Analysis of Rhm51, a DNA Recombinational Repair Gene in the Rice Blast Fungus

Table of contents

List of tables

List of figures

List of appendices

Chapter one

1. General overview
1.1. Rice
1.2. Rice blast disease
1.3. Life cycle of the rice blast fungus
1.4. Control of rice blast disease
1.5. Extent and mechanism of spontaneous genetic variation in the rice blast fungus
1.6. Genomic instability, recombinational repairs and pathogen variability
1.7. Functions and mechanism of homologous recombination in the fungi
1.8. Objectives

Chapter Two

2. Cloning, sequencing and expression analysis of Rhm51
2.1. Cloning and sequencing of Rhm51
2.1.1. DNA extraction from M. oryzae mycelia
2.1.2. Amplification and cloning of Rhm51
2.2. Determination of the open reading frame (ORF), the position and number of introns in Rhm51
2.2.1. RNA extraction from M. oryzae mycelia
2.2.2. Reverse transcriptase-PCR for Rhm51 cDNA amplification
2.3. Northern Hybridization after inducing expression under various stresses

Chapter Three

3. Disruption of Rhm51 and phenotypic analysis mutants
3.1. Disruption of the Rhm51
3.1.1. Construction of the disruption vector
3.1.2. Production of protoplast
3.1.3. Transformation of Ina168 protoplast with pDESTRRhm51inv and isolation of single conidium isolate
3.1.4. Screening for target Rhm51 knockouts
3.1.5. Screening of transformants by PCR
3.1.6. Confirmation of target knockouts by Southern hybridization
3.2. Phenotypic analysis of Rhm51 deletion mutants (Arhm51)
3.2.1 Growth analysis
3.2.2 Conidiation and appressoria induction
3.2.3 Conidia killing test
3.2.4 Use of pBARSTd^e^inv to check homologous recombination rate in M. oryzae
3.2.5 Stability of Rhm51 deletion mutants
3.2.6 Virulence test-
3.2.7 Complementation of rhm51 deletion mutants
3.2.7.1 Construction of pBARSTRhm51A vector for complementation of krhm51
3.2.7.2. Screening and confirmation of transformants
3.2.7.3. Phenotypic analysis of Ina168Rhm51A

Chapter Four

4. Mechanism of reduced pathogenicity in Rhm51 deletion mutants
4.1. Reactive oxygen species (ROS) generation in Rhm51 deletion mutants
4.1.1. Superoxide detection
4.1.2. Other reactive oxygen species detection
4.1.3. Reactive oxygen species inhibition experiments
4.1.4. Quantification of MgRac1, NOX1 & NOX2 mRNA expression
4.2. Cytogenetic analysis of Rhm51 deletion mutant
4.2.1. Enumeration of nuclei in conidia and during appressorium morphogenesis--
4.2.2. Nuclei distributtion and septum formation during vegetative growth
4.3. Detection of double strand breaks in M. oryzae during vegetative and infective growth
4.3.1. Neutral comet assay
4.3.2. Enumeration of double-strand breaks during vegetative and infective growth in M. oryzae using green fluorescent protein-Rhm51 (GFP-Rhm51) foci formation
4.3.2.1. Construction of pBARST-PPR-GFP-Rhm51A vector for Rhm51 foci detection
4.3.2.2. Screening and confirmation of transformants
4.3.2.3. In vitro quantification of Rhm51 foci using GFP-Rhm51 during appressoria morphogenesis-
4.3.2.4. In pianta quantification of Rhm51 foci using GFP-Rhm51 during infective growth on rice

Chapter Five

Summary
Summary in English
Summary in Japanese

Chapter six

References

Appemdices

List of abbreviations

Acknowledgements

LIST OF TABLES

Table 1. List of primers used for the cloning and sequencing Rhm51

Table 2. List of primers for Rhm51 disruption and checking of homologous recombination rate

Table 3. Homologous recombination rate in M. oryzae rhm51 deletion mutant

Table 4. Primers for Rac1, Nox1, Nox2 analysis, construction of vectors for complementation of rhm51 deletion mutants and Rhm51 foci detection

List of figures

Figure 1a. Life cycle of the rice blast fungus

Figure 1b. Pathways of DNA double-strand break repair in eukaryotes

Figure 2. Mechanism of DNA double-strand break repair by homologous recombination

Figure 3. Cloning of Rhm51. (a) Amplicon of Rhm51 using Ina168 genomic DNA as template (b) Plasmid DNA extracted from 8 different E. coli colonies with colony 6 having the desired insert (c) colony 6 digested with EcoR1 showing vector and insert

Figure 4. (a) Amplicons of Rhm51 resolved on 1 % agarose gel. (b) A Schematic presentation of Rhm51 locus. Two damage responsive elements (DRE1 and DRE2, striped box) and a Mlu-I consensus sequence (black box) in putative promoter region are shown. Open boxes above those elements shows the aligmnments of those elements and consensus sequences. A black arrow indicates the Rhm51 ORF, which contain two introns (white boxes)

Figure 5. Effect of different stress treatments on Rhm51 expression in Magnaporthe oryzae strain Ina168. RNA extracted from mycelia in liquid culture (control) and from liquid culture with different stress treatments: heat shock (HS), methyl viologen (MV), methy methanesulfonate (MMS) were electrophoresed, standarized by ethidium bromide staining of rRNA (rRNA) and then blotted onto nylon membrane and probed by Rhm51 cDNA (Rhm51)

Figure 6. Production of the Rhm51 disruption involved an inverse PCR step to produce Rhm51inv which was cloned into an entry vector pENTR-D/TOPO to produce pENTRRhm51inv. LR-Clonase reaction was performed with the destination vector, pDESTR, to produce pDESTRrhm51inv which was linearized with EcoR1 and used to transform Ina168 protoplast

Figure 7. Schematic representation of the Rhm51 knockout reaction and screening for target knockouts. (a) The primer set DCF/QR amplifies the 1.5 kb fragment while DCR/QF amplifies the 2 kb fragment. When both bands are amplified for a given transformant, that tranformant is considered a possible target knockout mutant (b) confirmation of target deletion by southern hybridization. Genomic DNA from possible target mutants was digested with BamH-I which cut the sequence at the H-site. Rhm51 gene was used as probe. D= target deletion mutants while T= non target transformants. Target deletion mutant number 2 for each strain was used for further analysis

Figure 8. Construction of pBARSTRhm51A vector for complementation of Rhm51 deletion mutant.

