Dr. Bo Zhou

Lr34 controls broad-spectrum resistance to multiple fungal pathogens in wheat and has been extensively utilized in wheat breeding program for its excellent durable resistance (Krattinger et al., 2009, ref. 1). Lr34 has been found to be functionally transferable into barley, where it conferred resistance against barley-specific diseases. in this study. we aim to test whether Lr34 can also be functional in rice. However, leaf tip necrosis (LTN) manifests in transgenic lines with strong expression of Lr34 probably due to insertion site effect. In this study, we aim to:

1) generate transgenic plants in IR64 genetic background;

2) validate the enhanced resistance to blast in the resultant transgenic lines as observed previously in Nipponbare transgenic plants;

3) validate the enhanced resistance to brown spot and false smut fungal diseases;

4) screen transgenic lines with enhanced resistance albeit no or low level of fitness cost, such as LTN and reduced yield. The generated transgenic plants will be used not only for destructive sampling but also for further studies.

The generation of transformation events will be conducted in the Genetic Transformation Laboratory (GTL) at IRRI. All transgenic plants will be grown exclusively in CL4 and the derived seeds will be kept in CL4 storage room. Both GTL and CL4 are previously approved transgenic facilities by the DOST-BC. Movement of all seeds and plant materials will be done in compliance with all relevant biosafety and phytosanitary requirements of the Philippines. Plants, soil and pots will be sterilized properly before the disposal. All other biosafety regulations and actions will be implemented and complied with.

a) All activities shall be conducted at the Genetic Transformation Laboratory and CL4 Transgenic Greenhouse of the International Rice Research Institute (lRRl) in Los Baños, 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 lRRl-IBC and the DOST-BC.

d) A biosafety contingency plan shall be submitted before the conduct of the experiment.

e) The proponent shall inform which activities would require the presence of the IRRI-IBC, DOST-BC and PEQS-BPI personnel.

f) The proponent shall ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities.

g) The DOST-BC and PEQS-BPI personnel should be informed in advance of any visitations by unauthorized persons.

h) The DOST-BC and the PEQS-BPI personnel shall be informed immediately of any intrusion by unauthorized persons.

i) The proponent shall ensure that stray animals are excluded from the experimental facilities while tests are being conducted.

j) 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.

k) 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.

l) All viable materials within the experiment area shall be accounted for.

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

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

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

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

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

Broad-spectrum resistance to multiple pathogens is an important component of durable resistance to pathogens that rapidly evolve new virulent races. In wheat, Lr34 confers race non-specific and partial adult plant resistance against four fungal diseases, i.e., leaf rust (Puccinia triticina), stripe rust (Puccinia striiformis f.sp. tritici) , stem rust (Puccinia graminis f.sp. tritici) and powdery mildew (Blumeria graminis f.sp. tritici). This gene has been extensively used in wheat breeding and agriculture worldwide for more than a century. Despite this extensive use of Lr34 in wheat production, no breakdown of disease resistance has been observed (Krattinger et al., 2009, ref. 1).

It is interesting that transgenic rice lines in the Nippone bare background receiving Lr34 is also confer enhanced resistance to blast, one of the devastating diseases in rice (Kattinger et al., unpublished). We want to validate this finding in the more popular rice variety IR-64. The objectives of this project are to: 1) generate transgenic plants in IR64 genetic background; 2) validate the enhanced resistance to multiple fungal diseases; and, 3) screen transgenic lines with enhanced resistance albeit no or low level of fitness cost, such as Leaf Tip Necrosis (LTN) and reduced yield.

In this study, we will generate 100 transgenic events of the wheat Lr34 gene in IR64 background using Agrobacterium-mediated transformation approach; identify a number of transgenic lines with only a single insertion site in the recipient genome by molecular characterization; screen the resistance of the shortlisted transgenic lines to multiple fungal pathogens including rice blast, brown spot and false smut; and, screen the resistant lines with least penalty for other agronomic traits, e.g. yield.

All the experiments will be strictly conducted in confined facilities including the Genetic Transformation Laboratory (GTL) and CL4 at IRRI, both of which are approved transgenic facilities.

All biosafety regulations will be properly implemented and complied with during the period of project.

This project is to be finished within 20 months (proposed from October 2014 to May 2016).

The generated transgenic plants will be used not only for destructive sampling but also for further studies of similar nature.