MENU

Project Information


Reference Number 2020-0326
Project Title CRISPR/Cas9-targeted modification in rice genes with resistance to rice sheath blight disease and abundance of key microbial hubs in rice leaf microbiome
Project Type Contained Use (Laboratory)
Status Ongoing
Name of Institution International Rice Research Institute
Cooperating Institution Iowa State University, USA
Supervising IBC International Rice Research Institute (IRRI)
Project Leader(s)

Dr. Ricardo Oliva and Dr. Inez Hortense Slamet-Loedin

Experimental Facility/Site IRRI
Purpose / Objectives

This project is conducted to validate the involvement of the ten candidate genes with resistance against rice sheath blight disease and abundance of key microbial hubs in the rice leaf microbiome. Specifically, it aims to:

  1. Develop CRISPR/Cas constructs with customized gRNAs targeting the R genes against sheath blight disease (Os11g0694125, Os11g0695800, Os11g0695000, Os05g0555600, Os06g0486900 and Os11g0641500) and rice leaf microbiome (Os02g02400, Os04g40990, Os05g44340, and Os01g48420) including several genes identified as related to microbiome composition based on genome-wide association study (GWAS) and network analysis (Annex 11).

  1. Generate mutant lines with at least 3 genes in each background line of IR64 and in the case where it is more convenient we will use IR64-derived varieties such as IRRI 154 and Ciherang (ΨLOC_ Os11g0694125, ΨLOC_ Os11g0695800, ΨLOC_ Os11g0695000, ΨLOC_Os05g0555600, ΨLOC_06g0486900, ΨLOC_ Os11g0641500, ΨLOC_ Os02g02400, ΨLOC_Os04g40990, ΨLOC_ Os05g44340, ΨLOC_ Os01g48420).

  1. Test the resistance of the mutant lines against corresponding rice pathogen and abundance of microbial hubs.

  1. Evaluate the association of loss of gene functions with the resistance of the generated mutant lines.

  1. Overexpress the R genes in the knockout lines and in the WT IR64 and IR64-derived varieties as complementary experiments.
Biosafety measures

1. Access to the organism, and other contaminated materials will be limited to those who will be granted permission to the restricted containment facilities in the Institute.

2. Only personnel trained to work under aseptic conditions and with sufficient knowledge of molecular techniques and/or properly supervised personnel will be allowed to take part in the experiment.

3. Seeds of the engineered rice lines will be handled in compliance to all relevant biosafety and phytosanitary requirements of the Philippines.

4. Engineered rice lines will be kept in isolation in Bay7, CL4 and transgenic screen houses (CS-01), respectively, to prevent access by unauthorized personnel.

5. Samples collected from the transformed plants will be placed in sealed plastic bags and then placed in a second sturdy, leak-proof, plastic container, which will be hermetically sealed.

6. Inoculated plants will be kept in isolation in the CL4 transgenic greenhouse to prevent access by unauthorized personnel.

7. Samples collected from the inoculated plants will be placed in sealed plastic bags and then placed in a second sturdy, leak-proof, plastic container, which will be hermetically sealed.

8. All the following other materials contaminated with the pathogen will be decontaminated by autoclaving at high temperature and high pressure to kill the inoculum prior to disposal according to standard biosafety protocol.

                  - pots and soil used to grow the uninoculated plants

                  - culture media, diluents and buffers

                  - syringe, gloves  and non-disposable laboratory materials

9. Plants and plant materials for disposal will be placed in sealed plastic bags and will be decontaminated by autoclaving and then disposed of following standard procedures prescribed by the biosafety committee.                              

10. Precautionary measures will be observed following guidelines set by IRRI’s Biosafety Office and IRRI’s Stewardship Implementation Team. Good laboratory practices will be stringently imposed to prevent cross-over and dissemination of transgenic plant materials. 

11. Researchers that will be working on transgenic materials are required to attend training and orientation on the proper handling of transgenic materials. Laboratory staff likewise need to pass the Good Laboratory Practice (GLP) Course. IRRI also implements stewardship practices that meet Excellence Through Stewardship standard meant to complement Biosafety. 

Conditions for Approval N/A
Date of Approval (DD-MM-YYYY) 22-08-2020
Date of Completion (DD-MM-YYYY) N/A
Executive Summary

Rice is an important agricultural crop that is constantly threatened by a number of biotic constraints and emerging virulent strains of pathogens. To meet the growing rice demand and improve yield stability, development of durable and broad spectrum disease resistance in rice is one of the major goals of plant research. There is a wide range of pathogens including bacteria, fungi, oomycetes, viruses and nematodes, attacking the rice plant in different pathosystems but only in a specific interaction result in disease. In the case of rice sheath blight, an important fungal disease caused by Rhizoctonia solani, it is important to understand and identify the core components of the host resistance and at the same time targets of pathogens for manipulation. More so in host-pathogen interaction, the microbial consortium surrounding the host, pathogen and environment also influence disease and yield stability of rice. Hence, it is vital to understand the abundance of microbial hubs and its mechanisms that are involved in stress response and metabolism in rice. The new tools in synthetic biology such as the CRISPR/Cas system, allow the development of targeted genome editing. This tool can be very promising to investigate the host-pathogen interactions and manage crop diseases.

In this study, we will validate the involvement of candidate genes related to (1) resistance to  the rice sheath blight disease and (2) abundance of key microbial hubs in leaf microbiome that influence crop disease. A total of six candidate genes associated with resistance to sheath blight (Os11g0694125, Os11g0695800, Os11g0695000, Os05g0555600, Os06g0486900 and Os11g0641500) and four candidate genes associated to composition of rice leaf microbiome (Os02g02400, Os04g40990, Os05g44340, and Os01g48420) will be used. The ten candidate genes were identified from the genomics and transcriptomics studies that were previously conducted by our team. The identified genes will be mutated using the CRISPR/Cas system in IR64 and IR64-derived varieties like IRRI 154 and Ciherang. CRISPR/Cas constructs will be developed with customized gRNAs for all of the candidate genes and generated mutants will be tested for resistance against the corresponding pathogens. We expect that engineered lines will allow pathogen multiplication after infections as a result of R gene knockouts from which we can validate the key genes of microbial hubs in the rice leaf microbiome that orchestrate the host-microbe interactions.  To support our findings, we will do a complementary experiment by overexpressing the candidate genes in the knockout lines and in susceptible varieties, IR64 and IR64-derived varieties. Our results will contribute to unravel some of the complex mechanisms underlying the rice-pathogen interactions. This study also aims to better understand the plant immune response system which may be of valuable information for breeders

.

This proof-of-concept project will be of interest to many rice researchers, thus the seeds that will be generated in this study will be stored for future collaborations. Transgenic facilities (CL4, Bay 7 and CS-01) at IRRI will be used for the growth of the engineered plants. The expected duration of the project is three (3) years.