Figure 9. Effect of loss of Rhm51 on (a) conidiation (b) conidia stability during treatment with 0.3 % methyl methanesulfonate in Magnaporthe oryzae Ina168 and Ina86-137. [illustration not visible in this excerpt] Ina168, # Ina86-137, Olna168Arhm51, [illustration not visible in this excerpt] Ina86-137Arhm51. The results are means of 3 independent measurements. * Significant difference at P < 0.05 using the Student’s t-test

Figure 10. Effect of loss of Rhm51 on mycelia growth. Each strain was inoculated on prune agar (PA), PA +0.0025 % methyl methanesulfonate (MMS) or PA + 0.01 % hydrogen peroxide plate. [illustration not visible in this excerpt] Ina168, • Ina86-137, x Ina86 137Rhm51Com, OIna168Arhm51, [illustration not visible in this excerpt] Ina86-137Arhm51. * Significant difference at P < 0.05 using the Student’s t-test.

Figure 11. Virulence of Magnaporthe oryzae strain Ina86-137, Ina86-137Arhm51 and Ina86-137Rhm51Com on two compatible rice cultivars Ishikari shiroke and Shin2. The photos were taken 7 days after inoculation.

Figure 12. Effect of loss of Rhm51 on appressoria formation in Magnaporthe oryzae strains Ina168 and Ina86-137. * Significant difference at P < 0.05 using the Student’s t-test. * Significant difference at P<0.05 using the Student’s t-test.

Figure 13. Stability of Arhm51 mutants following stress treatments

Figure 14. Homologous recombination (HR) rate in M. oryzae; Amplification of the Phosphinotricin acetyltransferase gene and absence of Ade gene demonstrated target Ade gene disruption

Figure 15. Localization of superoxide production sites at different stages of the infection process in Magnaporthe oryzae strains Ina168 and Ina86-137 and their respective Rhm51 deletion mutants using p-nitroblue tetrazolium chloride (NBT). (b, d, f, h, j, l) Hyphal growth, (a, c, e, g, i, k) Appressorium morphogenesis. Control means no treatment with NBT. Scale bars = 10 pm. (m) The intensity of the cyan color was estimated in the appressoria. Percentages are the means of 30 independent images. *Significant difference at P<0.05 using the Student’s t-test

Figure 16. Localization of other ROS production sites at different stages of the infection process of M. oryzae using 5,6-dicarboxy-2’,7’-dichlorodihydro- fluorescein diacetate (H2DCFDA)

Figure 17. Effect of reactive oxygen species (ROS) inhibition on growth of M. oryzae wild-type and krhm51 strain. * Significant difference at P<0.05 using the Student’s t-test

Figure 18. Quantification of (a) MgRacl, (b) NOX1 and (c) NOX2 by Real-Time PCR in krhm51 and wild-type strains of M. oryzae. * Significant difference at the 0.05 level using the Student’s t-test

Figure 19. Localization and enumeration of nuclei in conidia and appressorium 24 h post inoculation in two pathogenic strains of Magnaporther oryzae using a mixture of propidium iodide (PI) and calcofluor white (CW). BF= Bright field. Scale bar = 10 pm

Figure 20. Effects of loss of Rhm51 on nuclear distribution and septation in Magnaporthe oryzae. Ina86-137 and Ina86-137Arhm51 strains were grown (a) on frosted glass slides for 5 h or (b) in liquid media for 48 h and stained with a mixture of propidium iodide (PI) and calcofluor white (CW). BF= Bright field. Nuclei stained red under red fluorescence while septa stained white under blue fluorescence. (c) Strains were grown for 5 and 10 hrs on hydrophilic surface of Gelbond film and stained with CW to measure the rate of septum formation. Hpi (hours post inoculation). Scale bars = 10 pm

Figure 21. Neutral comet assay for double-strand break (DSB) quantification in M. oryzae during vegetative growth in liquid media. Bars with different letter show significant difference at P<0.05 using the Student’s t-test. Scale bars = 10 pm

Figure 22. Construction of pBARST'PPR-GFP-Rhm51 A vector for quantification of Rhm51 foci formation in vitro and in planta during the infection process

Figure 23: Detection of Rhm51-foci in M. oryzae during vegetative growth in liquid media. (a) Conidia were prepared from Ina86-137 GFP-Rhm51A. Conidia were inoculated in 2YEG and incubated at 27o C for 3 days and then treated with DAPI (4’, 6-diamidino-2- phenylindole). BF = Bright field. (b) The results presented are based on analysis from three independent samples and 150 different images. Arrows show points of foci. Scale bars = 10 pm

Figure 24: Detection of Rhm51-foci in M. oryzae during vegetative growth on hydrophobic surface. Conidia of Ina86-137- GFP-Rhm51A were spotted onto frosted glass slides and incubated at 27o C for 5-10 hours. Foci were detected in the germinating conidia, germ tubes and growing hyphae, with or without treatment using 0.1 gM mitomycin C. The results are means of three independent experiments. At least 50 images were analyzed at each stage of growth. Bar with different letter signify statistical difference at the 0.05 level of significance using the Student’s t-test. Arrows show points of foci. BF = Bright field. Scale bars = 10 gm

Figure 25: Detection of Rhm51-foci in M. oryzae during appressoria formation and plant infection. Conidia from Ina86-137-GFP-Rhm51A (1 x 104 /mL) were spotted on gelbond film and incubated at 27o C for 24 hours followed by DAPI staining. Some of the conidia (1.5 x 105 /mL) were inoculated on intact leaf sheath of compatible rice (cultivar: Shin2) and incubated at 25o C and 60 % relative humidity in a cultivation chamber for 48 hours. GFP-Rhm51 foci were detected during plant colonization. APP = Appressorium, PP = Penetration peck, HI = Infective hyphae. Arrows show points of foci. BF = Bright field. Scale bars = 10 gm

LIST OF APPENDICES

Appendix-I. Composition of solutions and reaction conditions

Appendix-II. Fungicidal compounds used for the control of rice blast disease

Appendix-III. Non- fungicidal compounds used for the control of rice blast disease

CHAPTER ONE

General Overview

Chapter 1

1. General Overview

1.1. Rice

Domesticated rice comprises two species in the Poaceae ("true grass") family, Oryza sativa and Oryza glaberrima. These plants are native to tropical and subtropical southern Asia and southeastern Africa (Crawford & Shen, 1998). Rice is a staple for a large part of the world's human population, especially in East, South and Southeast Asia, making it the most consumed cereal grain (FAOSTAT, 2006). It provides more than one fifth of the calories consumed worldwide by humans (Smith, 1998). Although its species are native to South Asia and certain parts of Africa, centuries of trade and exportation have made it commonplace in many cultures. Between 2005 and 2025, 1.5 billion new rice consumers will be added in the world. Feeding these people will require the greatest effort in the history of agriculture; rice production must be increased by one third from today’s 700 million tons to 933 million tons. However, efforts to increase rice production are hampered by less land, less water and decreasing soil fertility. Pests and diseases have further complicated these problems.

