Ethanol production in Knockdown and wild types of R. oryzae, detected by RI in HPLC

5.3 Growth Curves

The growth curves for the wild type of R. oryzae obtained according to the procedure described in section 5.34.6. Two fermentors were run with the same growth condition. Semi synthetic medium used for the flask fermentors and inoculated with the spores of R. oryzae. OD 600 (nm) was used and the Chart 5 and Chart 6 illustrate the changes of OD over time for both fermentors.

0,45 0,4 0,35 0,3 0,25 0,2 0,15 (600 nm) OD 0,1 0,05 0 -0,0500:00 04:48 09:36 14:24 19:12

Time (h)

Chart 5.Graphical representation of growth curve of R.oryzae in semi synthetic growth media in fermentor #1 according changes OD over Time

1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 (600 nm) OD 0,2 0,1 0 -0,100:00 04:48 09:36 14:24 19:12 Time (h)

Chart 6.Graphical representation of growth curve of R.oryzae in semi synthetic growth media in fermentor #2 according changes OD over Time

6. Chapter 6 –Discussion

Many studies have been performed in RNA interference (RNAi) in fungi, this story was began when in 1992 Machino and Romano could reduce the expression of the al-1,endogenous gene, responsible for carotenoid biosynthesis with the transformation of homologous al-1 sequences in the fungus Neurospora crassa (Nakayashiki and Nguyen, 2008b, Romano and Macino, 1992). As it was presented in Table 2 RNAi technique was used in various types of fungi except the Rhizopus oryzae strain. This is the first description of silencing ldhA in R. oryzae by using direct delivery of siRNA.

The objective of this study was to investigate whether RNAi technique can be used to silence the ldhA in R. oryzae. To determine it the siRNA was induced to the protoplasts of R. oryzae. We designed duplex siRNA with considering some specification associated with siRNA functionality and ordered it to the Invitrogen Company to construct it for us. Protoplast preparation of R. oryzae was the next step that we were dealing with in this experiment because of the specific characteristics of the fungal cell wall. Two enzymes were supplied for digestion of the cell wall of R. oryzae; Lysozyme and Yatalase.

The Calcium chloride /polyethylene glycol (CaCl2/PEG) method was used to transform R. oryzae with siRNA and then the fermentation process performed to reveal the silencing efficiencies of ldh gene. Analysis the results of transformed R. oryzae and compare them with the wild type was conducted by HPLC.

6.1 Labelling of siRNA

Acridine orange was used in this experiment to label the siRNA and trace it into the transformed R. oryzae cells under the Fluorescence microscope. The specimens were prepared from the spores on the surface of transformed agar plate that should be consists of siRNA but no specific red pieces were detected under the microscope. As it was described in section 4.3.1 the fluorochrome acridine orange (AO) can detect the RNA and DNA but this dye can differentially stain double strand DNA versus single strand RNA and this means that all double stranded RNA (dsRNA) should be denatured first and became to single stranded RNA (Darzynkiewicz, 1990). Unfortunately we did not know this fact and as a result we could not trace the siRNA in transformed cells. But in the current work, there was two possibilities suggested to distinguish between wild types and transformed cells with siRNA by using AO dye:

1. Labeling single stranded siRNA before transformation Since AO stains single stranded RNA so one of the following methods could be used to denature dsRNA to ssRNA then label them and use the ligase enzyme to reconstruct dsRNA and finally use them for transformation. First denaturation of double stranded RNA could be done by PCR and then the labeling should be performed by using AO. Second method which could be used for labeling siRNA was described by Darzynkiewicz et al. and Traganos et al. (Darzynkiewicz et al., 1974, Traganos et al., 1977). Chelating agents (EDTA, citrate) should be used to achieve the selective denatured RNA (Darzynkiewicz, 1990).

2. Labeling the mRNA after transformation To distinguish which cells were succeeding in the transformation process with siRNA, the labeling of mRNA after transformation could be performed. The spore cells of transformed R. oryzae 65

should be prepared from the surface of the agar plate, used the AO to stain the cells then compared the specimens from the wild type and transformed cells under the microscope. According to the RNAi technology described in section 3.3 (Mechanism) while double stranded RNA induced to the cell, the RISC complex sticks to siRNA and make it single strand, then the single stranded siRNA with the RISC complex bind to the single stranded mRNA and finally the RISC degrade both the mRNA and siRNA. So the lower amount of red single stranded mRNA should be visible in the transformed R. oryzae cells (with siRNA) under the microscope compare to the wild type cells.

6.2 Defining Growth Pattern of R. oryzae

Chart 5 and Chart 6 show the experimental growth curve we could illustrate for R. oryzae. As described in section 4.6 the growth curves were obtained by running two fermentors with the same condition just as a replicate. Results revealed main phases of the growth. At first by adding the inoculums to the fermentors the OD reduction in both fermentors was seen and it may be occurred because of the adaptation of the cells to new growth media until they reach to the lag phase. During the lag phase, the cell population growth is zero and fungi mainly synthesize ribosomes and enzymes and it takes around 5 hours. Then the exponential phase which starts from 7 hours after running the fermentors and continue for 5 hours. During this phase cells are undergoing primary metabolism, clearly those metabolic pathways that are essential for growth of the cell (Kavanagh, 2005).

Between the lag phase and the exponential phase there is a long transition period which is one of the characterizations of the fungi growth curve and the smooth graph is another characterization of the filamentous fungi growth curve which depends on the species and the growth medium (Meletiadis et al., 2001). The exponential phase is an important phase when the cell biomass production is required for producing interested products therefore the extension of this phase is desired for the industrial fermentations and it could be achieved by using fed batch or continuous culture instead of batch culture. In current work the exponential phase was followed by the death or logarithmic decline phase just 12 hours after running the fermentors and the stationary phase was not detected by the spectrophotometer in OD 600. Analysis the data by HPLC revealed that ethanol was produced by the R. oryzae exactly 12 hours after running the fermentors and this was the time that the growth curve continues with the death phase instead of stationary phase. It seems that exhaustion of an essential nutrient and beside that the existence of the produced ethanol in the medium provided a toxic media for the R. oryzae cells and they could not tolerate the new condition and died. As the stationary phase demonstrates the ability of fungi to survive for prolonged periods without added nutrients (Kavanagh, 2005) but based on the obtained results and lacking the stationary phase in the growth curve we could conclude that R. oryzae cannot survive for a long time in the batch culture because of the ethanol production along the fermentation process.

6.3 Reduction of Lactic Acid Production

The results show the success in reducing the lactic acid production and increasing the ethanol yield with silencing ldhA gene in R. oryzae. Production of lactic acid was decreased from 2 g/l in wild type to 0.2 g/l in silenced R. oryzae i.e. 10 folds reduction in best situation and 5.7 fold in average. We could not stop the lactic acid production because of: 66

The silencing process was not performed properly in RNase free condition. RNase free tips and water were used along the experiment but the flasks and other laboratory instruments may not be RNase free appropriately. Two types of genes are involved in lactic acid production in R. oryzae; ldhA and ldhB. ldhA encodes an NAD+-dependent L-LDH which is responsible for most of lactic acid production by R. oryzae. In current study just the silencing of ldhA was performed but still ldhB could be expressed and it was able to produce lactic acid (Skory, 2000). In 2000, Skory et al. described a hypothesis that at least three different LDH are produced by R. oryzae; LdhA and LdhB require the cofactor NAD+ and the third enzyme is probably a mitochondrial NAD+ -independent LDH that is used for oxidative utilization of lactate (Skory, 2000).

6.4 Increasing Ethanol Production

As a consequence of lactic acid reduction the glucose as a carbon source should convert to the other products. Figure 64 (Skory and Ibrahim, 2007) shows the main fermentation products by R. oryzae if the lactate dehydrogenase ( ―a‖ enzyme) was silenced then the yield of other products such as ethanol, glycerol and fumarate should be increased. According to the results (see Chart 4) the yield of ethanol production increased from 0.39 g/g to 0.45 g/g.

Figure 64. Production of major fermentation products by R. oryzae. Enzymatic activation shown as: a lactate dehydrogenase; b pyruvate decarboxylase; c alcohol dehydrogenase; d pyruvate carboxylase; e malate dehydrogenase; fumarase

Table 15.Comparision on different sample preparation with Lactic acid and ethanol production

Ethanol Cleaned Labelli Mixed Lactic precipitation V Transfor Cultured EtOH up ng with acid of Labelled (μl) mation on agar* (g/g) siRNA siRNA agar■ (g/g) RNA Knockdown N Y N 100 Y N Y 13.51 0.23 #1 Knockdown N Y N 100 Y Y N 13.41 0.29 #2 Knockdown N Y N 50 Y Y N 13.57 0.20 #3 Knockdown Y Y Y 50 Y N Y 13.37 0.84 #4 Knockdown Y Y Y 50 Y Y N 13.12 0.41 #5 Knockdown Y Y Y 100 Y Y N 13.25 0.20 #6 *Means that the transformed R. oryzae have been spread on the surface of the agar ■ Means that the transformed R. oryzae have been mixed with melted agar

Effect of Inoculum size Inoculum size was shown to influence the lactate, ethanol and biomass productions of R. oryzae in different studies (Büyükkileci et al., 2006). In this study and in the sample number 4 of knockdowns we observed a higher amount of lactic acid production even if the samples have been prepared in different ways as described in Table 15 but this difference in amount of lactic acid production that can be due to inoculum size.

When Rhizopus oryzae ferments glucose-1-C14, label appears predominantly in the methyl carbon of lactic acid and in the methyl carbon of alcohol. The labelling found indicates that R. oryzae converts glucose to lactic acid both aerobically and anaerobically in accordance with the Embden- Meyerhof- Parnas scheme. In 1951 Carson et al. indicated another pathway of lactic acid formation by Rhizopus where O2 is considered to play no role in the formation of lactic acid, except to suppress glycolysis, i.e., the Pasteur reaction. They reported that lactic acid could be synthesized oxidatively by Rhizopus via the 4-carbon dicarboxylic acids (Carson et al., 1951). But Gibs and Gastel claimed that under the oxidative conditions employed in these experiments the carbon atoms of glucose did not yield lactate via the 4-carbon dicarboxylic acids (Gibbs and Gastel, 1953).

