The ortholog of the Saccharomyces cerevisiae recombinational repair gene RAD51, Rhm51 was cloned from the genome of rice blast fungus Magnaporthe oryzae in order to clarify its role in the variability and pathogenicity. Rhm51 is a single copy gene, and is constitutively expressed at low levels during the cell cycle and at higher levels when the pathogen is grown in media supplemented with DNA damaging agents. We disrupted Rhm51 in two different strains of M. oryzae and both mutants showed reduced growth with conidia and colonies being highly sensitive to DNA double strand break-inducing agents. In addition, they lost the ability to undergo homologous recombination in adeA gene targeting. Mutants showed reduction in conidiation compared to the wild-type strains. Although mutants infected compatible rice cultivars, they produced significantly smaller and fewer lesions compared to wild-type strains and this might be due to the reduction in the amount of appressoria formed. DNA double-strand breaks (DSBs) quantified by the comet assay showed that loss of Rhm51 resulted in the accumulation of DSBs in mutant. Microscopic observation of nuclei and reactive oxygen species (ROS) production during hyphal growth and following appressoria formation revealed impaired mitotic entry, autophagic cell death and ROS production in Rhm51 deletion mutants. Rhm51-GFP foci were observed in all stages of the asexual life cycle including the invasive hyphae formed in an intact rice leaf sheath, demonstrating that M. oryzae suffers DSBs during vegetative and infective growth.These results for the first time show that Rhm51 is involved in growth and pathogenicity of M. oryzae through the accurate repair of DSBs that may arise during vegetative and infective growth.
Table of Contents
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
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
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 (Δrhm51)
3.2.1 Growth analysis
3.2.2 Conidiation and appressoria induction
3.2.3 Conidia killing test
3.2.4 Use of pBARBARSTAdeAinv 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 Δrhm51
3.2.7.2. Screening and confirmation of transformants
3.2.7.3. Phenotypic analysis of Ina168Rhm51A
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 planta quantification of Rhm51 foci using GFP-Rhm51 during infective growth on rice
Research Objectives and Thematic Focus
The primary objective of this thesis is to clone and characterize the Rhm51 gene, a homolog of the RAD51 DNA recombinational repair gene, in the rice blast fungus Magnaporthe oryzae, in order to evaluate its contribution to the pathogen's genomic variability, growth, and pathogenicity.
- Genomic stability and homologous recombination mechanisms in Magnaporthe oryzae.
- Identification of the open reading frame (ORF), intron positions, and expression patterns of Rhm51.
- Phenotypic characterization of Rhm51 deletion mutants regarding growth, conidiation, and virulence.
- Analysis of the relationship between homologous recombination, reactive oxygen species (ROS) production, and DNA double-strand break repair.
- In vivo quantification of double-strand breaks during the infection cycle using GFP-tagged Rhm51 foci.
Excerpt from the Book
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).
Summary of Chapters
1. General overview: Provides background information on the impact of rice blast disease, the life cycle of Magnaporthe oryzae, and the biological importance of DNA recombinational repair mechanisms.
2. Cloning, sequencing and expression analysis of Rhm51: Details the isolation, sequencing, and expression profiling of the Rhm51 gene, including the determination of its ORF and inducibility by various stress factors.
3. Disruption of Rhm51 and phenotypic analysis mutants: Describes the creation of Rhm51 deletion mutants and evaluates their phenotypic defects, including sensitivity to DNA-damaging agents and virulence reduction.
4. Mechanism of reduced pathogenicity in Rhm51 deletion mutants: Investigates the cytogenetic causes of reduced pathogenicity, focusing on ROS production, nuclear distribution, and the quantification of DNA double-strand breaks.
Keywords
Magnaporthe oryzae, Rhm51, RAD51, DNA repair, homologous recombination, double-strand breaks, rice blast disease, pathogenicity, reactive oxygen species, appressorium, genomic instability, gene disruption, phenotype analysis, cell cycle, virulence.
Frequently Asked Questions
What is the core subject of this research?
The work focuses on the functional analysis of the Rhm51 gene, which is a RAD51 homolog, and its role in the DNA recombinational repair pathway of the plant pathogen Magnaporthe oryzae.
What are the primary areas of research?
The key areas include genetic characterization of Rhm51, the creation and analysis of deletion mutants, the role of homologous recombination in genomic stability, and the impact of these factors on the fungus's pathogenicity.
What is the main objective of this study?
The primary goal is to clone and characterize Rhm51 to determine how this gene contributes to the pathogen's genetic variability and its ability to infect rice hosts.
Which methodologies are employed?
The research uses various molecular techniques, including DNA extraction, PCR-based gene disruption, Southern hybridization, Northern blot analysis, real-time PCR for gene expression, and fluorescent imaging of GFP-tagged Rhm51 foci.
What topics are covered in the main body?
The main body treats the molecular cloning of Rhm51, the construction of deletion vectors, the analysis of mutant phenotypes under different stress conditions, and a detailed cytogenetic study of double-strand break repair.
Which keywords define this work?
The work is defined by terms such as Magnaporthe oryzae, Rhm51, homologous recombination, DNA double-strand breaks, and pathogen virulence.
How does the loss of Rhm51 affect the fungus?
The deletion of Rhm51 leads to increased sensitivity to DNA-damaging agents, reduced vegetative growth, impaired conidiation and appressorium formation, and a significant decrease in overall virulence.
What did the GFP-Rhm51 foci experiments reveal?
These experiments provided in vivo evidence that M. oryzae suffers from double-strand breaks at various life cycle stages, and that Rhm51 is directly involved in repairing these lesions during both vegetative and infective growth.
- Citar trabajo
- Sali Atanga Ndindeng (Autor), 2010, Analysis of Rhm51, a DNA Recombinational Repair Gene in the Rice Blast Fungus, Múnich, GRIN Verlag, https://www.grin.com/document/184249
