Under herbivore attack, Plants release different blends of herbivore-induced plant volatiles (HIPVs). Therefore, when we identify a certain HIPV, we can roughly deduce which pest is attacking the plant.

Tea trees do not release benzaldehyde when they are not infested, whereas tea trees specifically release benzaldehyde when they are attacked by the Toxoptera aurantii. Therefore, by measuring the benzaldehyde concentration around the tea trees, we can decide if the tea trees are infested with the Toxoptera aurantii.

We plan to build the assay system on our engineering yeasts (S. cerevisiae, BY4741). G protein-coupled receptor (GPCR) HarmOR10 is one of the odorant receptors (ORs) isolated from Helicoverpa armigera, which is the specific receptor of benzaldehyde. In the project, we plan to introduce the encoding gene of HarmOR10 into engineering yeasts. Simultaneously, the original GPCR gene of the yeast α-factor receptor (Ste2) will be knocked out. When tea plants are infested by the Toxoptera aurantii and release benzaldehyde into the air, HarmOR10 will recognize the signal and evoke further intracellular GPCR pathways in the engineering yeast.

To verify whether the yeast can detect benzaldehyde and whether the GPCR pathway mentioned above is through, we introduce the reporter gene mCherry and the promoter fus1 into the yeast plasmid. HarmOR10 will couple to the yeast trimeric G protein, which consists of 3 subunits Gpa1(α), Ste4 (β), and Ste18 (γ). HarmOR10 activation causes βγ-dimer to dissociate from ɑ-subunit (shown in yellow) and induces mating-specific responses by activating the mitogen-activated protein kinase (MAPK) signaling cascade. Eventually, the translocation of the transcription factor Ste12 mediated by activation of the MAPK cascade further regulates the expression of numerous mating pathway target promotors including fus1.That’s to say, when we detect red fluorescence, we can confirm the successful coupling of HarmOR10 to the yeast GPCR pathway and the successful detection of benzaldehyde.

We use AJM3 as our chassis, which is a strain that can produce nepetalactol by fermentation with sugars. We introduce the MLPLA (major latex protein-like genes A) gene into it, which corresponds to an enzyme that increases the production of nepetalactol. During the fermentation process, the raw material is catalysed by the enzymes tHMGR, ERG20*, GPPS2, GES, G8H, GOR, ISY2 and MLPLA to produce nepetalactol. The galactose promoter is used during all the integration of the above genes and galactose medium is used for the induction of enzyme expression in the fermentation experiments to obtain more products.

NEPS1 is a short-chain dehydrogenase that catalyzes the conversion of nepetalactol to nepetalactone. We introduce NESP1 into AJM3 with MLPLA and it can change some of the nepetalactol in fermentation products into nepetalactone so that we can obtain the mixture of nepetalactone and nepetalactol.

There is a certain ratio of nepetalactone to nepetalactol in the sex pheromone of the Toxoptera aurantii. Because NEPS1 is quite efficient in catalyzing the formation of nepetalactone from nepetalactol, a weaker promoter is needed to regulate the ratio of nepetalactone to nepetalactol in the product to obtain a fermentation product with a ratio close to the certain ratio.

The detection system in the Teafender project aims to specifically identify Tea plant volatiles released by Tea aphids. To better achieve this goal, we plan to build a“And Gate” system to identify the gas to determine whether the tea plant was eaten by aphids. We plan to use CRISPRi-based systems to achieve“And gate” regulation.

Shown above is the rationale for the CRISPRa/i system, where the dCas9 protein is a mutant of the Cas9 protein, that is, both the RuvC1 and HNH nuclease active regions of the Cas9 endonuclease are mutated simultaneously. Thus, the endonuclease activity of the dCas9 protein is completely eliminated, leaving only the ability to be guided into the genome by the gRNA. The CRISPR-dCas9 system provides a platform for the study of site-specific transcriptional regulation. In this platform, dCas9 is mainly fused with other effector proteins (such as GFP, transcription factors, histone modifications, etc.) for gene regulation. Therefore, when dCas9 coexists with sgRNA, it can regulate the target gene. Based on this property, we constructed an“And gate” system. The diagram below shows the genetic circuitry.

As shown, we plan to modify two GPCR pathways in the yeast itself to sense two substances released by tea plants after being attacked by tea aphids, of which benzaldehyde is highly specific. Using the promoter of two genes downstream of GPCR as the input signal of“And gate”, sgRNA was transcribed and DCAS9 protein and MXI1 repressed transcription factor were expressed when two signals were present at the same time, both form a polymer that leads specifically to and silences the LACI gene via sgRNA, at which point the CMV promoter, which is originally bound by the Laci repressor protein, reactivates and ultimately outputs the target gene.

Compared with the traditional CRISP-Cas9 system, this method does not need to damage the original DNA and keeps the stability. Another reason not to regulate the target gene directly through the CRISPRA system is that the strictness of the ratio of the average output level in the on-and-off states can not be achieved, since it is essentially an activating system that plays a regulatory role. In addition, direct activation may lead to the release of the target gene, that is, the output of the input conditions do not meet the situation. After adding Laci repressor protein, the target gene can be guaranteed to not leak in the no-input state.

Considering the inhibitory effect, we plan to design multiple dCas9 binding sites in the TDH3p promoter controlling repressor genes to enhance the inhibitory effect.

Hydrophobins are secreted proteins with low molecular weights and they are about 100 amino acids long with low sequence homologies. Hydrophobins are special proteins produced by some filamentous fungi. They have both hydrophilic and hydrophobic parts, which means they are amphiphiles. Hydrophobins are able to lower the surface tension of water, and with the characteristics of amphiphiles, they can migrate to hydrophobic–hydrophilic interfaces (such as the air–water interface) . Moreover, hydrophobins can attach to some surfaces, in order to enhance the attaching ability of the engineering fungus.

Nakari and her team studied the biochemical characterization of the Trichoderma Reesei hydrophobin HFBⅠin 1996 and gave its genetic sequence and according amino acid sequence. HFBⅠ protein successfully expressed in their E.coli they discussed that hydrophobin mediates attachment of the fungus to some surfaces.

In 2002, Nalari and her team used yeast as their engineering fungi which successfully expressed hydrophobin HFBⅠ linked to Flo1 protein. (using 5’TCT AGC TCT AGA AGC AAC GGC AAC GGC AAT GTT 3’ as a 5’ primer, and 5’ TGC TAG TCG ACC TGC TAG CAG CAC CGA CGG CGG TCT G 3’ as a 3’ primer)

We hope our engineering yeast can suspend on the surface of the liquid/gel medium in order to obtain maximum efficiency of product release. Based on the characterizations of hydrophobin described above, we plan to have our engineering yeast express a certain hydrophobin and assemble it on the outer surface of the yeast for the purpose of immobilising the engineering yeast on the surface of the medium.

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