6.5 Statistical Analysis of Lactic Acid Production Results

R program used to illustrate the statistical graphs and define whether silencing ldh gene in R. oryzae could significantly reduce the lactic acid production or not. Following commands typed in the R and it calculated the mean and standard deviation for the lactic acid production by transformed (silenced) and wild type (non-silenced) samples. The mean of lactic acid production in transformed samples was equal to 0.36 g/l and the standard deviation was equal to 0.25 g/l. On the other hand the mean of lactic acid production in wild type samples was 2.06 g/l and the standard deviation was 0.09 g/l.

6.5.1 Commands: > x1<- c(0.23,0.29,0.2,0.84,0.41,0.2) > x2<-c(2.13,2.00) > mean(x1) [1] 0.3616667 > mean(x2) [1] 2.065 > sd(x1) [1] 0.2473392 > sd(x2) [1] 0.09192388 > hist(x1) > hist(x2) > boxplot(x1) > boxplot(x2) > qqnorm(x1) > qqline(x1) > qqnorm(x2) > qqline(x2)

6.5.2 Graphs: Histogram, Box plot and Normal Q-Q plot for the lactic acid production by the transformed (silenced) and wild type samples were displayed in Chart 7:

Chart 7.Statistical plots for lactic acid production in silenced samples (x1) and wild type samples (x2) taken from R

Results obtained in silenced samples (x1) and wild type samples (x2) have been illustrated in the above plots and because of the outliers at the both end of the Normal Q-Q plots we assumed data are not normally distributed.

6.5.3 Statistical test: The null hypothesis was that the lactic acid production in both silenced and wild type samples were not significantly different and the alternative hypothesis was that the lactic acid production in both silenced and wild type samples were significantly different. Since the calculated value was larger than the critical value then we rejected the null hypothesis at significance level 5%. Wilcox test of one-sided was performed in the R and the following data was achieved:

> wilcox.test(x1,x2,alternative="less",mu=0) Wilcoxon rank sum test with continuity correction data: x1 and x2 W = 0, p-value = 0.03257 alternative hypothesis: true location shift is less than 0

The p-value =0.03257 while the significant level α=0.05, this shows that the p-value is less than α (P-value=0.03257<α = 0.05) then we could conclude that the lactic acid production in silenced samples were significant lower than the produced lactic acid in wild type samples and we could reject the null hypothesis. Using the statistical test revealed that silencing ldh gene in R. oryzae could significantly reduce the lactic acid production.

6.6 Statistical Analysis of Ethanol Production Results

In this part R program was used to define whether silencing ldh gene in R. oryzae could significantly increase the ethanol production or not. Following commands typed in the R, mean and standard deviation was determined for transformed (silenced) and wild type (non-silenced) samples.

6.6.1 Commands: > x3<-c(13.51,13.41,13.57,13.37,13.12,13.25) > x4<-c(12.34,11.30) > mean(x3) [1] 13.37167 > sd(x3) [1] 0.1661826 > mean(x4) [1] 11.82 > sd(x4) [1] 0.735391 > op=par(mfrow=c(3,2)) > hist(x3) > hist(x4) 70

> boxplot(x3) > boxplot(x4) > qqnorm(x3) > qqline(x3) > qqnorm(x4) > qqline(x4)

Ethanol production in silenced samples: Mean =13.37 g/l Standard deviation = 0.17 g/l

Ethanol production in wild type samples: Mean = 11.82 g/l Standard deviation = 0.73 g/l

6.6.2 Graphs: Histogram, Box plot and Normal Q-Q plot for the ethanol production by the transformed (silenced) and wild type samples were displayed in Chart 8:

Chart 8.Statistical plots for ethanol production in silenced samples (x3) and wild type samples (x4) taken from R

In this case we also assumed non-normality distribution in silenced and wild type samples.

6.6.3 Statistical test: Because the data were not normal distributed so the Wilcox test of one-sided was performed in the R and the following results obtained:

> wilcox.test(x3,x4,alternative="greater",mu=0) Wilcoxon rank sum test data: x3 and x4 W = 12, p-value = 0.03571 alternative hypothesis: true location shift is greater than 0

The p-value =0.03 while the significance level was 5%; P-value=0.03 < α = 0.05 so reject the null hypothesis and accept the alternative hypothesis. This means that the ethanol production in silenced samples were significantly higher than the produced ethanol in wild type samples. The null hypothesis was rejected. Using the statistical test revealed that silencing ldh gene in R. oryzae could significantly increase the ethanol production.

7. Chapter 7 – Conclusions

7.1 Conclusions

RNA silencing approaches have been quickly developed and employed in plants, animals and many fungus species as a tool for exploring gene function. By conducting this study it was shown for the first time that RNA silencing is applicable to be employed in the filamentous fungus R. oryzae in order to trigger the metabolism to produce one specific product (here ethanol).

Accomplishing this experiment and studying several other experiments done by erstwhile researchers we also concluded that the RNA silencing approach has advantages and disadvantages over other gene knockout strategies. Its ability to suppress gene expression relatively easily and quickly can be one major advantage of RNA silencing. We were not able to silence the target gene 100% and this can be regarded as inefficiency for RNA silencing.

7.2 Limitations of the Study

Since we had not access to RT-PCR machine at the moment of performing this study we were not able to measure the gene transcript and reaction efficiencies for wild type and transformants.

7.3 Further Works

7.3.1 RT-PCR analysis Total cDNA can be generated by reverse transcription with the suitable kit. The amount of transcript in relation to gene transcript can be determined by real-time PCR, which is based on the high-affinity double stranded DNA-binding dye and can be performed in triplicate in a spectrofluorometric thermal cycler with the primers.

7.3.2 RNA extraction and Northern blot analysis Total cell RNA can be purified with a suitable kit to be separated by agarose-formaldehyde gel electrophoresis blotted onto a membrane and finally hybridized with a labelled probe. To make this probe, the bar gene fragment have to be PCR amplified with oligonucleotide primers and a template.

7.3.3 Protein extraction and determination of LDH activity The lactate dehydrogenase enzyme can be extracted and its amount can be measured.

7.3.4 Construction of silencing vectors Since this study proved that the RNA silencing is applicable in these filamentous fungi, Construction of silencing vectors is the next step complementary to this thesis work.

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1. Appendix A- Protocols

Protocol N0. 1: Acridine Orange Stain Recipe

Date of Preparation: 4 May 2010 Revision # 00 References: (theLabRat.com, 2005b) Estimated Time: 10 min

Acridine Orange Stain Recipe

Materials:

Acridine orange stain Distilled water

Procedure:

1. Dissolve 1g of acridine orange to 100 ml of distilled water. 2. Store in a foil wrapped container at 4°C. 3. Bring to room temperature prior to use.

Note:

Acridine Orange is a nucleic acid selective metachromatic stain useful for cell cycle determination.

We used solution of acridine orange in our experience for staining siRNA.

Protocol N0. 2: 1X TE Recipe (10mM Tris-Hcl, 0.1mM EDTA)

Date of Preparation: 23 Feb 2010 Revision # 00 References: (theLabRat.com, 2005a) Estimated Time: 20 min

1X TE Recipe (10mM Tris-Hcl, 0.1mM EDTA)

Materials:

Distilled H2O 1M Tris-HCl (See Protocol N0. 4 ) 0.5M EDTA (See Protocol N0. 5 )

Procedure:

Add the following to 990 ml distilled H2O:

1. 1M Tris-HCl ;pH = 7.6 10 ml 2. 0.5M EDTA 0.2 ml 3. Add distilled water. 4. Adjust the volume to 1 liter.

Protocol N0. 3: Preparation of Ice-Cold CaCl2 (25 ml , 0.1M)

Date of Preparation: 24 Feb 2010 Revision # 00 References: This work Estimated Time: 40 min

Preparation of Ice-Cold CaCl2 (0.1M, 25 ml)

Materials:

CaCl2

Distilled water

Procedure:

1. Scale 0.277 g of CaCl2 (powder) with the following molecular weight:

MW = 110.994 g/mol

2. Pour it to the Erlenmeyer flask.

3. Add distilled water to dissolve CaCl2.

4. Adjust the volume by using the volumetric flask to reach 25 ml.

5. Put it in the freezer and keep it there around 30 minutes.

Protocol N0. 4: 1M Tris-HCl Recipe (100 ml)

Date of Preparation: 23 Feb 2010 Revision # 00 References: (Biowww.net, 2009b) Estimated Time: 20 min

1M Tris-HCl (100 ml)

Materials:

Tris 1M HCl Distilled water

Procedure:

1. Weight 12.114 gr Tris.

2. Pour it in an Erlenmeyer flask.

3. Add 50 ml distilled water.

4. Adjust pH=7.6 by adding some drops of 1M HCl.

5. Add distilled water to reach the volume 100 ml.

MW of Tris: 121.14 121.14g 100ml 1M = 121.14 → X = = 12.114 g 1000ml

Protocol N0. 5: 0.5M EDTA; pH=8.0 Recipe (100 ml)

Date of Preparation: 23 Feb 2010 Revision # 00 References: (Biowww.net, 2009a, Sambrook et al., 1989) Estimated Time: 20 min

0.5M EDTA; pH=8.0 (100 ml)

Materials:

EDTA 1M NaOH Distilled water

Procedure:

1. Weight 18.612 g EDTA. 2. Pour it in an Erlenmeyer flask. 3. Add 50 ml distilled water. 4. Add some drops of 1M NaOH to adjust the pH to 8.0. 5. EDTA will dissolve at pH = 8.0. 6. Reach the final volume 100 ml by adding distilled water. 1M = 372.24 0.5M = 186.12 → x = = 18.612 g

Protocol N0. 6: TE Recipe (10mM Tris-HCl, 1mM EDTA)

Date of Preparation: 12 Mar 2010 Revision # 00 References: (Project, 1997 )

Estimated Time: 10 min

TE Recipe (10mM Tris-HCl, 1mM EDTA) (100 ml)

Materials:

1M Tris-HCl; pH = 8.0 (See Protocol N0. 4) 0.5M EDTA; pH = 8.0 (See Protocol N0. 5) Distilled water

Procedure:

1. TE solution should contain the final concentration of:

10mM Tris-HCl ; pH = 8.0 1mM EDTA ; pH = 8.0

2. To reach the solution with above final concentration, mix the following amount of each component: 1M Tris-HCl ; pH = 8.0 1 ml 0.5M EDTA; pH = 8.0 0.5 ml

3. Pour them in a volumetric flask. 4. Add distilled water to reach the final volume 100 ml.

Protocol N0. 7: Preparation of Ice cold 70% Ethanol (10 ml)

Date of Preparation: 15 Mar 2010 Revision # 00 References: Estimated Time: 10 min

Preparation of ice cold 70% Ethanol (10 ml)

Materials:

Ethanol 99% Distilled water

Procedure:

1. Pour 7ml of absolute Ethanol 99% in a tube. 2. Add 3 ml distilled water. 3. Mix it by pipetting up and down. 4. Keep the tube in freezer -20.

