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

Inhaltsverzeichnis (Table of Contents)

• Chapter one
  • Rice---
  • Rice blast disease--
  • Life cycle of the rice blast fungus----
  • Control of rice blast disease-
  • Extent and mechanism of spontaneous genetic variation in the rice blast fungus-------
  • Genomic instability, recombinational repairs and pathogen variability---------
  • Functions and mechanism of homologous recombination in the fungi--------
  • Objectives--
• Chapter Two
  • Cloning, sequencing and expression analysis of Rhm51-
  • Cloning and sequencing of Rhm51--
    • DNA extraction from M. oryzae mycelia---
    • Amplification and cloning of Rhm51-
  • Determination of the open reading frame (ORF), the position and number of introns in Rhm51--
    • RNA extraction from M. oryzae mycelia--
    • Reverse transcriptase-PCR for Rhm51 cDNA amplification---
  • Northern Hybridization after inducing expression under various stresses-----
• Chapter Three
  • Disruption of Rhm51 and phenotypic analysis mutants--
  • Disruption of the Rhm51-
    • Construction of the disruption vector-
    • Production of protoplast---
    • Transformation of Ina168 protoplast with pDESTRRhm51inv and isolation of single conidium isolate------
    • Screening for target Rhm51 knockouts--
    • Screening of transformants by PCR--
    • Confirmation of target knockouts by Southern hybridization--
  • Phenotypic analysis of Rhm51 deletion mutants (Arhm51)-
    • Growth analysis---
    • Conidiation and appressoria induction-
    • Conidia killing test---
    • Use of pBARSTAdeAiny to check homologous recombination rate in M. oryzae------
    • Stability of Rhm51 deletion mutants-
    • Virulence test--
    • Complementation of rhm51 deletion mutants-
      • Construction of pBARSTRhm51A vector for complementation of Arhm51-
      • Screening and confirmation of transformants---
      • Phenotypic analysis of Ina168Rhm51A-
• Chapter Four
  • Mechanism of reduced pathogenicity in Rhm51 deletion mutants--
  • Reactive oxygen species (ROS) generation in Rhm51 deletion mutants-------
  • Cytogenetic analysis of Rhm51 deletion mutant--
    • Enumeration of nuclei in conidia and during appressorium morphogenesis--
    • Nuclei distributtion and septum formation during vegetative growth-------
  • Detection of double strand breaks in M. oryzae during vegetative and infective growth------
    • Neutral comet assay--
    • Enumeration of double-strand breaks during vegetative and infective growth in M. oryzae using green fluorescent protein-Rhm51 (GFP-Rhm51) foci formation---
      • Construction of pBARST-PPR-GFP-Rhm51A vector for Rhm51 foci detection---
      • Screening and confirmation of transformants-
      • In vitro quantification of Rhm51 foci using GFP-Rhm51 during V

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This research aims to investigate the role of the Rhm51 gene in the rice blast fungus *Magnaporthe oryzae*, a significant plant pathogen causing significant economic losses in rice production worldwide. The work delves into the genetic mechanisms underpinning pathogen variability and explores the impact of the Rhm51 gene on the fungus's growth, conidiation, appressoria formation, and virulence. Key themes of the research include:

• The role of the Rhm51 gene in DNA recombinational repair in *M. oryzae* and its impact on pathogen variability
• The impact of Rhm51 deletion on the phenotypic characteristics of *M. oryzae* including growth, conidiation, appressoria formation, and virulence
• The potential involvement of reactive oxygen species (ROS) in the reduced pathogenicity observed in Rhm51 deletion mutants
• The cytogenetic analysis of Rhm51 deletion mutants focusing on nuclear dynamics and DNA damage detection
• The application of various molecular techniques, including gene cloning, sequencing, disruption, and complementation, for in-depth analysis of the Rhm51 gene

Zusammenfassung der Kapitel (Chapter Summaries)

Chapter One presents a comprehensive overview of rice, rice blast disease, and the life cycle of the rice blast fungus *M. oryzae*. This chapter also discusses various control strategies for rice blast disease and emphasizes the importance of understanding the genetic mechanisms behind pathogen variability. The chapter concludes with a focus on the role of homologous recombination in fungi and its implications for fungal pathogenicity. Chapter Two details the cloning, sequencing, and expression analysis of the Rhm51 gene in *M. oryzae*. The chapter describes the techniques used for DNA and RNA extraction, gene amplification, and sequencing. It also examines the expression of the Rhm51 gene under various stress conditions. Chapter Three focuses on the disruption of the Rhm51 gene in *M. oryzae* and the subsequent phenotypic analysis of the resulting mutants. This chapter discusses the construction of a disruption vector, the production of protoplasts, the transformation of *M. oryzae* protoplasts, and the screening and confirmation of Rhm51 deletion mutants. The chapter further examines the phenotypic effects of Rhm51 deletion on growth, conidiation, appressoria formation, conidia killing, and virulence. Chapter Four investigates the mechanism behind the reduced pathogenicity observed in Rhm51 deletion mutants. This chapter examines the role of reactive oxygen species (ROS) in the observed phenotype and delves into the cytogenetic analysis of Rhm51 deletion mutants. The chapter concludes by examining the generation of double-strand breaks in *M. oryzae* during vegetative and infective growth, using various techniques including the neutral comet assay and GFP-Rhm51 foci formation.

Schlüsselwörter (Keywords)

The main keywords and focus topics of this research include: rice blast fungus, *Magnaporthe oryzae*, Rhm51, DNA recombinational repair, pathogen variability, homologous recombination, gene cloning, sequencing, disruption, complementation, phenotypic analysis, virulence, reactive oxygen species (ROS), cytogenetic analysis, double-strand breaks, GFP-Rhm51 foci formation, and neutral comet assay.