1.2. Rice Blast disease

Rice blast disease, caused by the filamentous ascomycete fungus Magnaporthe oryzae (anamorph: Pyricularia oryzae) is one of the most economically devastating diseases worldwide. The disease is also commonly known as rice rotten neck, rice seedling blight, blast of rice, oval leaf spot of graminea, pitting disease, ryegrass blast, johnson spot, and Imochi-byo (Japanese). It can also infect a number of other agriculturally important cereals including wheat, rye, barley, and pearl millet causing diseases called blast disease or blight disease. Magnaporthe oryzae causes economically significant crop losses annually and it is estimated to destroy enough rice to feed more than 60 million people (Zeigler et al., 1994). Infection occurs when fungal conidia land and attach themselves to leaves using a special adhessive (mucilage) released from the tip of each conidium (Hamer et al., 1988). The germinating conidia develop an appressorium, a specialised infection cell, which generates enormous turgor pressure (up to 8 MPa) that ruptures the leaf cuticle, allowing invasion of the underlying leaf tissue (Dean, 1997; De Jong et al., 1997). Subsequent colonization of the leaf produces disease lesions from which the fungus conidiates and spreads to new plants (Dean et al., 2005). The pathogen attacks the aerial parts (stems, nodes or panicle) (Talbot, 2003) and roots (Sesma & Osbourn, 2004) of the plant at any stage of growth. When rice blast infects young rice seedlings the whole plant often dies, whereas its attack on older plant leads to total loss of the rice grain (Talbot, 2003).

1.3. Life cycle of the rice blast fungus

M. oryzae is an extremely effective plant pathogen as it can reproduce both sexually and asexually as it produces specialized infectious structures known as appressoria that infect aerial tissues and hyphae that can infect root tissues. The asexual life cycle (Fig.1a) begins when the hyphae of the fungus undergo conidiation to produce fruiting structures called conidia. When these conidia land on leaves and other aerial tissues of susceptible plants they germinate, developing the appressorium. The appressorium penetrates the plant cell by producing a penetration peg. Pressure in the appressorium increases and the structure explodes, forcing the penetration peg through the cell wall and into the plant cell. The fungus can then grow hyphae within the leaf and form lesions. Once established in the host plant the fungal hyphae can undergo asexual reproduction again. Sexual reproduction occurs when two strains of opposite mating types meet and form a perithecium in which ascoconidias develop. Once released, ascospores can develop appressoria and infect host cells. Conidia are transmitted between plants by the wind.

illustration not visible in this excerpt

Figúrela. Life cycle of the rice blast fungus (Dean et al., 2005)

1.4. Control of rice blast disease

Rice blast disease has been observed in almost every rice growing country and tremendous efforts have been put in place to control this disease. Chemical control and the breeding of blast resistant cultivars are the major strategies that have been employed to control the disease.

At moment, 16 chemicals have been developed and used to control rice blast disease (Yamaguchi, 2004). Seven of these chemicals are fungicidal while 9 are non-fungicidal (Appendix II & III respectively).

Benzylaminobenzene sulfonate of blasticidin-S, an antibiotic that contains calcium acetate and a harmless emulsion has been demonstrated to have high blast control efficacy and less toxic to plants and humans. In addition, several microbes can mobilize and detoxify blasticidin-S (Yamaguchi et al., 1975). Kasugamycin, another antibiotic formerly used for blast control has been discontinued due to reduction in efficacy and increase in the number of resistant strains (Yamaguchi, 2004).

Iprobenphos (IBP), edifenphos (EDDP) and isophothiolane have been used to practically control rice blast. IBP and isophothiolane are systemic while EDDP is non systemic. However, they all inhibit phospholipids biosynthesis in the membrane of the blast fungus by inhibiting the conversion of phosphatidylethanolamine to phosphatidylcholine (Kodama et al., 1979).

Ferimzone, a fungistatic blast control agent is a novel systemic fungicide that causes specific leakage of electrolytes from mycelia of the pathogen. It is thus used as a synergistic component in mixtures with other blast agents (Okuno et al., 1989). Metominostrobin is another systemic novel anti-blast agent that blocks fungal respiration and this is done with the aid of flavonoids from the host plant (Mizutani et al., 1996). Modern anti-blast chemicals are non-fungicidal and act on secondary metabolism of the pathogen such as melanin biosynthesis, or they induce plant defense mechanisms referred to a systemic acquired resistance (SAR) (Yamaguchi, 2004). Fthalide, tricyclazole, pyroquilon, carpropamid, diclocymet and fenoxanil exert their anti-blast activity by inhibiting melanin biosynthesis (Yamaguchi & Kubo, 1992). Melanin is important for the maturation of appressoria, which is necessary for pathogen infectivity. Pyroquilon has also been shown to inhibit secondary infection under field conditions by reducing conidiation of M. oryzae.

Probenazole (Oryzemate®) is a systemic plant defense activator that has been used for the control of blast in Japan since 1975 and no development of resistance in the blast fungus has been observed (Iwata et al., 2004). This is possible since probenzole and its metabolites neither affect the growth nor infectivity of the fungus. Probenzole activates (1) the defense-related phenylpropanoid pathway in the host plant, (2) accumulation of fungicidal substances of plant origin mostly hydroxylated unsaturated fatty acids derived from a-linoleic acid, (3) signal transduction systems that allow the plant to quickly respond to pathogen attack and thus prevent infection, and (4) amplifies superoxide production, which is part of a hypersensitivity reaction, a powerful defense mechanism against pathogen attack (Iwata et al., 2004). A similar compound, acibenzolar-s-methyl also induces systemic acquired resistance but its mode of action is different from that of probenzole. These plant defense activators are environmentally safe, not biocidal, act against a wide range of plant pathogens and have a low risk of inducing pathogen resistance.