Protocol N0. 8: 5M NaCl Recipe (100 ml)

Date of Preparation: 15 Mar 2010 Revision # 00 References: (Sambrook et al., 1989) Estimated Time: 1 h and 10 min (see the note)

5M NaCl Recipe (100 ml)

Materials:

NaCl Distilled water

Procedure:

1. Weight 29.2 g of NaCl. 2. Pour it in an Erlenmeyer flask. 3. Add 80 ml distilled water to dissolve NaCl powder. 4. Adjust the volume to 100 ml. 5. It is recommended to sterilize it by autoclaving at 121°C for 20 min. 6. Keep the sterilized NaCl solution at room temperature.

Note:

• It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 9: Preparation of Salt Solutions

Date of Preparation: 20 Apr 2010 Revision # 00 References: (Karimi et al., 2005, Lennartsson et al., 2009) Estimated Time: 30 min

Preparation of Salt Solutions

Materials:

(NH4) 2 SO4 Distilled water

KH2PO4

CaCl2.2H2O

MgSO4.7H2O

Procedure:

Following salt solutions are used in semi –synthetic growth medium for fungi:

1. (NH4) 2 SO4 : (25 ml) MW: 132.14 Concentration: 375g/l for 20 ml solution per litre (i.e. 7.5g / 20 ml).

Preparation: Weight 9.375g of (NH4) 2 SO4, add to adjust the volume to 25 ml.

2. KH2PO4 : (25 ml) MW: 136.09 Concentration:175g/l for 20 ml solution per litre (i.e. 3.5g / 20 ml)

Preparation: Weight 4.375g of KH2PO4, add water to adjust the volume to 25 ml.

3. CaCl2.2H2O: (10 ml) MW: 147.02 Concentration:200g/l for 5 ml solution per litre (i.e. 1g / 5 ml)

Preparation: Weight 2g of CaCl2.2H2O, add water to adjust the volume to 10 ml.

4. MgSO4.7H2O: (10 ml) MW: 246.50 Concentration:150g/l for 5 ml solution per litre (i.e. 0.75g / 5 ml)

Preparation: Weight 1.5g of MgSO4.7H2O, add water to adjust the volume to 10 ml.

Protocol N0. 10: Preparation of Trace Metal Solution

Date of Preparation: 20 Apr 2010 Revision # 00 References: (Karimi et al., 2005, Lennartsson et al., 2009) Estimated Time: 1 h and 30 min (see the note)

Preparation of Trace Metal Solution (1 L)

Materials:

EDTA (C10H14N2Na2O8·2H2O)

CaCl2·2H2O

ZnSO4·7H2O

FeSO4·7H2O

H3BO3

MnCl2·4H2O

Na2MoO4·2H2O

CoCl2·2H2O

CuSO4·5H2O KI Distilled water NaOH

Procedure:

1. Dissolve the following compounds in 600 ml water:

EDTA (C10H14N2Na2O8·2H2O) 3 g

CaCl2·2H2O 0.9 g

ZnSO4·7H2O 0.9 g

FeSO4·7H2O 0.6 g

H3BO3 0.2 g

MnCl2·4H2O 0.19 g

Na2MoO4·2H2O 0.08 g

CoCl2·2H2O 0.06 g

CuSO4·5H2O 0.06 g KI 0.02 g

2. Adjust pH to 4 with NaOH. 3. Add water to 1 litre. 4. Autoclave at 121 °C for 20 minute. 5. Store in refrigerator at 4ºC.

Note:

• It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 11: Preparation of Vitamin Solution

Date of Preparation: 20 Apr 2010 Revision # 00 References: (Karimi et al., 2005, Lennartsson et al., 2009) Estimated Time: 2 h

Preparation of Vitamin Solution (500 ml)

Materials:

D-Biotin 0.1 M NaOH Distilled water 0.1M HCl p-amino benzoic acid (PABA) Nicotinic acid Ca-Panthothenate Pyroxidine HCl Thiamine HCl 2 M NaOH m-Inositol

Procedure:

1. Dissolve 25 mg of d-Biotin in 10 ml 0.1M NaOH. 2. Add the dissolved biotin to approximately 300 ml water. 3. Adjust the pH to 6.5 by using 0.1M HCl. 4. Add the following vitamins:

p-amino benzoic acid (PABA) 100 mg Nicotinic acid 500 mg Ca-Panthothenate 500 mg Pyroxidine HCl 500 mg Thiamine HCl 500 mg

5. Adjust pH to 6.5 by using 2M NaOH. 6. Add 12.5 g of m-Inositol. 7. Adjust pH to 6.5 with 2M NaOH. 8. Adjust the volume to 500 ml with water. 9. Transfer the solution into autoclaved blue cap bottles through sterile filter (0.45 μm filter). 10. Keep the bottles in refrigerator 4 ºC or divide the solution in eppendorfs via the following instruction: 11. Pour the solution into 1.5 ml eppendorf tubes for the next usage. It needs 333 eppendorf tubes. 12. Store the tubes in freezer at -20 ºC.

Note:

As it was mentioned above for preparing 500 ml of vitamin solution the required amounts of vitamins are very small (mg) therefore it is recommended to prepare the solution one time and store it in eppendorf tubes at -20 ºC for the next usage.

Protocol N0. 12: Preparation of Semi-Synthetic Growth Medium for Cultivation of Zygomycetes Fungi

Date of Preparation: 20 Apr 2010 Revision # 00 References: (Karimi et al., 2005, Lennartsson et al., 2009) Estimated Time: 2 h and 30 min

Preparation of Semi-Synthetic Growth Medium for Cultivation of Zygomycetes Fungi

Materials:

Glucose Yeast extract

(NH4)2SO4 (See Protocol N0. 9)

KH2PO4 (See Protocol N0. 9)

CaCl2.2H2O (See Protocol N0. 9)

MgSO4.7H2O (See Protocol N0. 9) Vitamin solution (See Protocol N0. 11) Trace metal solution (See Protocol N0. 10) Spore solution (See Protocol N0. 33) 2M NaOH

Procedure:

For preparing different volume of synthetic medium the following table (Table 1) and instruction can be used:

Table 1: The amounts of salt and glucose solutions required for preparation of different volumes of semi- synthetic growth medium

Type of Components Unit 100 (ml) 150 (ml) 1 ( l) 1.5 ( l) Cfinal (g/l) Solution

Salt (NH4)2SO4 ml 2 3 20 30 7.5

Solution KH2PO4 ml 2 3 20 30 3.5

CaCl2.2H2O ml 0.5 0.75 5 7.5 1

MgSO4.7H2O ml 0.5 0.75 5 7.5 0.75 Deionized water ml 45 67.5 450 675 - Glucose Glucose g 3 4.5 30 45 30 Solution Yeast extract g 0.5 0.75 5 7.5 5 Deionized water ml 50 75 500 750 -

Instruction: 1. Prepare glucose solution in an Erlenmeyer flask which is used for the cultivation of fungi. Glucose is the only carbon source in this media. 2. Seal the flask with cotton wool plug and aluminium foil. 3. Prepare the salt solution in a separate blue cap bottle.(see the notes) 4. Autoclave the bottle and flask at 121°C for 20 minutes. 5. Take them out from the autoclave and let them to be cooled. 6. Pour the salt solution into the flask. (Becareful: This and next steps should be done near the flame to avoid the contamination and keep the media sterile.) 7. Let the flask to be cooled enough to keep with the hand. 8. Add 1 ml/l vitamin solution. 9. Add 10 ml/l trace metal solution. 10. Adjust the pH=5.5 (±0.07) by using 2M NaOH. (Usually the pH of the media is 5.5, so it is not necessary to adjust the pH) 11. Add around 20 ml/l spore solution. 12. Cultivate the flask in a shaker incubator at 30°C and 140 rpm.

Notes: If the trace metal solution has not been autoclaved before then you should use the sterile filter to sterilise the trace metal solution and mix it with salt solution and autoclave them together in step 2. Pour the salt solutions in the blue cap bottle with the following order otherwise you could see some crystals in the salt solution after autoclaving: • Deionized water

• (NH4)2SO4

• KH2PO4

• CaCl2.2H2O

• MgSO4.7H2O

Protocol N0. 13: Preparation of Potato Dextrose Agar Plate (PDA)

Date of Preparation: 28 Apr 2010 Revision # 00 References: (Acumedia, 2008, Laboratory, 2005) Estimated Time: 1 h and 10 min (see the note)

Preparation of Potato Dextrose Agar Plate (1 litre)

Materials:

Potato extract Glucose Agar Distilled water or RNase free water (See Protocol N0. 21)

Procedure:

1. For preparing 1 litre of potato dextrose agar (PDA) which is one of the general media for the fungi you need the following components: Potato extract 4 g Glucose 20 g Agar 15 g

2. Weight the above components and pour them in a blue cap bottle. 3. Add distilled water (or RNase free water) to adjust the volume to 1 litre. 4. Autoclave the media at 121 ºC for 20 minutes.

