Overview

Our project to treat rice sheath blight includes three stages: prevention, detection and treatment. In the prevention stage, we use engineered Trichoderma atroviride as bio-control fungus, and the engineering modification of Trichoderma atroviride involved the expression of three encoded proteins and a condition-triggered suicide switch; in the detection stage, we designed specific LAMP primers for Rhizoctonia solani AG1-IA for accurate diagnosis; in the treatment stage, we used engineered E. coli to produce shRNA molecules against Rhizoctonia solani, and transferred R-body to promote the self-cracking of engineered bacteria and the release of shRNA. In addition, we came up with tentative idea of timed suicide switch.

All parts involved in our rice sheath blight control system were integrated to form a complete parts-collection. We have classified the parts according to the different functions involved in each stage. The following are the part used by 2022 SZU-China in RiceAide.

Prevention

I. Proteins encoded by engineered Trichoderma atroviride

For better prevention of rice sheath blight, we utilized the following parts to modify our engineered Trichoderma atroviride.

• 1.BBa_K4286202 encodes Prb1, which is a type of serine protease playing great importance in mycoparasitism through hydrolyzing the cell wall of R.solani.
• 2.BBa_K4286204 encodes hydrophobic protein Epl 1, which promotes T.atroviride to float at the aquatic interface of rice so as to prevent R.solani from infesting the rice.
• 3.BBa_K4286205 encodes Snakin 1, a potato-derived cysteine-rich antimicrobial peptide inhibiting the growth of a variety of plant pathogens, especially R.solani.
• 4.BBa_K4286203, BBa_K4286206, BBa_K4286207 are composite parts that are based on CaMV 35S promoter and NOS terminator. CaMV 35s is a constitutional promoter that makes high expression in plants. 6x His affinity tag at the end of each CDS is used for detection and purification of the protein.

II. Condition-triggered suicide switch

Considering safety, we designed a condition-triggered suicide switch, which prevents engineered T.atroviride from escaping and bringing negative effects to the field ecological environment.

• 1.The circuit of our condition-triggered suicide switch (BBa_K4286907) is based on cbh1 biobricks and MazEF system.
• 2.cbh1 promoter (BBa_K4286408) is one of the strongest inducible promoters in the fungal kingdom.
• 3.MazEF (BBa_K2292002) is a common toxin-antitoxin system.

Detection

III. ITS sequence and LAMP primers of Rhizoctonia solani

With the following primers (BBa_K4286001, BBa_K4286002, BBa_K4286003, BBa_K4286004, BBa_K4286005), LAMP reaction rapidly amplifies ITS sequence of R.solani (BBa_K4286404, BBa_K4286406). The successfully amplified product can be detected by LAMP-LFD detection strip, indicating that the corresponding sample contains the pathogen R. solani, thus achieving a rapid, convenient and sensitive detection of rice sheath blight.

Treatment

IV. RNAi molecules

After theoretical research and experimental verification, we found that the most stable form of RNAi molecules is shRNA. Therefore, we submitted the 10 shRNA molecules targeting 7 mRNAs of R.solani as our parts crucial to the treatment of rice sheath blight.

• 1.In order to prevent the infection process, we have identified PG1, RPMK1-1 and RPMK1-2, which are critical to the infectivity of Rhizoctonia solani, as our first batch of RNAi targets. The corresponding shRNA parts are BBa_K4286107, BBa_K4286125, BBa_K4286130, BBa_K4286230, BBa_K4286223, BBa_K4286322.
• 2.In addition, new batch of shRNAs is required for killing R.solani. Thus, we have identified RNAPolIII subunit C6, cohesin complex subunit Psm1, Ubiquitin ligase E3,and Importin β1, which are critical to the survival of Rhizoctonia solani, as our second batch of RNAi targets. The corresponding shRNA parts are BBa_K4286115, BBa_K4286505, BBa_K4286815, BBa_K4286909.
• 3.E.coli HT115(DE3) is our bacterial factory producing shRNAs. T7 promoter (BBa_R0187) and T7 terminator ( BBa_M50060) together with each pre-shRNA sequence constitute the gene circuit for shRNA production.

V. Improved R-body gene cluster

In the first step of production, we induced E. coli HT115(DE3) to produce RNAi molecules by IPTG as the above states; in the second step, we used arabinose to induce E. coli to produce R-body, then we acidify the bacterial medium below pH6.5 to crack E. coli, leading to the release of shRNAs.

R-body is a part first submitted by 2019 SZU-China. This year, based on previous parts of R-body, we modified the R-body gene cluster and obtained the improved version, in which the genes are arranged in the order of RebB-RebA-RebC.

See more about the modified R-body gene cluster in Improvement.

Outlook

VI. A tentative timed suicide switch

2022 SZU-China attaches great importance to the safety of engineered microorganisms and hopes to design a suicide switch activated by endogenous factors. Based on the classical gene oscillator, we propose a concept of timed suicide switch, aiming to kill the engineered microorganism population at a predictable time.

We have designed the timed suicide switch, exploring the properties through a mathematical model. See more about oscillator1.0 in BBa_K4286099 and effector1.0 in BBa_K4286100.

Some improvements have been made to make sure the timed suicide switch more regular and reliable. See more about oscillator2.0 in BBa_K4286101 and effector2.0 in BBa_K4286103.