Downstream

Introduction

When our T.atroviride is activated by the signals which have been described above, they will produce the corresponding effects to save our little plants. Those can be zwittermycin A, chitinase, serine protease and anything you need to protect your lovely plants. Zwittermycin A is an antibiotic which can inhibite the growth of P. nicotianae. Chitinase is a lytic enzyme that breaks down fungal cell walls. Serine protases plays an important role in hydrolyzing the eggshell of root-knot nematodes. With these fungal growth inhibitors our engineered T.atroviride will be able to better protect our plants.

Zwittermycin A

Zwittermicin A is an antibiotic that has the potential to suppress plant disease due to its broad spectrum activity against certain gram positive and gram negative prokaryotic micro-organisms. Since T.atroviride does not produce Zwittermycin A by itself, a gene cluster obtained from Bacillus cereus UW85 was introduced into T.atroviride. The genes responsible for the production of zwittermicin A are located on a 16 kb cluster containing nine orfs, from orf1 to orf9, and a self resistant gene zmaR, a gene that encodes an acylation enzyme that deactivate zwittermicin A.[1]

Serine protease

Root-knot nematodes (Meloidogyne spp.), which are one of the most destructive nematodes, cause the loss of crop about 10%, serious as high as 75%. [2] In the present, the egg-parasitic fungus P.lilacinum is the main biocontrol material of root-knot nematodes.P.lilacinum. secretes protease and chitinase to hydrolyze the nematode eggshell, so that the root knot nematodes cannot grow normally.[3] However, because P.lilacinum can live in the cornea, the usage of P.lilacinum is still dangerous. Therefore, in this part, the purpose of our project is to give our harmless T.atroviride the ability to kill the root-knot nematodes by overexpressing serine protease which plays an important role in hydrolyzing the eggshell of root-knot nematodes.

Chitinase

Chitinase is a hydrolytic enzyme that breaks down hydrolytic bonds in chitin. As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals (including worms and arthropods), chitinase has been shown to be useful in biological control against fungi.[4] Therefore, in order to inhibit fungl growth, our T.atroviride can produce chitinase when the plants are infested by fungi and the signal conversion systems work well.

Anything else

Your lovely plants will face many challenges in the complex and dangerous soil condition, so that, the little plants must be protected by the strong T.atroviride which can product corresponding effects to inhibite the fungi. The following table can help you choose the right inhibitor to help your plants.

Inhibitor	Part	Function
dimethyldisulfide(DMDS) and dimethyltrisufide(DMTS)	BBa_K1493300	DMTS was shown to have an inhibitory effect on F. oxysporum. DMDS is used as plant growth promoter and at the same time also has been shown a slight inhibition to F. oxysporum.
Omega-hexatoxin-hv1a	BBa_K1974001	Hv1a can bind on insect voltage-gated Calcium channels (CaV1) in the central nervous system, making it paralyze and die eventually
μ-segestritoxin-Sf1a	BBa_K1974003	OAIP can bind on the voltage-gated sodium channel in the insect’s nervous system, making it paralyze and die eventually.
Orally Active Insecticidal Peptide (OAIP)	BBa_K1974003	OAIP can bind on the voltage-gated sodium channel in the insect's nervous system, making it paralyze and die eventually.
Art-175	BBa_K1659000	Art-175 is a fusion protein that kills Gram-negative bacteria, such as Pseudomonas aeruginosa by means of bypassing their outer membranes and catalysing the hydrolysis their cell walls.
Microcin S	BBa_K1659100	tdMicrocin S (MccS) is a narrow-spectrum antibacterial protein that has been shown to exhibit high-potency killing of select strains of E. coli and P. aeruginosa.
Plu1537	BBa_K1668007	Plu1537 is a 14kDa insecticidal toxic protein, which has strong toxicity against termites.

Result

Because of lacking of time, we only did the experience of serine protease. We structured two plasimads: one could work in yeast and the other could work in T.atroviride. Before constructing the plasmid expressed in yeast, we did codon optimization based on the codon preference of yeast and the gene was synthesized by Genscipt. In the contrast, the serine protease that producted in the T.atroviride was cloned from the genome of P.lilacinum because they have high homology.

Reference

[1]Stohl E A, Milner J L, Handelsman J. Zwittermicin A biosynthetic cluster[J]. Gene, 1999, 237(2):403-411.

[2]Wang J P, Wang J X, Liu F, et al. Enhancing the virulence of Paecilomyces lilacinus against Meloidogyne incognita eggs by overexpression of a serine protease. Biotechnology Letters, 32, 1159-1166[J]. Biotechnology Letters, 2010, 32(8):1159-1166.

[3]Brand D, Roussos S, Pandey A, et al. Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita.[J]. Applied Biochemistry & Biotechnology, 2004, 118(1-3):81-88.

[4]Sámi L, Pusztahelyi T, Emri T, et al. Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.[J]. Journal of General & Applied Microbiology, 2001, 47(4):201.

Team:ZJU-China/Project/Downstream