Note:

• It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time. Protocol N0. 14: Weighting the Harvested Mycelia Recipe

Date of Preparation: 4 May 2010 Revision # 00 References: This Work Estimated Time: 10 min

Weighting the Harvested Mycelia Recipe

Materials:

Mycelia pad (See Protocol N0. 19 and Protocol N0. 20) Water

Procedure:

In protoplast preparation of Rhizopus oryzae you should know the wet weight of harvested mycelia which was accumulated on the filter paper placed into Buchner funnel. In this regard just follow the next steps:

1. Select one empty watch glass, you need it in further steps. 2. Rinse the whatman filter paper with water. 3. Put wet filter paper in a watch glass that was selected in first step. 4. Weight the watch glass containing wet filter paper. 5. Wash the watch glass and dry it. 6. Put the mycelia pad, which is the filter paper in the Buchner funnel containing mycelia, in the previous selected watch glass. 7. Weight the watch glass containing mycelia pad. 8. Calculate the wet weight of mycelia just by minus the number that was achieved in step 4 from the number measured in step 7. Protocol N0. 15: Preparation of Digestion Buffer by Using Lysozyme

Date of Preparation: 3 May 2010 Revision # 00 References: (Latterich, 2008) Estimated Time: 30 min

Preparation of Digestion Buffer (100 ml)

Materials:

Na2HPO4

MgSO4 Lysing enzyme Nuclease-free water (See Protocol N0. 21 and Protocol N0. 22)

Procedure:

The procedure should be carried out in RNase free condition:

1. Digestion buffer should contain the final concentration of:

10mM Na2HPO4

1.2M MgSO4 10 mg/ml Lysing enzyme 2. To reach the solution with above final concentration, mix the following amount of each component:

Na2HPO4 (FW = 142) 0.142 g

MgSO4 (M = 120.37) 14.44 g Lysing enzyme 1 g 3. Add nuclease-free water (RNase free water which have been autoclaved) to dissolve the components. Each component should dissolve in water separately then mix them together (Becarful: Final volume should be 100 ml).

4. MgSO4 is highly exothermic so do not mix it with lysozyme to avoid degradation of the enzyme.

5. Use the hotplates magnetic stirrer to dissolve Na2HPO4.

6. Mix dissolved MgSO4 and Na2HPO4 together and let them to be cooled at room temperature. 7. Add lysozyme. 8. Adjust the volume to 100 ml; the solution was containing foam which made it difficult to adjust the volume. 9. Filter the solution on 0.22 µm filter.

Notice: Lysozyme, from chicken egg white was the lysing enzyme which was used in this experiment. (Fluka Biochemika, 62970-5G-F, Lot # BCBB3704)

Protocol N0. 16: Preparation of Digestion Buffer by Using Yatalase

Date of Preparation: 10 Jun 2010 Revision # 00 References: (TAKARA, 2010) Estimated Time: 20 min

Preparation of Digestion Buffer (50 ml)

Materials:

0.2M Maleate (See Protocol N0. 37)

0.6M MgSO4 Yatalase (digestive enzyme of cell walls of filamentous fungi) 0.2N NaOH RNase free water (See Protocol N0. 21)

Procedure:

The procedure should be carried out in RNase free condition:

1. Digestion buffer should contain the final concentration of: 0.05M maleate buffer; pH = 5.5

0.6M MgSO4 2% Yatalase 2. To reach the solution with above final concentration, mix the following amount of each component: 0.2M Maleate 12.5 ml Yatalase 1 g

0.6M MgSO4 3.611 g 0.2N NaOH Some drops 3. Dissolve Yatalase separately in 15 ml water then add it to the other dissolved compounds.

4. Add 15 ml RNase free water to dissolve the MgSO4 and Maleate. 5. Mix them together. 6. Adjust the pH = 5.5 by using some drops of 0.2N NaOH. 7. Add water to reach the volume 50 ml.

Protocol N0. 17: Preparation of STC Buffer

Date of Preparation: 3 May 2010 Revision # 00 References: (Latterich, 2008) Estimated Time: 1 h and 20 min (see the note)

Preparation of STC Buffer (500 ml)

Materials:

Sorbitol

CaCl2 1M Tris-HCl; pH = 8 (See Protocol N0. 4) RNase free water (See Protocol N0. 21)

Procedure:

The procedure should be carried out in RNase free condition:

1. STC buffer should contain the final concentration of: 1M Sorbitol

50mM CaCl2 50mM Tris-HCl; pH = 8

2. To reach the solution with above final concentration, mix the following amount of each component: Sorbitol (M =182.18) 91.09 g

CaCl2 (M =110.994) 2.774 g Tris-HCl 1M ;pH = 8 25 ml

3. Add RNase free water to dissolve the components. Each component should dissolve in water separately then mix them together (Becarful: Final volume should be 500 ml). Using the hotplates magnetic stirrer can be helpful for dissolving the components. 4. Adjust the volume to 100 ml. 5. Pour into the blue cap bottle. 6. Autoclave the STC buffer at 121°C for 20 minutes. 7. Store it at room temperature.

Note:

• It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 18: Preparation of PEG Solution

Date of Preparation: 3 May 2010 Revision # 00 References (Latterich, 2008) Estimated Time: 1 h and 20 min (see the note)

Preparation of PEG Solution (25 ml)

Materials:

PEG 4000

CaCl2 1M Tris-HCl; pH = 8 (see Protocol N0. 4) RNase free water (see Protocol N0. 21)

Procedure:

The procedure should be carried out in RNase free condition:

1. PEG solution should contain the final concentration of: 60% PEG 4000 (Sigma) in 50mM Tris-HCl; pH = 8

50mM CaCl2 2. To achieve the solution with above final concentration, mix the following amount of each component: PEG 4000 15 g

CaCl2 (M =110.994) 0.13 g 3. Pour them in an Erlenmeyer flask and add 15 ml of 50mM Tris-HCl (See Protocol N0. 31). 4. Add RNase free water and adjust the volume to 25 ml. 5. Autoclave PEG solution at 121°C for 20 minutes. 6. Store it at room temperature.

Note: • It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time. Protocol N0. 19: Protoplast Preparation of Rhizopus oryzae by Using Lysozyme

Date of Preparation: 17 June 2010 Revision # 00 References: (Latterich, 2008, Vollmer and Yanofsky, 1986, Nakayashiki, 2005, Kadotani et al., 2003, Nakayashiki et al., 2005) Estimated Time: 4 days and 4 h (see the note)

Protoplast Preparation of Rhizopus oryzae by Using Lysozyme

Materials:

Semi synthetic media (See Protocol N0. 12) Potato dextrose agar plate (See Protocol N0. 13) STC Buffer (See Protocol N0. 17) Digestion buffer (See Protocol N0. 15) RNase free water (See Protocol N0. 21)

Procedure:

Inoculation:

1. Transfer 150 ml of semi-synthetic media to an Erlenmeyer flask. 2. Add 20 ml RNase free water to a cultured potato dextrose agar plate and take 3ml of inoculum and add it to the semi synthetic medium.

Incubation:

3. Incubate it at 30 °C and 140 rpm, for 3-4 days.

Protoplast preparation:

4. Harvest the mycelia by filtration through a Buchner funnel containing filter paper (Whatman N0.1), this was named mycelia pad. 5. Weight the harvested mycelia. (See Protocol N0. 14) 6. Put the filter paper containing mycelia in a sterile plastic tube (size 50 ml). 7. Add 10 ml of digestion buffer (See Protocol N0. 15) per gram wet weight of mycelia in plastic tube. 8. Incubate for 3 hours at room temperature with gentle inversion on a horizontal shaker. 9. Pass the containing of the tube from four layers sterile gauze. 10. Centrifuge the filtrate in a swing bucket rotor at 800 g (2400 rpm with our facility) for 5 min. 11. Pour off supernatant, and resuspend the collected protoplasts in 50 ml STC buffer. (See Protocol N0. 17) 12. Centrifuge swing bucket rotor at 800 g (2400 rpm with Megafuge 1.0R, Heraeus Sepatech machine) for 5 min. 13. Pour off supernatant; just leave a little STC buffer in the bottom of tube. 14. Vortex the tube for 15 sec. 15. Use the protoplast or keep it in -20°C.

Note:

• It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 20: Protoplast Preparation of Rhizopus oryzae by Using Yatalase

Date of Preparation: 10 Jun 2010 Revision # 00 References: This Work Estimated Time: 4 days and 4 h (see the note)

Protoplast Preparation of Rhizopus oryzae by Using Yatalase

Materials:

Semi synthetic media (See Protocol N0. 12 ) Potato dextrose agar plate (See Protocol N0. 13) Digestion buffer prepared by Yatalase (See Protocol N0. 16) RNase free water (See Protocol N0. 21)

Procedure:

Inoculation:

1. Transfer 150 ml of semi-synthetic to an Erlenmeyer flask. 2. Add 20 ml water to a cultured potato dextrose agar plate and take 3ml of inoculum and add it to the semi synthetic medium.

Incubation:

3. Incubate it at 30 °C and 140 rpm, for 3-4 days.

Protoplast preparation:

4. Harvest the mycelia by filtration through a Buchner funnel containing filter paper (Whatman N0.1), this was called mycelia pad. 5. Weight the harvested mycelia. (See Protocol N0. 14) 6. Put the filter paper containing mycelia in a sterile plastic tube (size 50 ml). 7. Add 10 ml of digestion buffer prepared by Yatalase (See Protocol N0. 16) per gram wet weight of mycelia in plastic tube. 8. Incubate in shaking water bath for 3 hours at 30°C and 90 rpm. 9. Filtrate the containing of the tube from four layers sterile gauze. 10. Centrifuge the filtrate in a swing bucket rotor at 4°C and 3500 rpm (with Megafuge 1.0R, Heraeus Sepatech machine) for 5 min. 11. Pour off supernatant; just leave a little of that over the pellet. 12. Vortex the tube for 15 sec. 13. Use the protoplast or store it in freezer -20°C.

Note: • The inoculation step takes around 5 min but the incubation time is around 3-4 days. The protoplast preparation step needs 4 hours.