Chemical control of plant pathogens is most effective. However, the use of chemicals is not generally desired because it is not labor-saving and also due to the serious environmental threat it poses sometimes mainly as a result of misuse (Sasahara &

Koizumi, 2004). In developing countries, poor farmers cannot afford to control blast disease by the application of chemicals. Besides, their continuous use leads to the resurgence of resistant races of the pathogen under selection pressure.

The use of resistant cultivars is the best alternative to overcome yield losses. However, the variability of the pathogen and the history of resistance breakdown have led to the development of different plant breeding approaches to achieve durable blast resistance (De Waard et al., 1993). One approach has been the breeding of isogenic and multilines that are resistant to the rice blast disease (Zhu et al., 2000; Sasahara & Koizumi, 2004; Correa-Victoria et al., 2004; Kojima et al., 2004). Another approach has been the introduction of the plant defensin gene in to rice to control rice blast (Kawata et al., 2004; Kanzaki et al., 2004). The transformants are highly resistant and this resistance has been inherited over several generations. However, most of these studies are being carried out under greenhouse condition or on small field trials and the durability of these resistances under extensive field conditions remains to be tested.

1.5. Extent and mechanism of spontaneous genetic variation in the rice blast fungus

Although the sexual stage of M. oryzae can be produced between highly selected isolates in the laboratory (Leung & Williams, 1987), clonality remains the natural mode of reproduction (Chen et al., 1995). Despite apparent clonality, M. oryzae displays a high level of genetic variability. Tremendous variation in virulence has been documented in field populations of the blast fungus (Correa-Victoria & Zeigler, 1993; Ou, 1980; Ou, 1985; Zeigler et al., 1995) and, to some extends, among asexual derivatives of single- conidia isolates (Valent et al., 1991). Parasexual recombination in the blast pathogen occurs at a detectable frequency in field populations (Zeigler et ai., 1996) thus providing an important mechanism for generating genetic variation and eliminating deleterious mutations accumulated in clonal lineages. Based on these results, the blast fungus can be described as having a predominantly asexual mode of reproduction in which a recombination system is active.

Resistance conditioned by a single major resistance (R) gene is typically effective against races of M. oryzae containing the corresponding avirulence (AVR) gene (Silué et ai., 1992). Management of rice blast through breeding of blast-resistant varieties has had only short-term success due to frequent breakdown of resistance under field conditions (Valent & Chumley, 1994). Frequent appearance of new races (or pathotypes) of the fungus that are capable of infecting previously resistant varieties has been proposed as the principal cause for the loss of resistance (Ou 1980). Several mechanisms have been proposed for the appearance of new pathotypes. Transposon insertion into AVR genes has been shown to convert avirulent isolates into virulent isolates and may be a distinct molecular mechanism by which the pathogen defeats the resistance gene (Kang et ai., 2001; Zhou et ai., 2007). Reports of diverse mutations in AVR genes have been reported in several laboratory strains of M. oryzae (Orbach et ai., 2000; Kang et ai., 2001) resulting in a race-shift from avirulence to virulence on rice cultivars previously exhibiting resistance to M. oryzae in a gene-for-gene fashion (Flor, 1971; Correll et ai., 2000). Dean et ai., (2005), report that 9.7 % of M. oryzae genome assembly is made up of repetitive DNA sequences longer than 200 bp. Furthermore, Thon et ai., (2006) reported that 14 % of chromosome 7 of M. oryzae alone is made up of transponsable elements (TE) with 8.8 % being the long terminal repeat (LTR)-type of retrotransposons. They went ahead to demonstrate that a significant correlation exist between chromosome rearrangement rate and TE content not only for chromosome 7 but for the entire genome suggesting that TE clusters are the major contributors of the genesis and evolution of new genes in M. oryzae genome.

In certain strains of M. oryzae, locus-specific genetic instability has been identified, including SMO1 (Hamer et ai., 1989), BUFI (Chumley & Valent, 1990), PWL2 and AVR2-YAMO (Valent & Chumley, 1994) and MGR586-associated RFLP, an LTR-type retrotransposon that appears to be hypervariable and undergoes recurrent changes during asexual propagation. In certain instances (e.g., AVR2-YAMO), this mutability has been attributed to the association of the locus adjacent to telomere (Shull & Hamer, 1996).

1.6. Genomic instability, recombinational repairs and pathogen variability

Cells proliferate mainly by meiosis and mitosis and these processes need to be tightly coordinated to preserve genome integrity and favor faithful genome propagation. Coordination of DNA replication with DNA-damage sensing, repair and cell-cycle progression ensures, with a high probability, genome integrity during cell divisions, thus preventing mutations and DNA rearrangements (Aguilera & Gomez-Gonzalez, 2008). Mutation and rearrangement in M. oryzae have been shown to drive genetic variation and evolution at the molecular level (Dean et al., 2005). Instability leading to mutations (base substitutions, micro-insertions and micro-deletions), are mainly associated with replication errors, impairment of base excision repair (BER) and mismatch repair (MMR) or error-prone translesion synthesis (Aguilera & Gomez-Gonzalez, 2008). Instability leading to rearrangements refers to events that involve changes in genetic linkage of two DNA fragments. Increases in homologous recombination (HR)-mediated events (such as unequal sister-chromatid exchange (SCE) and ectopic HR between non-allelic DNA fragments) or in end-joining between non-homologous DNA fragments can result in gross chromosomal rearrangements such as translocations, duplications, inversions or deletions (Fig. 1b). The common substrate of these rearrangement events are DNA double-stranded breaks (DSBs) (Aguilera & Gomez-Gonzalez, 2008) since unrepaired DSB can cause loss of chromosome /or cell death while misrepaired DSB can give rise to mutations and chromosomal rearrangements (Ljungman & Lane, 2004).

