IDENTIFICATION AND CHARACTERIZATION OF PATHOGEN EFFECTOR- HOST TARGET INTERACTIONS INVOLVED IN RICE BLAST DISEASE

Open Access
Author:
Chen, Yueying
Graduate Program:
Plant Pathology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 06, 2016
Committee Members:
  • Yinong Yang, Dissertation Advisor
  • Yinong Yang, Committee Chair
  • Timothy W Mcnellis, Committee Member
  • Seogchan Kang, Committee Member
  • Surinder Chopra, Outside Member
  • Yanming Wang, Committee Member
Keywords:
  • Rice Blast disease
  • Effectors
  • plant immunity
  • rice
  • plant development
  • Disease regulator
Abstract:
During the plant-pathogen interaction, plants have evolved a multi-layered immune system to overcome pathogen infection, whereas pathogens have gained broad capabilities to defeat host defense by secreting effector proteins into plant cells to interfere with host physiology. Rice blast disease, caused by fungal pathogen Magnaporthe oryzae, is an important plant disease and the model pathosystem for studying the monocot plant-fungus interactions. However, little is known regarding how M. oryzae effectors interact with rice proteins for host defense suppression and fungal infection. This study aims to shed light on molecular and biochemical mechanisms of the rice-M. oryzae interaction by functionally characterizing some M. oryzae effectors and their host target proteins. In this study, Yeast two-Hybrid (Y2H) screenings were performed using four fungal effectors as baits to identify their interacting rice proteins. These pathogen effector-host protein interactions were further confirmed using in vitro GST pull-down assay and in vivo bimolecular fluorescence complementation (BiFC) assay. An M. oryzae effector-rice protein interaction map was then established by integrating the data from this study and previously published information. These putative host targets include several interesting rice proteins such as chromatin remodeler OsHIRA, ubiquitin-like protein OsNPI, and transcription factor OsVOZ1. Functional prediction and categorization of host target proteins reveal that M. oryzae effectors appear to favorably target host Ubiquitin Proteasome System (UPS) and nuclear processes. Two M. oryzae Zinc Finger Effectors (MoZFEs) were individually expressed in transgenic rice lines (MoZFEs-OX) to investigate their function in plant cells. Interestingly, MoZFEs-OX lines exhibited growth and developmental defects and increased susceptibility to M. oryzae infection. Furthermore, transcriptome analysis with RNA-seq reveals a amount of differentially expressed genes (DEGs) in MoZFE1-OX lines vs. wildtype plants. Many of these DEGs are enriched in primary metabolism and defense pathways that correlated with developmental and susceptible phenotypes shown in transgenic rice lines. To elucidate the mode of action of MoZFEs in rice cells, BiFC was used to demonstrate that MoZFEs interact directly with OsHIRA in the nucleus and cytoplasm. HIRA forms gene silencing complex with AS1 and AS2 transcription factors which bind to the promoters of Class I Knotted1-like homeobox (KNOX) genes and regulate plant growth and development in an epigenetic fashion. Bioinformatics analysis of rice gene promoters reveals proximal nucleotide binding motifs that are potentially bound by MoZFE1 and OsHIRA/AS1/AS2. As shown by gene expression analysis with quantitative RT-PCR, the silencing status of KNOX genes maintained by the OsHIRA complex in wild-type leaves was disrupted in MoZFE-OX lines. These bioinformatics predictions and experimental evidence suggest that MoZFEs likely interact with HIRA to interfere with AS1/AS2 binding, and negatively modulate rice growth and defense responses. To further characterize the biological function of the HIRA/AS1/AS2 complex in rice development as well as innate immunity, oshira, osas1, and osas2 mutants were generated by targeted mutagenesis using the CRISPR/Cas9 mediated genome editing. Strikingly, these mutants all exhibited developmental defects during the vegetative and reproductive stages, with some of these phenotypes resembling that of MoZFEs-OX lines. Moreover, oshira mutant also shows enhanced disease resistance to M. oryzae infection, suggesting its role in negative regulation of rice immunity. In summary, thesis studies reveal some novel interactions between M. oryzae effectors and rice protein targets, demonstrates the functions of MoZFEs and OsHIRA/AS1/AS2 silencing complex in rice development and immunity, and improves our understanding of the molecular and biochemical, molecular mechanisms involved in the rice-M. oryzae interaction.