Protocol N0. 21: RNase Free Water Recipe

Date of Preparation 28 Apr 2010 Revision # 00 References: (ambion, 2010) Estimated Time: 1 h and 30 min (see the note)

RNase Free Water Recipe (1 litre)

Materials:

DEPC MiliQ-water

Procedure:

1. Add 0.05-0.1 % (v/v) DEPC (diethyl pyrocarbonate) to MiliQ-water. For instance 1 ml DEPC should be mixed with 999 ml water to reach the final volume of 1000 ml. 2. Prepare it in a blue cap bottle. 3. Stir the water for 20-30 min with a magnetic stirrer. 4. Autoclave the water at 121°C for 15-20 min.

Notes: Increasing the concentration of DEPC can inactivate the high amounts of RNase A contamination by the way 0.1% DEPC probably seems to be sufficient to inhibit most RNases with minimal effect on reactions. On the other hand some investigation shows that the solutions which contain Tris cannot be treated with DEPC because Tris contains an amino group which takes up DEPC and makes it unavailable to inactivate RNase. But this hypothesis is not true for all molecular biology reagents so to be in a safe side you can buy pre-made nuclease-free solutions which are provided by some companies such as Ambion. In this project we used RNase free water which had been made by using DEPC. It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 22: DNase Free Water Recipe

Date of Preparation: 28 Apr 2010 Revision # 00 References: This Work Estimated Time: 1 h (see the note)

DNase Free Water Recipe (1 litre)

Materials:

MiliQ-water

Procedure:

1. Pour 1 litre of MiliQ-water in blue cap bottle. 2. Autoclave it at 121°C for 20 minutes.

Note:

It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 23: Calculating the Volume of dsRNA

Date of Preparation: 4 May 2010 Revision # 00 References: (ambion, 2009) Estimated Time: -

Calculating the Volume of dsRNA

Materials:

Molecular Weight of Cytosine Molecular Weight of Uracil Molecular Weight of Adenine Molecular Weight of Guanine Sequence of duplex siRNA Molarity of the siRNA solution Desired mass amount

Procedure:

Molecular weight (MW) of dsRNA

1. For determining the molecular weight of desired dsRNA, you should know the molecular weight of each bases made dsRNA: Molecular Weight of Cytosine (C): 111.102 g/mol Molecular Weight of Uracil (U): 112.086 g/mol Molecular Weight of Adenine (A):135.128 g/mol Molecular Weight of Guanine (G):151.13 g/mol 2. The following sequence shows our desired dsRNA which was made by Invitrogen: Sense: GGAGGCAGGGCAGGCAGAUAUUGUU Anti-Sense: AACAAUAUCUGCCUGCCCUGCCUCC MW of dsRNA=(14C)+(11U)+(11A)+(14G) =6390.602 g/mol

Molarity of the solution

3. We ordered 80 nmol of desired dsRNA to Invitrogen Company. Then we diluted it (See Protocol N0. 24) and made a 20 µM stock solution for next usage. Calculating the concentration 4. The concentration of the RNA oligonucleotide solution can be calculated via the following formula: Concentration = (MW) × (Molarity of the solution) Concentration = (6390.602 µg/µmol) × (20 µmol/l) =127812.04 µg/l =1.27×105µg/l

Calculating the volume

5. Divide the mass amount desired by the concentration of the solution to calculate the volume of a molar solution of duplex siRNA needed for 5 µg: =3.91×10-5 l = 39.1 µl This means that 39.1 µl of a 20 µmol/l stock solution of duplex siRNA contains 5µg RNA.

Protocol N0. 24: Storage and Resuspension of Duplex siRNA

Date of Preparation: 4 May 2010 Revision # 00 References: (Invitrogen, 2010, amaxa, 2004) Estimated Time: 15 min

Storage and Resuspension of Duplex siRNA to Create a 20 µM Working Stock

Materials:

Dry pellet of siRNA RNase free water (see Protocol N0. 21)

Procedure:

1. Invitrogen provide a dry pellet of siRNA so you should store it at -20°C until you decide to use it. 2. Dry pellet of siRNA is stable at least 6 months. 3. Before the first usage, thaw a tube containing siRNA at 4°C. 4. Centrifuge the tube briefly at 4°C to allow the siRNA to settle at the bottom of the tube. 5. Put on the gloves. 6. Use RNase free tips. 7. Resuspend the duplex siRNA (80 nmol) in 800 µl DEPC treated water supplied by Invitrogen to make a 100 µM solution. 8. Dilute 1:5 to create a 20 µM working solution. To obtain the desired concentration we added 3200 µl RNase free water made by ourselves (see Protocol N0. 21) to the tube. 9. Store at -20°C and avoid contacting with RNase.

Notes: Repeated freeze-thaw cycles will not have any effect on duplex siRNA until performing the experiment was done in RNase free condition. Duplex siRNA was dried in a buffer solution. Resuspension to 20µM will reconstitute the buffer to 10mM Tris-HCl; pH=8, 20mM NaCl, 1mM EDTA.

Protocol N0. 25: Cleaning up siRNA Before Labelling

Date of Preparation: 4 May 2010 Revision # 00 References: (ambion, 2009) Estimated Time: 1 h and 10 min

Cleaning up siRNA Before Labelling

Materials: siRNA Phenol: chloroform: isoamyl alcohol (25:24:1) Chloroform: isoamyl alcohol (24:1) 3M sodium acetate 100% ethanol RNase free water (See Protocol N0. 21) Procedure:

1. Put on the gloves. 2. Use RNase free tips in the whole procedure. 3. Pour 100 µl siRNA from working stock (see Protocol N0. 24) in an RNase free eppendorf tube. 4. Add an equal volume (100 µl) of phenol: chloroform: isoamyl alcohol (25:24:1). 5. Vortex 30 seconds. 6. Centrifuge (≥10,000× g) at room temperature for 5 min to separate the aqueous and organic phases. 7. Transfer the aqueous phase to a new RNase free eppendorf tube. (Becarful: you should know how many volume of the aqueous phase you can transfer to the new tube. It is important for the next step.) 8. Add an equal volume of chloroform: isoamyl alcohol (24:1). 9. Vortex 30 seconds. 10. Centrifuge (≥10,000× g) at room temperature for 5 min to separate the phases. 11. Transfer the aqueous phase to a new RNase free eppendorf tube. The volume that you transfer to a fresh tube is important for the next step. 12. Add 0.1 volume (the volume that transferred to fresh tube in previous step) 3M sodium acetate (See Protocol N0. 26). For instance if you transfer 70 µl aqueous phase to new tube in previous step then you should add 7 µl of 3M sodium acetate. 13. Add 2.5 volumes (transferred volume in step 11) of 100% ethanol (molecular biology grade). 14. Mix thoroughly. 15. Keep in -20°C or colder for more than 20 min. 16. Centrifuge at ≥10,000× g for at least 15 min. 17. Discard the supernatant carefully. 18. Respin the tube briefly. 19. Remove the last traces of ethanol. 20. Resuspend the siRNA to 20 µM in RNase free water. Protocol N0. 26: 3M Sodium Acetate Recipe

Date of Preparation: 4 May 2010 Revision # 00 References: This Work Estimated Time: 10 min

3M Sodium Acetate Recipe (10 ml)

Materials:

Sodium acetate anhydrous RNase free water (See Protocol N0. 21)

Procedure:

1. Use the sodium acetate anhydrous (C2H3NaO2) with the following molecular weight: M = 82.03 g/mol 2. Weight 2.46 g of sodium acetate anhydrous: 3M = 3 × 82.03 = 246.09 g/mol X = = 2.46 g 3. Pour it to the Erlenmeyer flask. 4. Add RNase free water to dissolve the sodium acetate. 5. Adjust the volume to 10 ml.

Protocol N0. 27: Labeling siRNA from 20 µM Working Stock Solution

Date of Preparation: 5 May 2010 Revision # 00 References: (ambion, 2009) Estimated Time: 1 h and 10 min

Labeling siRNA from 20 µM Working Stock Solution

Materials:

Acridin Orange dye solution (See Protocol N0. 1) siRNA Nuclease free water (See Protocol N0. 21 and Protocol N0. 22 ) Ice

Procedure:

1. Work in RNase free condition. 2. Put on the gloves. 3. Use sterile RNase free tips and tubes in the whole procedure. 4. Take out the 20µM working stock solution siRNA from the freezer. (See Protocol N0. 24) 5. Thaw tube on ice (at 4°C). 6. Centrifuge the tube briefly at 4°C. 7. Pour 6.9 µl of nuclease free water to the sterile nuclease free eppendorf tube. 8. Add 39.1 µl of duplex siRNA. 9. Add 4 µl acridin orange dye solution. (Make sure to add dye solution in last step.) 10. The final volume should be 50 µl. 11. Mix by pipetting up and down several times. 12. Vortex the tube briefly. 13. Wrap the tube in the foil and let the siRNA to be labeled in dark. 14. Cultivate the tube in shaking incubator for 1 hour at 37°C and 140 rpm. 15. Labeled siRNA is ready for transformation. (See Protocol N0. 30)

Protocol N0. 28: Labeling cleaned up siRNA

Date of Preparation: 5 May 2010 Revision # 00 References: (ambion, 2009) Estimated Time: 1 h and 10 min

Labeling cleaned up siRNA

Materials:

Acridin Orange dye solution (See Protocol N0. 1) siRNA Nuclease free water (See Protocol N0. 21) Ice

Procedure:

1. Work in RNase free condition. 2. Put on the gloves. 3. Use sterile RNase free tips and tubes in the whole procedure. 4. Take out cleaned up siRNA from the freezer. (See Protocol N0. 25) 5. Thaw tube in ice (at 4°C). 6. Centrifuge the tube briefly at 4°C. 7. Pour 6.9 µl of nuclease free water to the sterile nuclease free eppendorf tube. 8. Add 39.1 µl of cleaned up siRNA. 9. Add 4 µl acridin orange dye solution. (Make sure to add dye solution in last step.) 10. The final volume should be 50 µl. 11. Mix by pipetting up and down several times. 12. Vortex the tube briefly. 13. Wrap the tube in the foil and let the siRNA to be labeled in dark. 14. Cultivate the tube in shaking incubator for 1 hour at 37°C and 140 rpm. 15. Labeled siRNA is ready for the next step which is the ethanol precipitation of labeled RNA.(See Protocol N0. 29) 16. After ethanol precipitation the siRNA is ready for transformation.(See Protocol N0. 30)

