Team:Korea U Seoul/Project/Description

     Bacterial leaf blight disease (BLB) is one of the prominent vascular diseases in irrigated rice. This disease can cause up to 50% yield reduction . Bacterial leaf blight in rice is caused by infection of bacteria known as X. oryzae pv. oryzae (Korean specie: Xanthomonas oryzae KACC10331), a yellow, slime-producing, motile, gram negative rod with a polar flagellum . It reaches the vascular tissue, particularly the xylem, from where it multiplies and spreads throughout the plant. An infected leaf has yellow watersoaked lesions at the margin of its leaf blade. The lesions run parallel along the leaf and when they join together may cover the whole leaf. The infection also accompanies notable symptoms such as seeding wilt, yellow leaf, and panicles sterile.

Figure.1: bacterial leaf blight rice infected by Xanthomonas oryzae KACC10331

     Bacterial leaf blight severely damages agriculture and nation’s economy. BLB is a major concern in countries where the staple food is rice. Bacterial blight is reported to have reduced Asia's annual rice production by as much as 60%. For example, in Japan, about 300,000 to 400,000 hectares of rice were affected by the disease in recent years. There were 20% to 50% yield losses reported in severely infected fields. In Indonesia, losses were higher than those reported in Japan. In India, millions of hectares were severely infected, causing yield losses from 6% to 60%.

     Because of its devastating impact on rice production, researchers in Korea sequenced full genomic map of some strains in X. oryzae pv. oryzae species, and one of them is Xanthomonas oryzae KACC10331. Still majority of protein functions and mechanisms are unknown, previous researches give us insight on how X. oryzae pv. oryzae causes BLB. Based on previous researches, it was proven that bacterial rax genes (rax A, B, C, P, Q, R, H) and its protein products are responsible for BLB. In fact, RaxP and Q proteins modify proteins called Ax21, a short oligopeptide with a sulfate group, inducing disease and provoking immune response. Then Ax21 is secreted by RaxA,B and C complex. It is known that Ax21 also acts not only as a ligand for plant receptor Xa21, but also as a quorum sensing molecule. Ax21 is then detected by RaxR and H proteins informing any changes in environment. Yet, the mechanism how Ax21 induces BLB in rice remains unclear.

Figure 2. Genes responsible for Ax21 production, a main source of pathogen inducing BLB.

     Unfortunately, there is no way to stop BLB. Only several precautionary measures were given to farmers. Several attempts to tackle spreading of disease were ineffective; for example, genetically modified rice was not effective . It is evident that solution for reducing BLB is in killing pathogen, not in rice. Thus we decided to create bacteria that are able to detect and kill X. oryzae KACC10331(Korean specie). Since Ax21 is major pathogen that causes BLB and ever present molecule that signifies presence of X. oryzae KACC10331, we decided to make synthetic bacteria that detects Ax21 and furthermore kill them. We will use rax R and H gene promoters to detect Ax21. As a result of transcription activation, gene will synthesize bacteriocin to kill the bacteria. We hope our project can minimize the damage caused by the bacteria.

Figure 3. Plasmid construction of Rice Guardian

          Logic circuit is an operation system consists of sets of various logic gates such as AND, OR, XOR, NOR etc.. that interact with each other to draw certain outputs from input signals. Attempts to make biological logic gates have been tried by a lot of researchers due to their lots of applications and some meaningful results have been introduced. Our project adopted some methods to build biological XOR, AND, and OR gates, and by arranging the gates in proper order with certain supplementary system our team has designed, we are expecting a working full adder to complete biologically operating binary adder.

          In our project, a kind of major quorum sensing molecules, AHLs, were used as signaling molecules mediating delivery of information or data. For different input signals 3OC6 - 3-oxo-C6 HSL, 3OC12 - 3-oxo-C12 HSL and products of hrpR and S genes were used. Also, as a common mediator in signal transduction between logic gates, C4 AHL was used. For the project, our team used 9 types of bricks and semi-permeable membranes to complete the circuit. We made a model for the project adopting Hall equation and setting some key variables. Then we proceeded simulation for the model. Due to conditions in our laboratory and time limit, we were unable to do actual wet-lab experiments that show how well our model predicted the results and give some key variables which could provide us more sophisticated information to elaborate our model.