DNA double-Standed breaks (DSB) arise as intermediates during meiosis and mitosis, DNA replication, transposition of certain mobile elements, transduction, transformation and conjugation in bacteria, mating-type switching in yeast and V(D)J- joining in vertebrate immune system. DSBs are also induced by exogenous agent (such as ionizing radiations (IR) and a wide range of chemicals) and endogenous agent such as free radicals generated during metabolic processes (Dudas and Chovanec, 2004). Homologous recombination involves the exchange of DNA between sequences of perfect or near perfect homology over several hundreds of base pairs. The process of homologous recombination plays essential roles in the mitotic and meiotic cell cycles of most eukaryotic organisms. In meiosis, the primary function of recombination is to establish a physical connection between homologous chromosomes to ensure their correct disjunction at the first meiotic division. In addition, meiotic recombination contributes to diversity by creating new linkage arrangements between genes, or parts of genes. It is now widely recognized that the primary function of homologous recombination in mitotic cells is to repair DSBs that form as a result of replication fork collapse, from processing of spontaneous damage, and from exposure to DNA-damaging agents.

Homologous recombination is an accurate pathway for the repair of DSB and studies on RAD52 epistasis group (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11 and XRS2; originally identified by their importance in the repair of ionizing radiation-induced DNA damage) has provided information on how these gene participate in DNA repair processes. Mutation in these genes leads to defects in meiotic/or mitotic recombination, providing evidence for a link between DSB repair and homologous recombination (Symington, 2002). Two recombinational repair genes Rhm52 and Rhm54 from M. oryzae, both homolog of the S. cerevisiae RAD52 and RAD54 respectively have been cloned and expression of both genes, as is the case with RAD52 epistasis group members, were induced by the methyl methanesulfonate (MMS) and ultraviolet (UV) light treatment. Expression was induced even at higher levels by Methyl viologen dichloride hydrate (MV) and heat stress (HS) (Elegado et al., 2006). Genetic studies place the RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54, and RFA1 genes in the homologous recombination pathway. Within this group, the RAD51, RAD52, RAD54, RAD55, and RAD57 genes are essential for conservative DSB repair, resulting in gene conversion (and associated crossing over), while RAD52 and RAD59 have additional functions in the non-conservative break induced replication (BIR) and single-strand annealing (SSA) pathways (Symington, 2002). In E. coli, RecA is the most important gene for homologous recombination and based on protein sequence, RAD51 is considered to be its structural and functional homologue (Dudas and Chovanec, 2004). The predicted mouse and human Rad51 protein differ only by 4 amino acids. They have 80 % similarity to S. cerevisiae or S. pombe Rad51 proteins. Saccharomyces cerevisiae RAD51 shows 81 % similarity to the N. crassa mei-3 proteins. However, the similarity of E coli RecA protein is only 30 % and is confined to the ATP-binding domains. Like RecA, Rad51 has an ATP-dependent DNA-binding activity and polymerizes on both ssDNA and dsDNA forming helical filaments, only the RAD51-ssDNA nucleoprotein filament is functionally relevant for DNA strand exchange during homologous recombination (Namsaraev & Berg, 2000).

1.7. Functions and mechanism of homologous recombination in the fungi

Homologous recombination (HR), the exchange of genetic material between allelic sequences, has essential roles in meiosis and mitosis. In meiosis, HR mediates the exchange of information between the maternal and paternal alleles within the gamete precursor cells and thus generates diversity among progeny derived from common parents. HR has a second critical function in meiosis; it ensures the proper segregation of homologous chromosome pairs at the first meiotic division through the formation of crossovers, resulting in gametes with the correct number of chromosomes (Neale & Keeney, 2006; Aguilera & Gomez-Gonzalez, 2008). These functions of HR ensure the stability of the organism karyotype. Meiotic HR is initiated by Spo11-mediated DNA DSBs (Neale & Keeney, 2006). HR maintains somatic genomic stability by promoting accurate repair of DSBs induced by ionizing radiations and other agents, repair of incomplete telomeres that arise when the enzyme telomerase is nonfunctional, repair of DNA interstrand crosslinks, and the repair of damaged replication forks (Ljungman & Lane, 2004). Although cells have alternate DNA repair pathways such as non homologous end-joining (NHEJ), these may not be operative at all phases of the cell cycle, they do not always act on injured replication forks, nor are they as precise as HR in the repair of broken chromosomes (Filippo et al., 2008).

After a DSB is formed and recognized by the Mre11-RAD51-Xrs2 (MRX) complex, Sae2, in collaboration with MRX, trims the ends to create a minimally resected intermediate that is ‘compatible’ for processive resection by the 59-39 exonucleolytic activity of Exol or Sgsl helicase and a single-strand-specific nuclease (Fig. 2.). Cells lacking both the nuclease and the helicase activity accumulate the intermediates from MRX/Sae2 cleavage. Because Amrell and Asae2 mutants still show DSB processing, Exol and Sgsl must be able to access unprocessed DNA ends, but with reduced efficiency (Mimitou & Symington, 2008). Rad51 forms a nucleoprotein filament on processed DNA ends at the site of DNA breaks. This filament invades the intact homologous chromosome to search for homologous sequences that leads to D-loop formation. Agmon et al. (2009) showed that resection and homology search occur simultaneously and the resection process ends once homology is found. Following D- loop formation, Rad54 binds the junction and promotes branch migration either toward or away from the site of DNA damage. Depending on the direction of branch migration, Rad54 can contribute to the mechanisms of HR described by the DSBR or SDSA models that lead to crossover and non-crossover products (Fillips et al., 2008; Rossi & Mazin, 2008)

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Figurelb. Pathways of DNA double-strand break repair in eukaryotes.

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Figure 2. Mechanism of DNA double-strand break repair by homologous recombination (Filippo et ai., 2008; Mimitou & Symington, 2008; Rossi & Mazin, 2008; Agmon et ai., 2009)

Double-strand breaks (DSBs) can be repaired by distinctive homologous recombination (HR) pathways, such as synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR) (Fig. 2). (a) After DSB formation, the DNA ends are resected to yield 3’single-strand DNA (ssDNA) overhangs, which become the substrate for the HR protein machinery to execute strand invasion of a partner chromosome. After a successful homology search, strand invasion occurs to form a nascent D-loop structure. DNA synthesis then ensues. (b) In the DSBR pathway, the second DSB end can be captured to form an intermediate that harbors two Holliday junctions (HJ)s, accompanied
by gap-filling DNA synthesis and ligation. The resolution of HJs by a specialized endonuclease can result in either non-crossover or crossover products (c). Alternatively, the D-loop is unwound and the freed ssDNA strand anneals with the complementary ssDNA strand that is associated with the other DSB end. The reaction is completed by gap-filling DNA synthesis and ligation. Only non-crossover products are formed.