Protocol N0. 29: Ethanol Precipitation of Labeled siRNA

Date of Preparation: 5 May 2010 Revision # 00 References: (ambion, 2009) Estimated Time: 3 h and 15 min

Ethanol Precipitation of Labeled siRNA

Materials:

Labeled siRNA (See Protocol N0. 28) 5M NaCl (See Protocol N0. 8: 5M NaCl Recipe (100 ml)) Ice cold 100% ethanol 70% ethanol (See Protocol N0. 7) Nuclease free water (See Protocol N0. 21 and Protocol N0. 22)

Procedure:

Follow the next steps for the labeled siRNA prepared via the Protocol N0. 28: 1. Work in RNase free condition. 2. Put on the gloves. 3. Add 5 µl of 5M NaCl (0.1 volume) to the 50 µl labeled siRNA.(See Protocol N0. 28) 4. Add 125 µl of cold 100% ethanol (2.5 volumes). 5. Mix NaCl and ethanol with labeled siRNA well by using vortex. 6. Store the eppendorf tube in freezer -20°C or colder for 30-60 min. (preferably 2hours and 30 minutes). The siRNA can be incubated overnight or longer at -20°C if desired. The labeled siRNA precipitate during this step. 7. Centrifuge at ≥ 8000 × g for 20 min. 8. Discard the supernatant; be careful not to disrupt the pellet. Dark orange siRNA pellet should be visible at the bottom of the eppendorf tube. 9. Add 175 µl of 70% ethanol gently, it is recommended to pour ethanol at the wall of the eppendorf tube to avoid disrupting the pellet. 10. Centrifuge at ≥ 8000 × g for 5 min. 11. Remove the supernatant with a pipet carefully. (Use RNase free tips) 12. Respin the tube briefly to discard the last traces of ethanol. 13. Discard supernatant. 14. Let the siRNA to dry at room temperature for just 5-10 minutes not longer. 15. Resuspend the siRNA pellet in 39.1 µl nuclease free water. 16. The labeled siRNA is ready for transformation. (See Protocol N0. 30)

Note: Do not dry the pellet longer than 5-10 minutes in step 14, because the siRNA will be difficult to solubilize.

Protocol N0. 30: Transformation of Rhizopus oryzae with siRNA by the Calcium Chloride/Polyethylene Glycol Method

Date of Preparation: 5 May 2010 Revision # 00 References: (Latterich, 2008, Vollmer and Yanofsky, 1986) Estimated Time: 1 h and 10 min

Transformation of Rhizopus oryzae with siRNA by the Calcium Chloride/Polyethylene

Glycol (CaCl2/PEG) Method

Materials:

Prepared protoplasts of Rhizopus oryzae (See Protocol N0. 19 and Protocol N0. 20) Labeled siRNA (See Protocol N0. 27 and Protocol N0. 28) Ice PEG solution (See Protocol N0. 18) STC buffer (See Protocol N0. 17) Potato dextrose agar medium (See Protocol N0. 13) Prepared potato dextrose agar plate

Procedure:

The following procedure should be carried out in RNase free condition:

1. Pour 100 µl diluted protoplasts of Rhizopus oryzae in a 15 ml sterile plastic tube. 2. Add 100 µl of labeled siRNA prepared by Protocol N0. 27 or Protocol N0. 28. 3. Mix gently by pipetting up and down. 4. Incubate the tube on ice for 15 min. 5. Add PEG solution to the tube in three steps. Start to add 200 µl of PEG. 6. Mix gently by pipetting up and down. 7. Pour 400 µl of PEG solution to the tube. 8. Mix gently by pipetting up and down. 9. Add 600 µl of PEG solution. 10. Mix gently by pipetting up and down. 11. Incubate on ice for 10 min. 12. Centrifuge the tube at 2000 × g (3700 rpm with our centrifuge) for 3 min to collect the protoplasts. 13. Discard the supernatant. 14. Add 1 ml of STC buffer to resuspend the protoplasts. 15. Incubation can be done by two following ways: Add 10 ml potato dextrose agar medium (when it is still in liquid form but not too warm that irritates your hand) to the tube prepared in step 14, mix it and then spread it in a sterile petri dish. Pour the whole protoplast solution prepared in step 14 on the surface of a prepared potato dextrose agar plate. 16. Incubate the plates in the incubator at 30°C. 17. Wait until the growth of transformed Rhizopus oryzae will be visible. (We could see them after 20 hours.)

Protocol N0. 31: 50mM Tris-HCl; pH=8 Recipe

Date of Preparation: 3 May 2010 Revision # 00 References: This work Estimated Time: 10 min

50mM Tris-HCl; pH=8 (15 ml)

Materials:

1M Tris-HCl; pH=8 (See Protocol N0. 4) RNase free water (See Protocol N0. 21)

Procedure:

The procedure should be carried out in RNase free condition:

1. Pour 750 µl of 1M Tris-HCl; pH=8 to the volumetric flask. 2. Add RNase free water to adjust the volume to 15 ml.

Protocol N0. 32: Cultivation of Rhizopus oryzae in the Flask Fermentors

Date of Preparation: 7 May 2010 Revision # 00 References: (Karimi et al., 2005, Lennartsson et al., 2009) Estimated Time: 2 h and 30 min (See the note)

Cultivation of Rhizopus oryzae in the Flask Fermentors

Materials:

Glycerol Glucose solution Salt solutions (See Protocol N0. 9) Vitamin solution (See Protocol N0. 11) Trace metal solution (See Protocol N0. 10) 2M NaOH Spore solution (See Protocol N0. 33)

Procedure:

Batch cultivation could carry out in 500 ml Erlenmeyer flask containing 150 ml semi-synthetic growth media (See Protocol N0. 12).

1. Prepare the shake flask fermentor that consists of: Erlenmeyer flask Silicon cap glass fermentation lock Sampling pipe Hose Needle Clamp

2. Pass a glass fermentation lock and a sampling pipe through silicon cap. It is very hard to make a hole in the silicon cap and you need an instrument to do that. 3. Use some drops of glycerol in the hole to make it easier to pass the glass fermentation lock and sampling pipe through the cap. 4. Wash the cap to clean the remained glycerol. 5. Attach the hose with a needle to the end of sampling pipe. The hose should be closed by the clamp while sampling is not performed from the fermentor. 6. Make the glucose solution in 500 ml Erlenmeyer flask. Glucose is used as the only carbon source. For preparation of 150 ml media the following amounts of glucose solution is essential (See Protocol N0. 12): Glucose 4.5 g Yeast extract 0.75 g RNase free water 75 ml

7. Prepare the salt solution in a blue cap bottle, mix them with the following order (See Protocol N0. 9 and Protocol N0. 12): Deionized water 67.5 ml

(NH4)2SO4 3 ml

KH2PO4 3 ml

CaCl2.2H2O 0.75 ml

MgSO4.7H2O 0.75 ml

8. Fit the Erlenmeyer flask (shake flask fermentor) with washed silicon cap. 9. Pour 1 ml water in the glass fermentation lock to provide anaerobic condition for fermentation process. 10. Cover the glass fermentation lock with aluminum foil. 11. Autoclave both flask and bottle at 121°C for 20 minutes. 12. Let the flask to be cooled enough to keep in your hands. 13. The following steps should be done near the flame and quickly to provide aseptic and anaerobic condition for fermentation of Rhizopus oryzae. 14. Pour the autoclaved salt solutions to the flask. 15. Add 150 µl of vitamin solution. 16. Add 1.5 ml of trace metal solution. 17. Adjust the pH=5.5 (±0.07) prior to the inoculation by using 2M NaOH. (Usually the pH of the media is 5.5, so it is not necessary to adjust the pH) 18. Take 1 ml sample of the media for calculating the glucose concentration, pour it in an eppendorf tube and keep in freezer -20°C. 19. Inoculate the media with 3 ml of spore solution. 20. Close the cap of fermentor very tight and clamp the hose. 21. Cultivate the shake flask fermentor in a shaker incubator for 62 hours at 30°C and 140 rpm.

Note: • It has been considered 1 hour for autoclaving by using Certoclav autoclave (CV-EL 12 L GS) in estimated time.

Protocol N0. 33: Preparation of Spore Solution

Date of Preparation: 7 May 2010 Revision # 00 References: This work Estimated Time: 5 min

Preparation of Spore Solution

Materials:

Nuclease free water (See Protocol N0. 21) Rhizopus oryzae agar plate (See Protocol N0. 30)

Procedure:

1. Put on the gloves. 2. Work beside the flame to avoid contamination. 3. Add 20 ml nuclease free water on the surface of each agar plate covering with the spores. The agar palate can be contained transformed Rhizopus oryzae (See Protocol N0. 30) or wild type of Rhizopus oryzae. 4. Use the disposable sterile microbiological spreader. Scrub the surface of agar to get spores and hyphae suspended in water. 5. Use the disposable sterile pipette (5 ml) to transfer 3 ml of the spores and hyphae suspension from the agar plate to the shake flask fermentor containing semi-synthetic growth medium.(See Protocol N0. 12)

Protocol N0. 34: Sampling from Fermentor for Analysis by HPLC

Date of Preparation: 10 May 2010 Revision # 00 References: This work Estimated Time: 15 min

Sampling from Fermentor for Analysis by HPLC

Materials:

Shake flask fermentor

Procedure:

1. Fit the syringe to the needle at the end of sampling pipe. 2. Release the hose clamp. 3. Take in 1.5 ml of the sample from the shake flask fermentor to the syringe.(see the note) 4. Pour the sample containing the Rhizopus oryze cells and hyphae to the eppendorf tube. 5. Centrifuge at 10,000 rpm for 10 min. 6. Pour the supernatant to the fresh eppendorf tube. Try to fill the tube more than 1 ml because you have to fill the HPLC vial with at least 1 ml of the sample. 7. Collect all the sample tubes for analysis by HPLC or store them in freezer -20 °C for another day.

