Safety

Background

According to the grading of ATCC(American Type Culture Collection), T. atroviride is a grade 1 organism, which means that it is safe to use in almost all conditions. Besides, there is no documented report of T.atroviride infection on human species. However, since we made genetic transformation on T.atroviride, there still remains some uncertain safety risks.

As most filamentous fungi does, T.atroviride produce conidia, which enables the engineered Trichoderma to spread and get out of control. The conidia producing ability may cause an escape of exogenous genes.Thus, we made effort to interfere the production of conidia, trying to limit such influence to a minimum level.

Interestingly, during the culture of T.atroviride, we found an exciting phenomenon. As Figure 1 shows, when the T.atroviride grew in the dark condition, the colony is white. After being transferred to the incubator with light on, some green dots appeared around the primitive white colony. Faced with this puzzling fact, we turned to Prof.Zhang for explanation. He told us that the green dots were developed Trichoderma conidia. That is to say, with the light stimulating the T.atroviride, the conidia developed! In other words, if we inhibit the response of T.atroviride towards light, the conidia may fail to come out.

Fig.1 The development of conidia in the dark and light

Inspired by this surprising phenomenon, we searched for some information about the mechanism of light-response in T.atroviride. Luckily, we found a key gene named hda-2 in the paper, which plays a significant role in the regulatory network. According to the paper, Δhda-2 mutant strain showed slow growth and the absence of conidia when exposed to blue light, which is the main light stimuli causing the development of conidia. Therefore, we chose the hda-2 as our target to knock out.

Design

In this part, we used a site-specific recombination to delete the gene hda-2, which is indispensable for the development of conidia of T.atroviride. To delete this gene, a deletion element was constructed to assemble into a vector used for transformation into Agrobacterium. As is shown in the Figure 2, this element consists of fragments which are ~1.5 kb long corresponding to each 5′- and 3′-flanking regions for the hda-2 open reading frame, with the hygromycin-resistant gene(HygR) inserted between them. When the element is absorbed by T.atroviride, the flanking sequence in the element complements to the sequence near hda-2 gene, which is replaced by the HygR.

Fig.2 Pko1 with deletion construct

Future work

Due to the lack of time, we did not fulfill the construction of the plasmid used for transformation into Agrobacterium. Previously, we successfully ligated the flanking fragments and the hygromycin-resistant gene by overlap PCR. Furthermore, we confirmed the identity of the whole construct. Therefore, the remaining work is to insert the construct into the vector pko1 and transformation of this plasmid. And we will try to optimize the condition of experiment such as enzyme digestion to make sure our system works.

References

[1] Osorio-Concepción M, Cristóbal-Mondragón G R, Gutiérrez-Medina B, et al. Histone Deacetylase HDA-2 Regulates Trichoderma atroviride Growth, Conidiation, Blue Light Perception, and Oxidative Stress Responses[J]. Applied and environmental microbiology, 2017, 83(3): e02922-16.

[2] Casas-Flores S, Herrera-Estrella A. 2013. The influence of light on the biology of Trichoderma, p 43–66. In Mukherjee PK, Horwitz BA, Singh US, Mukherjee M, Schmoll M. (ed), Trichoderma: biology and applications. CABI, Wallingford, United Kingdom.