1.8. Objectives

Double-Stranded Breaks that may result from cell metabolism or genotoxic substances from both endogenous and exogenous sources are the substrates of homologous recombination (Aguilera & Gomez-Gonzalez, 2008). Although pathogenic variability in M. oryzae has been attributed to; parasexuality (Zeigler et al., 1996), mitotic recombination (Sukhodolets, 2006), locus-specific instabilities (Hamer et al., 1989; Chumley & Valent 1990; Valent & Chumley, 1994; Shull & Hamer, 1996) and repetitive DNA sequences that can generate new genes (Dean et al., 2005) and creates “hot spots” for homologous recombination at the transposon donor sites (Hagemann & Craig, 1993), the role played by homologous recombination in pathogenicity and variability in M. oryzae is not clear. Rhm51, the RAD51 homolog gene in M. oryzae, which is expected to play a crucial role both in mitotic and meiotic recombination (Haber, 1999; Dudas & Chovanec, 2004), has not been characterized. Unlike in higher eukaryotes where RAD51 is indispensable (Lim & Hasty, 1996; Markmann-Mulisch et al., 2007), in yeast, the null mutant is viable but accumulates meiosis-specific DSB, partially defective in the formation of recombinants, poor sporulation efficiency and very low conidia viability (Dudas & Chovanec, 2004). RAD51 is essential as RAD52 for some processes like DSB- induced gene conversion involving two homologous chromosomes but less important for other type of events like spontaneous recombination, DSB-induced DNA replication and DSB-induced SSA. Based on the crucial role RAD51 plays in DNA DSB-induced homologous recombination, we set out to clone and characterize Rhm51 in view to shed light on its contribution to the pathogens’ variability and pathogenicity.

CHAPTER TWO

2 Cloning, Sequencing and Expression analysis of Rhm51

Chapter 2

2.1. Cloning and sequencing of Rhm51

Magnaporthe oryzae Ina168 (Ina from Inabu) has been reported to have a mutation rate of 12.5 % (Sone et al., 1997) and the RAD51 homolog for this strain, Rhm51 has not been sequenced. Information relating to the conservation of this gene is unknown. Faced with the growing interest in pathogenic variability and the unknown role of homologous recombination in generating such variability, it was necessary to clone and sequence Rhm51 to see if this gene has been conserved in the face of this high rate of mutation in M. oryzae.

Materials and Methods

2.1.1. DNA extraction fTom M. oryzae mycelia

Ina168 stored on prune agar slant was inoculated in 2 YEG (0.2 % yeast extract and 1 % glucose) and incubated at 27o C for a week while shaking at 100 rpm. Fifty milligram (50 mg) of freeze-dried mycelia was powdered using a cell disruption centrifuge (Multi-Beads Shocker, Yusui Kikai, Japan). After cell disruption, 500 pL of DNA extraction buffer was added and vortexed for about 20 seconds to ensure that the buffer mixes well with the powdered mycelia. The DNA was then extracted using the phenol/ chloroform method and precipitated using isopropanol. The samples were vacuum-dried and 50 pL of TE buffer added and stored at 4o C. The next day, the DNA concentrations were checked.

2.1.2. Amplification and cloning of Rhm51

The M. grisea hypothetical protein similar to N. crassa mei-3 gene sequence was downloaded online from Broad Institute (MG11350.6, in the genome database http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/GeneDetails.htm). This sequence is located in Chromosome I, supercontig196 and spanning from 3795000­3800000 i.e. 5kb. Forward and reverse primers for the amplification of Rhm51 (Table 1) were designed respectively 50 nucleotides from the beginning and before the end of this sequence. Primers were synthesized (Invitrogen, Carlsbad, CA) and M. oryzae Ina 168 genomic DNA was used to amplify Rhm51 by polymerase chain reaction (PCR) using Platinum® PCR SuperMix High Fidelity kit (Invitrogen). The reaction mixture and PCR conditions were done according to the manufacturers’ guidelines (Invitrogen) with slight modifications using the GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA). Amplification was confirmed by running PCR products on 1 % agarose gel (125V for 35 min), staining with ethidium bromide and visualizing under a UV-Transilluminator before taking a photograph. The PCR products were then cleaned using a MicroSpin Column (GE Healthcare, Buckinghamshire, UK), and ligated to pGEM®-T Easy vector (TA cloning) by the standard reaction protocol (Promega, Madison, WI) with slight modifications. The ligation product was concentrated to 5 pL by ethanol precipitation using Ethachinmate (Wako, Osaka, Japan) and used to transform 40 pL of E.coli TOP10 cells (Invitrogen) by electroporation using a gene pulser (BIO-RAD, Hercules, CA).

After electroporation, the cells were recovered in 1 mL SOC medium (see appendix-I for composition) and incubated at 37oC in an incubation shaker (140 /min) for 30 min. Two hundred microlitres of recovered cells were plated on five LB Agar/Ampicillin/Isopropyl-ß-D-thiogalactopyranoside/5-bromo-4-chloro-3-indolyl- ß- d- galactoside (LB-AgarAmp/IPTG/Xgal) plates. The concentrations of Ampicillin, Isopropyl-ß- D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl- ß- D-galactoside (Xgal) in this medium were 100 pg/mL, 100 nM and 40 pg/mL respectively. The plates were incubated at 37oC overnight and transformants were selected by Blue/White screening.

The white colonies that were probably carrying the plasmid with the insert of interest were transferred on to an LB-AgarAmp master plate and incubated overnight at 37oC. Each colony was picked with a tooth pick and transferred into 5 mL of LBAmp in a test tube and incubated in an incubator shaker at 37oC overnight. A plasmid was extracted from each culture using the centrifugation protocol of the Quantum Prep® Plasmid Miniprep Kit (BIO-RAD). In order to determine which plasmid had the right insert, the plasmid was digested with EcoRl in a final reaction mixture of 10 pL and resolved on 1 % agarose gel. Plasmid with sum total size of digested product equivalent to the size of the plasmid (3 kb) plus insert (5 kb) were selected for sequencing using the BigDye Terminator V1.1 cycle sequencing kit and M13 universal primers. The reaction mixture and PCR cycling conditions were done according to manufacturer’s guidelines (Applied Biosystems). After the sequencing reaction, the products were purified by filtration using the Performa® DTR Gel Filtration Cartrige (Edge BioSystems, Gaithersburg, MD), freeze dried in a Vacuum Freeze dryer (Freeze Trap, VA-500F, TAITEC, Saitama,