Note:

Taking sample from the fungi fermentor becomes hard by passing the time. If sampling by syringe comes to be impossible then take out the needle from the hose and use the sterile pipet.

Protocol N0. 35: Standard Solutions for HPLC

Date of Preparation: 10 May 2010 Revision # 00 References: This Work Estimated Time: 30 min

Standard Solutions for HPLC (High Performance Liquid Chromatography)

Materials:

Lactic acid Pyruvic acid Citric acid Succinic acid Fumaric acid Ethanol Glycerol Glucose Distilled water

Procedure:

For analysing the data obtained by HPLC, you should run standard solutions as well as samples by HPLC. According to the component that you are interested to analyse by HPLC you should also prepare the standard solution for that compound.

In this project the aim was to compare the concentration of the following compounds between the fermentors inoculated with wild type Rhizopus oryzae and the others that inoculated with transformed Rhizopus oryzae. (See Protocol N0. 30)

The chemical compounds used in this project have been shown in Table 2:

Table 2: Chemical compounds specification

Name Formula Molecular Weight Supplier (g/mol)

L(+)-Lactic acid, 98% C3H6O3 90.08 Sigma

Pyruvic acid, 98% C3H4O3 88.06 Acros Organics

Citric acid anhydrous, 99.5% C6H8O 192.13 Scharlau

Succinic acid, 99% C4H6O4 118.09 Sigma-Aldrich

Fumaric acid C4H4O4 116.07

Ethanol absolute, 99.5% C2H5OH 46.07 Scharlau

Glycerol, 99.5% C3H5(OH)3 92.09 BDH laboratory

D(+)-Glucose anhydrous C6H12O6 180.16 Scharlau

Standard solution of each compound should be diluted 4 times. Table 3 shows the final concentration of components in four diluted standard solutions:

Table 3: Final concentration of compounds in four diluted standard solutions

Name Concentration in Concentration in Concentration in Concentration in St. solution No.1 St. solution No.2 St. solution No.3 St. solution No.4 (g/l) (g/l) (g/l) (g/l) Lactic acid 2.4 1.2 0.6 0.3 Pyruvic acid 2 1 0.5 0.25 Citric acid 7 3.5 1.75 0.87 Succinic acid 10 5 2.5 1.25 Fumaric acid 10 5 2.5 1.25 Ethanol 12 6 3 1.5 Glycerol 1.8 0.9 0.45 0.22 Glucose 30 15 7.5 3.75

Preparation

Prepare 10 ml standard solution No.1 according to the next instructions:

1. Put the volumetric flask in a scale and push the reset button to show the zero. 2. Then add the following amounts of the compounds to the flask: Lactic acid 0.024 g Pyruvic acid 0.02 g Citric acid 0.07 g Succinic acid 0.1 g Fumaric acid 0.1 g Ethanol 0.12 g Glycerol 0.018 g Glucose 0.3 g 3. Add distilled water to reach the volume 10 ml. 4. Use the magnetic stirrer to mix the compounds. 5. Transfer 5 ml of the standard solution No.1 to another volumetric flask. 6. Add water to reach the volume 10 ml. 7. The standard solution No.2 becomes ready. 8. Transfer 5 ml of standard solution No.2 to other volumetric flask to prepare standard solution No.3. 9. Add water to obtain the volume 10 ml. 10. Transfer 5 ml of standard solution No.3 to the fresh volumetric flask. 11. Add water to adjust the volume to 10 ml. 12. The standard solution No.4 becomes ready. 13. Pour 1.25 ml of the standard solution No.1 in each eppendorf tube. Then you can prepare 4 tubes of standard solution No.1. 14. Do the same for standard solution No.2, 3 and 4.then you have 8 tubes of each standard solution. 15. Mark the tubes. 16. Store them in freezer -20 °C.

If you want to run the HPLC, you need one tube of each standard solution. So continue with next steps:

17. Take them out from the freezer. 18. Centrifuge the tube for 10 min. 19. Vortex the tube to mix all the compounds. 20. Pour each standard solution in separate vial of HPLC. (Becareful: The volume of standard solutions and samples in the HPLC vials should not be less than 1 ml.) 21. Place them in the first fourth positions in HPLC carousel and then put the samples (See Protocol N0. 34) after the standard solutions. 22. Run the HPLC.

Protocol N0. 36: HPLC Retention Time

Date of Preparation: 29 Apr 2010 Revision # 00 References: (Karimi et al., 2005, Lennartsson et al., 2009) Estimated Time: -

HPLC Retention Time

Materials:

Lactic acid Pyruvic acid Citric acid Succinic acid Fumaric acid Ethanol Glycerol Glucose

Procedure:

After running the samples by HPLC, it is time to analysis the obtained results. Each component in the sample will have a different retention time so for analyzing the data it is essential to know the retention time of the interested compounds in the sample.

We run our samples by the HPLC, the hydrogen-based HPX-87H column (Bio-Rad, USA) was used for detecting the metabolites. A refractive index (RI) detector (Waters 410, Millipore, Milford, USA) was used for determining the concentration of Ethanol, Glycerol, Glucose, Fumaric acid, while the concentration of Succinic acid was determined by UV absorbance detector at 210 nm (Waters 486).The concentration of Citric acid, Lactic acid and Pyruvic acid were determined by both UV detector and RI detector.

We analyzed our results according to the following retention times come in Table 4:

Table 4: Retention times of different compounds

Item Compounds Retention time (min) Retention time (min) (RI column) (UV column) 1 Ethanol 21.7 5,784 2 Glycerol 12,975 5,782 3 Glucose 8,807 6,129 4 Fumaric acid 13.004 - 5 Citric acid 7,854 7,655 6 Lactic acid 12,205 12,004 7 Pyruvic acid 9,619 9,437 8 Succinic acid 11,072 10,871

Protocol N0. 37: 0.2 M Maleate Buffer Recipe

Date of Preparation: 10 Jun 2010 Revision # 00 References: This Work Estimated Time: 10 min

0.2 M Maleate Buffer Recipe (250 ml)

Materials:

Maleic acid 1N NaOH (See Protocol N0. 38) RNase free water (See Protocol N0. 21)

Procedure:

The procedure should be carried out in RNase free condition.

1. Mix the following compounds to prepare 0.2M maleate stock solution: Maleic acid 5.8 g 1N NaOH 50 ml 2. Add RNase free water to reach the volume 250 ml.

Protocol N0. 38: 1N NaOH Recipe (10 ml)

Date of Preparation: 12 Mar 2010 Revision # 00 References: This Work Estimated Time: 10 min

1N NaOH (10 ml)

Materials:

NaOH Distilled water

Procedure:

1. Weight 0.4 g NaOH powder. 2. Pour it in an Erlenmeyer flask. 3. Add distilled water to reach 10 ml. NaOH: MW= 40, EW= 40, 1N= 40 g/l X= = o.4 g

Appendix B: Information about ldhA gene from ncbi

LOCUS AB111547 963 bp DNA linear PLN 01-SEP-2004 DEFINITION Rhizopus oryzae ldh A gene for lactate dehydrogenase A, complete cds, strain: NBRC5414. ACCESSION AB111547 VERSION AB111547.1 GI:51775758 KEYWORDS . SOURCE Rhizopus oryzae ORGANISM Rhizopus oryzae Eukaryota; Fungi; Fungi incertae sedis; Basal fungal lineages; Mucoromycotina; Mucorales; Mucoraceae; Rhizopus. REFERENCE 1 AUTHORS Saito,K., Saito,A., Ohnishi,M. and Oda,Y. TITLE Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes JOURNAL Arch. Microbiol. 182 (1), 30-36 (2004) PUBMED 15278242 REFERENCE 2 (bases 1 to 963) AUTHORS Oda,Y. and Saito,K. TITLE Direct Submission JOURNAL Submitted (05-JUN-2003) Katsuichi Saito, National Agricultural Research Center for Hokkaido Region, Department of Upland Agriculture Research; Shinsei, Memuro, Kasai, Hokkaido 082-0071, Japan (E-mail:[email protected], Tel:81-155-62-9280, Fax:81-155-62-9281) FEATURES Location/Qualifiers source 1..963 /organism="Rhizopus oryzae" /mol_type="genomic DNA" /strain="NBRC5414" /db_xref="taxon:64495" gene 1..963 /gene="ldh A" CDS 1..963 /gene="ldh A" /codon_start=1 /product="lactate dehydrogenase A" /protein_id="BAD38918.1" /db_xref="GI:51775759" /translation="MVLHSKVAIVGAGAVGASTAYALMFKNICTEIIIVDVNPDIVQA QVLDLADAASISHTPIRAGSAEEAGQADIVVITAGAKQREGEPRTKLIERNFRVLQSI IGGMQPIRPDAVILVVANPVDILTHIAKTLSGLPPNQVIGSGTYLDTTRLRVHLGDVF DVNPQSVHAFVLGEHGDSQMIAWEAASIGGQPLTSFPEFAKLDKTAISKAISGKAMEI IRLKGATFYGIGACAADLVHTIMLNRKSVHPVSVYVEKYGATFSMPAKLGWRGVEQIY EVPLTEEEEALLVKSVEALKSVEYSSTKVPEKKVHATSFSKSSC" ORIGIN 1 atggtattac actcaaaggt cgccatcgtt ggagctggtg cagtaggagc ctccactgct 61 tatgcactta tgtttaaaaa catttgtaca gaaatcatta ttgtggatgt taatcctgac 121 atcgttcaag ctcaagtcct tgaccttgca gatgctgcca gtataagtca cacgcccatc 181 cgagcaggta gcgcagagga ggcagggcag gcagatattg ttgtcatcac ggccggtgcg 241 aaacaaaggg aaggtgagcc tcggacaaag ctcattgaac gaaacttcag agtgttgcaa 301 agtatcattg gtggcatgca acccattcga ccagacgcag tcatcttggt ggtagcaaat 361 ccagtcgata tcttgacaca cattgcaaag accctctctg gactgcctcc aaaccaggtc 421 attggctccg gtacctacct tgacacgacc cgtcttcgcg tccatcttgg cgatgtcttt 481 gatgtcaatc ctcaatcggt ccatgctttt gtcttgggtg aacatgggga ttcccagatg 541 atcgcttggg aggctgcttc gattggtggg cagccgttga caagtttccc ggaattcgca 601 aagctggata aaacagcaat ttcaaaagcg atatcaggta aagcgatgga gatcattcgt 661 ttgaaaggag ccacgtttta tggaattggt gcctgtgcag cggatttagt gcacactatc 721 atgttgaata ggaaatcagt acatccagtt tctgtttatg ttgaaaagta tggagccact 781 ttttctatgc ctgctaaact tggatggaga ggtgttgaac agatctatga agtaccactg 841 acggaagaag aagaagcgct gcttgtaaaa tctgtagaag cactgaaatc agttgaatat 901 tcatctacaa aagtcccaga aaaaaaagtt catgctactt ccttttctaa aagtagctgt 961 tga //

