Part:BBa_K5169005
A symbiotic gene from Rhizophagus irregularis that expresses growth beneficial protein for plants
Facilitating the symbiosis between previously pathogenic fungi and plants is an innovative approach, with relatively few studies conducted in this area before. The RiCLE gene, derived from arbuscular mycorrhizal fungi Rhizophagus irregularis, is a symbiotic gene that encodes CLE peptides, which are beneficial proteins for most plants. By providing advantages to the host and utilizing the hyphae of the original pathogenic fungus, we can enhance the competitive edge of this fungus and ensure its ecological niche within the plant. Here, we propose a strategy for promoting microbial symbiosis for future iGEM participants.
Usage and Biology
Promoting symbiosis between previously pathogenic fungi and plants represents an innovative frontier in biotechnology, with relatively few studies exploring this field. Our approach involves the introduction of the CLE gene, which is derived from arbuscular mycorrhizal fungi. The CLE gene encodes CLE peptides, a group of beneficial proteins known to support plant growth and health by facilitating symbiotic relationships between fungi and their plant hosts. By harnessing the hyphae and competitive advantages of a previously pathogenic fungus, such as Fusarium oxysporum, we can provide this fungus with a competitive ecological niche within the plant, transforming it to a beneficial partner. This strategy not only enhances the fungus's role in symbiosis but also contributes to a more sustainable agricultural system. Rhizophagus irregularis is a type of arbuscular mycorrhizal fungi that is found to symbiosis with bananas. Thus, we choose to use RiCLE as the target symbiosis gene to be introduced into Fusarium oxysporum f. sp. cubense tropical race 4(FocTR4)[1][2].
Since the CLE gene originates from Rhizophagus irregularis , a non-model organism which does not naturally express well in heterologous systems, including Fusarium oxysporum. To overcome the challenge of coden preference leading to low protein expression, we employed codon optimization to the RiCLE gene by changing the original sequence into Fusarium fungi optimal sequence， ensuring the gene can be accurately and efficiently expressed in the new host. Codon Optimization was done with the following parameters: 15% cut-off was used for codon efficiency; any codon below 15% was removed except for positions with strong secondary structures (in this case, codons of lower frequency were used to alleviate the problem). By opimizing the codon usage, we provide a pathway to facilitate the expression of beneficial genes in a wide range of organisms, thus broadening the scope of genetic manipulation in fungi and other microorganisms.
We inserted the CLE gene into the aforementioned plasmid to achieve strong expression via the use of trpC promoter system. Then, we amplified the whole linear DNA part from trpC promotor to terminator using PCR, followed by fusion it with the toxin gene FUB1 upstream and downstream. After transforming the protoplasts, we conducted the following PCR verification to confirm the success of the transformation, in which the dark stripe indicated successful transformation.
And to ensure expression on the protein's level, SDS-Page and Comassie blue stain were used to examine the existence of RiCLE peptide within the engineered strains. Compared with other strains, our engineered strain showed a deep band at about 7kDa against the control strain, showing the evidence of the CLE peptide's existence and hence successful insertation.
After obtaining the strain, we tested the growth and toxicity effect of engineered FocTR4 in bananas. Compared to wild type FocTR4, our new strain obviously declined toxicity and the banana growth showed no inhibition compared to no infection, indicating the RiCLE can promote banana growth and providing a symbiosis environment.
Besides low toxicity, we further studied the prevention effects of our genetic engineered ∆FUB1+RiCLE strain compared to a control strain Fusarium oxysporum Cucumerinium that proved not to infect bananas. After incubation with spore suspension of ∆FUB1+RiCLE strain and Cucumerinium strain for 10 days, we reinfected the bananas with wilt type FocTR4 to check whether our strain can serve as an effective vaccine of FocTR4. Our results proved that ∆FUB1+RiCLE strain infected bananas gained immunity towards FocTR4 and can strongly reduce wilt disease symptom.
This strategy offers a novel framework for engineering beneficial relationships between plants and microbes, presenting significant opportunities for future research and biotechnological applications, especially in the context of synthetic biology and sustainable agriculture. Through this approach, iGEM participants and other researchers can contribute to advancing plant-microbe symbiosis, offering potential solutions for agricultural challenges such as disease resistance, nutrient deficiency, and environmental stress.
Reference：
1. Le Marquer M, Bécard G, Frei Dit Frey N. Arbuscular mycorrhizal fungi possess a CLAVATA3/embryo surrounding region-related gene that positively regulates symbiosis. New Phytol. 2019 Apr;222(2):1030-1042. doi: 10.1111/nph.15643. Epub 2019 Jan 12. PMID: 30554405.
2. LU Jingshi, ZHAO Yuanzheng, WANG Dong, LI Xiaojie, PENG Jingwen, ZHANG Longmei, ZHANG Xiaoming. Analysis of genome-wide codon preference and characterization of Fusarium oxysporum[J]. Journal of Northern Agriculture, 2022, 50(4): 11-17.
3. Shang, S., Wang, P., & Chen, C.-Y. (n.d.). Signal recognition and transduction in the arbuscular mycorrhizal symbiosis. Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences; College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, China.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 94
| None |