Japan). Dried samples were mixed with 25 pL of Hi-Di-Formamide, heated at 95 C for 2 min and then rapidly cooled on ice, transferred into sequence analysis tubes and analyzed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). One colony with the right insert was selected, grown in a large quantity (100 mL of LBAmp) in a round bottom flask using the same incubation conditions as above, and plasmid extracted using the Quantum Prep® Plasmid Maxi Prep Kit (BIO-RAD). Ten forward and reverse Rhm51 sequencing primers (Table 1) were designed 500 bp apart using the online sequence. These primers were used to sequence the entire insert. A total of 20 sequencing reactions were done as describe above. After analyzing the sequence reactions, a consensus sequence was built using Factura and Auto assembler version 2.0 (Applied Biosystems). The plasmid was named as pGEMRhm51

Results and discussions

Genomic DNA from M. oryzae Ina 168 was successfully used as template for the amplification of Rhm51 (Fig. 3a). After transforming the ligated product into E. coli TOP10 cells, 8 white colonies were selected by blue/white screening. Analysis of these colonies by separating EcoR1-digested products on 1 % agarose gel and Sequencing with M13 universal primers revealed one colony (colony 6) (Fig. 3b) which was selected for plasmid extraction. After full length sequencing, a consensus sequence that had 4950 bp was built. This consensus sequence had 100 % identity with the M. grisea hypothetical protein similar to mei-3 gene sequence in the National Centre for Biotechnology Information (NCBI) database.

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Figure 3: Cloning of Rhm51. (a) Amplicon of Rhm51 using Ina168 genomic DNA as template (b) Plasmid DNA extracted from 8 different E. coli colonies with colony 6 having the desired insert (c) colony 6 digested with EcoR1 showing vector and insert.

2.2. Determination of the open reading frame (ORF), the position and number of introns in Rhm51

The putative Rhm51 transcript size and sequence reported at the Broad Institute database are estimated by computer simulation. It was thus necessary for us to get RNA from Magnaporthe oryzae Ina 168, perform cDNA cloning and sequencing to identify the ORF, introns and exons.

Materials and Methods

2.2.1 RNA Extraction from M. oryzae mycelia

M. oryzae Ina168 was grown on oatmeal agar for 2 weeks in the dark at 27oC and then transferred and grown for 3 days under light at 25oC for conidia production.

Colonies were flushed with 2YEG scrapped lightly; conidia filtered with gauze and counted using a haemocytometer. In 2YEG, 105 conidias were treated with 1.5 % methyl methanesulfonate (MMS, Sigma-Aldrich, St Louis, MO) for an hour, washed to remove of MMS and grown at 27oC for 3 days in fresh 2YEG while shaking at 100 rpm on a Recipro Shaker (NR-10, TAITEC). Mycelia was collected after 3 days growth and immediately stored on -80oC until RNA was extracted. Total RNA was extracted from mycelia that were collected at the log phase of growth using RNAiso Plus according to manufacturer’s instruction (Takara, Shiga, Japan).

2.2.2. Reverse transcriptase-PCR for Rhm51 cDNA amplification

The M. grísea hypothetical protein similar to N. crassa mei-3 gene sequence was used to query the National Centre for Biotechnology Information (NCBI) database for mRNA sequences that were closely aligned to the hypothetical protein mRNA sequence using the blast basic local alignment search tool (Altschul et al., 1997). This information was used to determine the possible start and stop site of the Rhm51 transcript. Primers were designed for reverse transcriptase-PCR (RT-PCR) (Table 1) using Superscript High Fidelity RT-PCR kit (Invitrogen). The template was total RNA extracted from 1.5 % MMS treated M. oryzae Ina 168 mycelia treated with DNase. The procedure for cloning and sequencing of the RT-PCR product was similar to that of cloning and sequencing of Rhm51 described in section 2.1. Three forward and reverse Rhm51cDNA primers (Table 1) were designed 500 nucleotides apart and used to sequence the entire Rhm51cDNA. The Rhm51 cDNA consensus sequence was aligned with the Rhm51 consensus sequence using GENETYX version 10 software maximum match options to determine the ORF, introns and exons.

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Results and discussions

The M. grisea hypothetical protein similar to N. crassa mei-3 transcript sequence report in the NCBI database has 3132 bp with 5 exons and 4 introns. Primers designed at the estimated start and stop sites did not yield the expected amplification product after RT-PCR. Therefore, new forward and reverse primers were designed at a putative start and stops sites (position 3220 and 4440 respectively) of the hypothetical sequence estimated from the alignment between mei-3 of N. crassa. These primers yielded a clear and highly induced 1.2 kb band using RNA extracted from MMS treated mycelia (Fig. 4a) as template. The complete nucleotide sequence Rhm51 is published at EMBL/GenBank/DDBJ databases, accession number AB562330. The proposed Rhm51 has an ORF of 1054 bp with a start site at position 3232 and the stop site at position 4425 of the Rhm51 sequence (Fig. 4b). The ORF has three exons (96, 148 and 810 bp) and two introns (64 and 73 bp). RAD 51 gene of S. cerevisiae has a transcript size of 1.6 kb but its homolog have shown some degree of variation from one organism to the other; 1.6 kb for N. crassa; 1.2 kb for Pneumocystis; 1.4 kb for Aspergillus nidulans and 1.9 kb, 1.6 kb and 1.3 kb for S. pombe with possible reason being differences in transcription initiation and termination sites. The Mlu-I consensus sequence 5’-ACGCGT-3’ is present at 172 bp to the ORF. The 5’ flanking region of this Mlu-I sequence is absolutely essential for cell cycle-regulated expression of genes (Gordon & Campbell, 1991). Two damage responsive elements (DREs), which are important for DNA-damage expression, are also present at position 2593 and 2723 bp to the ORF although only the 3’end (TGAAA) is highly conserved in M. oryzae Ina168 and their position is more distal than in other organisms (Fig.4b). These DREs with conserved 3’end have also been reported in the upstream region of RAD51 of S. cerevisiae (Silva et al., 2005) while the Mlu-I consensus sequence has been reported in the promoter regions of RAD51 of S. cerevisiae, uvsC of A. nidulans and mei-3 of N. crassa (Verma et al., 1992; Hatakeyama et al., 1995). The predicted Rhm51 protein contains 351 amino acids and show 97 % and 66 % similarity to the N. crassa mei-3 and S. cerevisiae Rad51 proteins respectively.