FASTA

>gi|51775758|dbj|AB111547.1| Rhizopus oryzae ldh A gene for lactate dehydrogenase A, complete cds, strain: NBRC5414 ATGGTATTACACTCAAAGGTCGCCATCGTTGGAGCTGGTGCAGTAGGAGCCTCCACTGCTTATGCACTTA TGTTTAAAAACATTTGTACAGAAATCATTATTGTGGATGTTAATCCTGACATCGTTCAAGCTCAAGTCCT TGACCTTGCAGATGCTGCCAGTATAAGTCACACGCCCATCCGAGCAGGTAGCGCAGAGGAGGCAGGGCAG GCAGATATTGTTGTCATCACGGCCGGTGCGAAACAAAGGGAAGGTGAGCCTCGGACAAAGCTCATTGAAC GAAACTTCAGAGTGTTGCAAAGTATCATTGGTGGCATGCAACCCATTCGACCAGACGCAGTCATCTTGGT GGTAGCAAATCCAGTCGATATCTTGACACACATTGCAAAGACCCTCTCTGGACTGCCTCCAAACCAGGTC ATTGGCTCCGGTACCTACCTTGACACGACCCGTCTTCGCGTCCATCTTGGCGATGTCTTTGATGTCAATC CTCAATCGGTCCATGCTTTTGTCTTGGGTGAACATGGGGATTCCCAGATGATCGCTTGGGAGGCTGCTTC GATTGGTGGGCAGCCGTTGACAAGTTTCCCGGAATTCGCAAAGCTGGATAAAACAGCAATTTCAAAAGCG ATATCAGGTAAAGCGATGGAGATCATTCGTTTGAAAGGAGCCACGTTTTATGGAATTGGTGCCTGTGCAG CGGATTTAGTGCACACTATCATGTTGAATAGGAAATCAGTACATCCAGTTTCTGTTTATGTTGAAAAGTA TGGAGCCACTTTTTCTATGCCTGCTAAACTTGGATGGAGAGGTGTTGAACAGATCTATGAAGTACCACTG ACGGAAGAAGAAGAAGCGCTGCTTGTAAAATCTGTAGAAGCACTGAAATCAGTTGAATATTCATCTACAA AAGTCCCAGAAAAAAAAGTTCATGCTACTTCCTTTTCTAAAAGTAGCTGTTGA

Appendix C: Information about ldhB gene from ncbi

LOCUS AB281557 1046 bp DNA linear PLN 17-JUN-2008 DEFINITION Rhizopus oryzae ldhB gene for lactate dehydrogenase, complete cds, strain: CBS 112.07. ACCESSION AB281557 VERSION AB281557.1 GI:139005932 KEYWORDS . SOURCE Rhizopus oryzae ORGANISM Rhizopus oryzae Eukaryota; Fungi; Fungi incertae sedis; Basal fungal lineages; Mucoromycotina; Mucorales; Mucoraceae; Rhizopus. REFERENCE 1 AUTHORS Abe,A., Oda,Y., Asano,K. and Sone,T. TITLE Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers JOURNAL Mycologia 99 (5), 714-722 (2007) PUBMED 18268905 REFERENCE 2 (bases 1 to 1046) AUTHORS Abe,A., Oda,Y., Asano,K. and Sone,T. TITLE Direct Submission JOURNAL Submitted (09-NOV-2006) Contact:Teruo Sone Hokkaido University, Research Faculty of Agriculture; Kita-9 Nishi-9 Kita-ku, Sapporo, Hokkaido 060-8589, Japan FEATURES Location/Qualifiers source 1..1046 /organism="Rhizopus oryzae" /mol_type="genomic DNA" /strain="CBS 112.07" /db_xref="taxon:64495" Gene 22..930 /gene="ldhB" CDS 22..930 /gene="ldhB" /codon_start=1 /product="lactate dehydrogenase" /protein_id="BAF52488.1" /db_xref="GI:139005933" /translation="MVLHSKVAIIGAGAVGASTAYALMFKNICTEIIIVDINPDIVQA QVLDLADAASVSNTPIRAGSAEEAGQSDIIVITAGAKQKEGEPRTKLIERNYRVLKNI IGGMQPIRSDAIILVVVNPVDILTHIAQTLSGLAPNQVIGSGTYLDTTRLRVHLGDIF DVNPQSIHAFVLGEHGDSQMIAWEAASIGGQPLTSFPEFAELDKKAISKAISGKAMEI IRLKGATFYGIGACAADLVHTIMLNRKSVHPVSVYVEKYGVTFSMPAKLGWRGVEKIY EVPLTEEEEALLLKSVEALKAVEYLS" ORIGIN 1 ttttcttttc tatataattc catggtacta cattcaaagg ttgccatcat tggagctggg 61 gcagtagggg cctccactgc ttatgcactt atgtttaaaa acatttgtac agaaatcatt 121 attgttgata ttaatcccga catcgttcaa gctcaagtcc ttgaccttgc agacgcagcc 181 agtgtaagca acacgcccat ccgagcaggc agtgcagagg aggcagggca gtcagatatt 241 attgtcatca cggccggtgc gaaacaaaag gaaggtgagc ctcggacaaa gctcattgaa 301 cgaaattaca gagtgttgaa aaatatcatt ggtggcatgc aacctattcg atcagacgca 361 atcatcttgg tggtagtaaa tccagtcgat atcttgacac acattgctca gacactctct 421 ggactggcac caaaccaggt gattggctcc ggtacctacc ttgacacgac ccgccttcgt 481 gtccatcttg gtgatatctt tgatgtcaat cctcaatcga tccatgcttt tgtcttgggt 541 gagcacgggg attcacagat gatcgcttgg gaggctgctt cgattggtgg gcagccgttg 601 acaagttttc cagaattcgc agagctggat aaaaaagcaa tttcaaaagc gatatcaggc 661 aaagcgatgg agatcattcg tttgaaagga gccacgtttt atggaattgg tgcctgtgca 721 gcggatttag tgcatactat catgttgaat aggaaatcag tacatccagt ttctgtttat 781 gttgaaaaat atggagtcac tttttctatg ccagctaaac ttggatggag aggtgttgaa 841 aagatatatg aggtaccact gacggaagaa gaggaagcgc tgcttttaaa atctgtagag 901 gcactgaaag cagttgaata tttatcataa tttatactca taaaaataaa aatggtgtca 961 ttcatctata aaaatcccag aaaaaaaagt ccatactact tccttttcca aaagtaaatg 1021 ttgataattt acaaataaca aatcat //

Fasta

GenBank: AB281557.1

>gi|139005932|dbj|AB281557.1| Rhizopus oryzae ldhB gene for lactate dehydrogenase, complete cds, strain: CBS 112.07 TTTTCTTTTCTATATAATTCCATGGTACTACATTCAAAGGTTGCCATCATTGGAGCTGGGGCAGTAGGGG CCTCCACTGCTTATGCACTTATGTTTAAAAACATTTGTACAGAAATCATTATTGTTGATATTAATCCCGA CATCGTTCAAGCTCAAGTCCTTGACCTTGCAGACGCAGCCAGTGTAAGCAACACGCCCATCCGAGCAGGC AGTGCAGAGGAGGCAGGGCAGTCAGATATTATTGTCATCACGGCCGGTGCGAAACAAAAGGAAGGTGAGC CTCGGACAAAGCTCATTGAACGAAATTACAGAGTGTTGAAAAATATCATTGGTGGCATGCAACCTATTCG ATCAGACGCAATCATCTTGGTGGTAGTAAATCCAGTCGATATCTTGACACACATTGCTCAGACACTCTCT GGACTGGCACCAAACCAGGTGATTGGCTCCGGTACCTACCTTGACACGACCCGCCTTCGTGTCCATCTTG GTGATATCTTTGATGTCAATCCTCAATCGATCCATGCTTTTGTCTTGGGTGAGCACGGGGATTCACAGAT GATCGCTTGGGAGGCTGCTTCGATTGGTGGGCAGCCGTTGACAAGTTTTCCAGAATTCGCAGAGCTGGAT AAAAAAGCAATTTCAAAAGCGATATCAGGCAAAGCGATGGAGATCATTCGTTTGAAAGGAGCCACGTTTT ATGGAATTGGTGCCTGTGCAGCGGATTTAGTGCATACTATCATGTTGAATAGGAAATCAGTACATCCAGT TTCTGTTTATGTTGAAAAATATGGAGTCACTTTTTCTATGCCAGCTAAACTTGGATGGAGAGGTGTTGAA AAGATATATGAGGTACCACTGACGGAAGAAGAGGAAGCGCTGCTTTTAAAATCTGTAGAGGCACTGAAAG CAGTTGAATATTTATCATAATTTATACTCATAAAAATAAAAATGGTGTCATTCATCTATAAAAATCCCAG AAAAAAAAGTCCATACTACTTCCTTTTCCAAAAGTAAATGTTGATAATTTACAAATAACAAATCAT

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