Project Information

Reference Number 20160304
Project Title CRISPR/Cas9-targeted modification in rice genes associated with resistance to plant diseases
Project Type Contained Use (Greenhouse)
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
Dr. Inez Hortense Slamet-Loedin

Experimental Facility/Site N/A
Purpose / Objectives

This project is conducted to validate the involvement of the five candidate genes in resistance against rice diseases. Specifically, it aims to:

1.     Develop CRISPR/Cas9 constructs with customized gRNAs targeting the R genes against bacterial blight (LOC_Os07g01560, LOC_Os07g15460 and LOC_Os09g29820), sheath blight (LOC_Os11g46900 and LOC_Os11g46860) and sheath brown rot (LOC_Os02g39330) diseases,

2.     Generate IR64 (?LOC_Os11g46900 and ?LOC_Os11g46860), IRBB8 (?LOC_Os07g01560, ?LOC_Os07g15460 and ?LOC_Os09g29820) and MI GU (?LOC_Os02g39330) mutant lines,

3.     Test the resistance of the mutant lines against corresponding rice pathogens,

4.     Evaluate the association of loss of resistance to the level of transcripts of the knockout genes in the generated mutant lines and

Overexpress the R genes in the knockout lines and in the wt IR64, as complementary experiment.

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 inoculumprior 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 always be observed and good laboratory practices will be stringently imposed to prevent cross-over and dissemination transgenic plant materials

Conditions for Approval

a) Project activities shall be conducted at the Genetic Transformation and Plant Pathology Laboratories, CL4 transgenic glasshouse (Bay7) and at CS-01 transgenic screen house located at the International Rice Research Institute in Los Banos, Laguna.

b) The proponent shall adhere as closely as possible to the schedule of activities reflected in the submitted Gantt chart.

c) Any modifications in the schedule of activities shall be made with the concurrence of the IRRl-IBC and the DOST-BC.

d) Signage should be in place indicating that the facility is restricted/limited access.

e) The proponent must provide a logbook to record access of personnel to the experimental facility.

f) The proponent must take measures to ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities .

g) The experimental facilities must be kept under lock when not in use.

h) In case of undue destruction of experimental materials resulting from unauthorized entry of personnel or breach of containment of experimental facilities, the proponent shall implement measures to prevent the inadvertent escape of any viable material within the facility.

i) The proponent and the supervising IBC shall be held accountable for the undue destruction of the experimental materials and the consequences that their inadvertent escape may cause to the surrounding environment.

j) The DOST-BC should be informed in advance of any visitations by unauthorized persons.

k) The DOST-BC shall be informed immediately of any intrusion by unauthorized persons.

I) The proponent shall strictly observe proper disposal procedures for all materials.

m) Movement of all materials will be done in compliance with all relevant biosafety and phytosanitary requirements of the Philippines.

n) Any additional requirement that the DOST-BC may impose, as necessary, for the duration of the experiment shall be complied with.

o) The proponent shall submit through the IBC a completion report within 90 days after completion of the experiment.

p) The IBC shall submit to the DOST-BC a report on the completion of this project, in the prescribed format.

Date of Approval (DD-MM-YYYY) 21-01-2017
Date of Completion (DD-MM-YYYY) N/A
Executive Summary

Rice is constantly threatened by many damaging emerging virulent strains of pathogens. Thus, one of the major goals of plant research is to improve yield stability through the development of durable, long-lasting and broad-spectrum disease resistance in rice. 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 this result in disease. Therefore, it is important to understand the plant immune system and identify core components of resistance which will be the key players and at the same time targets of pathogens for manipulation. The new tools in synthetic biology such as the CRISPR/Cas9 system, allow the development of targeted genome editing. This tool can be very promising to exploit for understanding the host-pathogen interaction and comprehend what lies behind the susceptibility and resistance in plants. In this study, we have identified a total of six candidate genes associated with resistance to bacterial blight (LOC_Os07g01560, LOC_Os07g15460 and LOC_Os09g29820), sheath blight (LOC_Os11g46900 and LOC_Os11g46860) and sheath brown rot (LOC_Os02g39330). The data that we gathered from our genomics and transcriptomics studies allow us to pinpoint these six R genes and to validate our findings, we will mutate these genes using the CRISPR/Cas9 system in the respective resistant varieties (IR64 for sheath blight, IRBB8 for bacterial blight and MI GU for sheath brown rot). CRISPR/Cas9 constructs will be developed with customized gRNAs for the six R 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.  To support our findings, we will do a complementary experiment by overexpressing the R genes in the knockout lines and in a susceptible variety, IR64. Our results will contribute to unravel some of the complex mechanisms underlying the rice-pathogen interactions and also this study will help to better understand the plant immune response system which serves as valuable information for breeders.

This project of proof-of-concept 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 two (2) years.