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Figure 4. (a) Amplicons of Rhm51 resolved on 1 % agarose gel. (b) A Schematic presentation of Rhm51 locus. Two damage responsive elements (DRE1 and DRE2, striped box) and a Mlu-l consensus sequence (black box) in putative promoter region are shown. Open boxes above those elements shows the aligmnments of those elements and consensus sequences. A black arrow indicates the Rhm51 ORF, which contain two introns (white boxes).

2.3. Northern Hybridization after inducing expression under various stresses

In order to understand if Rhm51 will be induced by genotoxic substances and various stresses like other RAD52 epistasis group member, we set out to study the effect of genotoxic substances and other stresses on the induction of Rhm51.

Materials and methods

M. oryzae Ina168 was grown on oatmeal agar for 2 weeks in the dark at 27oC and then transferred and grown for 3 days under light at 25oC for conidia production.

Colonies were flushed with 2YEG scrapped lightly; conidia filtered with gauze and counted using a haemocytometer. Conidias (105) were treated with 0.1 and 0.15 % methyl methanesulfonate (MMS, Sigma-Aldrich, St Louis, MO) for 1 h; 0.1, 1 and 10mM 1,1’- dimethyl-4,4’-bipyridinium dichloride [methyl viologen dichloride hydrate (MV), Sigma- Aldrich] for 18 h; and heat stressed (HS) at 42°C for 45 min. The conidias were grown at 27oC for 3 days in fresh 2YEG while shaking at 100 rpm on a Recipro Shaker (NR-10, TAITEC). Mycelia were collected after 3 days growth and immediately stored on -80oC until RNA was extracted. Total RNA was extracted from mycelia that were collected at the log phase of growth using RNAiso Plus according to manufacturer’s instruction (Takara, Shiga, Japan). DNase treated total RNA extracted from Ina 168 mycelia that had been subjected to the following stresses; heat (HS), methyl viologen dichloride hydrate (MV), methyl methanesulfonate (MMS) were dissolved in 5 pL dethyl pyrocarbonate double distilled water (DEPC, see Appendix-1 for preparation) and stored at -80oC until needed. Ten microlitres of loading buffer (65.79 % (V/V) formamide, 13.16 % (V/V) 10X MOPS [41.2 g 3-(V-morpholino) propanesulfonic acids (MOPS); 10.9 g

CH3COONa'3H2Ü; 3.7 g EDTA sodium salt per litre DEPC water, pH 7.0] and 21.05 % (V/V) formaldehyde) were added to 5 pL (10 pg) of RNA samples, heated at 65oC for 15 min. After heating, 2 pL of 1 mg/mL Ethidium Bromide and 3 pL of 6x loading dye (0.25 % (W/V) bromophenol blue; 0.25 % (W/V) xylene cyanol FF; 30 % (W/V) Glycerol) were added to the samples and ran on a denaturing gel. In order to prepare the gel, 0.30 g agarose (Seakam-GTG) was first dissolved in 25 mL DEPC water, boiled and maintained at 55oC in a water bath. Next, a denaturant was prepared by mixing 1.75 mL formaldehyde with 3 mL of 1x MOPS and maintained at 55oC. The gel and denaturant were mixed and cast in a flame hood. Electrophoresis was performed at 50 V for 60 min with 1x MOPS as running buffer.

After electrophoresis, the gel was stained with ethidium bromide and observed under a UV-transilluminator. The gel was then soaked twice in DEPC water for 15 min and then once in 10x SSC (0.3 M Sodium Citrate; 3 M sodium chloride). Blotting was done according to Sambrook and Russell (2001) overnight on to a Hybond-N+ nylon membrane (GE Healthcare) using 20x SSC as the transfer solvent. Following blotting, the membrane was carefully removed and soaked in 2x SSC for 5 min and the RNA on it was cross-linked using a UV spectrolinker (Spectronics Corp., Japan), applying maximum cross-linking conditions (1200 x 100 pJ/cm2). Rhm51 cDNA that was used to produce the probe was derived by digesting pGEMRhm51 cDNA with EcoR1, running the digested products on 1 % Agaraose (Seakam GTG) gel and purifying the Rhm51 cDNA from the gel using the Wizard® SV Gel and PCR Clean-Up System (Promega) by the centrifugation procedure. The Amersham Gene Images Alkphos Direct Labelling and Detection System (GE Healthcare) procedures was used for the preparation of labeled probe, hybridization, post hybridization stringency washes, signal generation and detection. The X-ray film (Fuji Photo Film) was exposed for 1 h after which they were processed using Rendol and Renfix solutions (Fuji photo film).

Results and discussions

Rhm51 was induced by MMS and MV and the level of induction increased with increasing dose of treatment. Although Rhm51 was not induced in heat-treated samples, the level of induction was measurable to that of untreated samples (Fig. 5). This goes to support the proposal by Hatakeyama et al., (1995) that this gene is constitutively expressed at low levels during the cell cycle and at a higher level following treatment with mutagens. Methyl viologen dichloride hydrate (MV) generates reactive oxygen species (ROS) and this mimics ROS produce by the rice plant as a component of a hypersensitivity reaction against the attack of an incompatible pathogen (Elegado et al., 2006). In addition, the pathogen may also come in contact with a wide range of chemicals used against the blast disease necessitating the induction of Rhm51. Two RAD52 epistasis group members of M. oryzae, Rhm52 and Rhm54, RAD52 and RAD54 homologues had already been characterized. These genes were induced by the methyl methanesulfonate and ultraviolet light treatment. Expression was induced even at higher levels by methyl viologen and heat stress (Elegado et al., 2006). Thus RAD52, RAD51 and RAD54 are involved in homologous recombination and their increase induction following certain stresses indicates that this pathway is very active in M. oryzae Ina 168 and may function both in the repair of DSBs and for generating genetic diversity.

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Figure 5. Effect of different stress treatments on Rhm51 expression in Magnaporthe oryzae strain Ina168. RNA extracted from mycelia in liquid culture (control) and from liquid culture with different stress treatments: heat shock (HS), methyl viologen (MV), methy methanesulfonate (MMS) were electrophoresed, standarized by ethidium bromide staining of rRNA (rRNA) and then blotted onto nylon membrane and probed by Rhm51 cDNA (Rhm51).

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