Team:BNU-CHINA/Project

Team:BNU-CHINA - 2015.igem.org

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Background

Nematode is a kind of lower invertebrates distributed widely which can survive anywhere on the earth. According to Hyman (1950) and Poinar (1983)’s estimation, there are over half a million kind of nematodes all over the world. Most soil nematodes are beneficial insects whereas some of them are important plant causative agents so-called plant-parasitic nematodes. Plant-parasitic nematodes have been up to over 5000 species of 200 genus[1]. Among them, over 100 species of nematodes are damaging the agriculture, forestal and economic crops of China who widely parasitize the roots, stems, leaves, flowers, buds and seeds of manifold plants, harming the development of agriculture and forestry seriously.

According to incomplete statistics, the losses caused by pathogenic nematodes can reach \($\)100 billion every year worldwide[2]. Meanwhile a newest research by USA indicates that the plant-parasitic nematodes’ damage leads to about \($\)8 billion dollars’ loss to croppers, which takes 12% of the whole value of the crop output. Chinese researchers have done some relevant surveys as well. According to incomplete statistics, 17 provinces such as Anhui, Hainan, Hubei, Gansu, Zhejiang and Fujian have reported root knot nematode (Meloidogyne spp.) disease once, among which the morbidity of some severe regions in Shandong province can up to 2/3[3]. Pine wood nematode (Bursaphelenchus xylophilus) dose severe harm to forestry and have caused billions yuan losses directly and indirectly. As Fig.1 shown below, 3rd announcement of China’s State Forestry Administration (SAF) in 2015 shows the pine wood nematode disease distribution in China in 2014. Therefore we can note that plant-parasitic nematodes have brought out severe loss to global agriculture and forestry already.

Loss the Fig
Fig. 1 Pine wood nematode disease distribution in China (2014)

Plant-parasitic nematodes are usually too tiny to catch sight of with naked eyes. In addition, the present surveys about the agricultural damage of nematodes are so limited that people often fail to notice and prevent nematodes in time. The plant-parasitic nematodes are furnished with scalpellus functioning to stab into the plant cells to obtain nutrients after seeking out the host with the amphids on their forehead. As a result, nematodes could damage the host as well as bring in pathogenic fungus and therefore causing complex harm to them. Up to now, the pathogenesis of plant-parasitic nematodes can be concluded in theory as follow:

  1. The nematodes give raise to severe mechanical injury to the host plants when feeding on them with the scalpellus.
  2. Other pathogens accompanied with the nematodes induce plant disease.
  3. Some nematode secretion is toxic to the plants which as a consequence damages them.

In most cases, the mechanical damage by nematodes to host is a drop in the bucket, therefore the latter two theories are relatively common[4].

The majority of harmful nematodes in agriculture and forestry belong to Tylenchida while a little belongs to Dorylaimida. Meloisogyne spp., Heterodera spp., Aphelenchoides composticola, Ditylenchus dipsaci are nematodes that gravely damage plants around the world. Among them, Meloisogyne spp. mainly destroy the roots of plants by forming root-knot to hinder their development, which in turns, causes the roots rot.(Fig. 2a) According to statistics, a slight Meloisogyne spp. disease can lead to a 20%~30% output reduction, and 50%~70% even a total loss if it’s severe. Heterodera spp. also target the roots of plants primarily, weakening and decomposing the roots.(Fig. 2b) Ditylenchus dipsaci damage the underground part such as the tuber, tuberous root, bulb of the plants which results in their rot and malformation while some local part over ground would also be influenced to turn to malformation,(Fig. 2c) Aphelenchoides composticola such as Bursaphelenchus xylophilus and Aphelenchoides besseyi generally aim at the overground part of plants who infect the leaves and causing lesion and leaf tip drying, and more than that, infect the trunks to kill the whole plant rapidly.(Fig. 2d)

Fig. 2 The damage to the plants of different plant parasitic nematodes
a.Root-kont (Meloisogyne spp.) galls on vegetable roots
b.Segment of soybean root infected with soybean cyst nematode (Heterodera spp.).
c.Ditylenchus dipsaci damage of sweet potatoes
d.Austrian pine with pine wilt (Bursaphelenchus xylophilus)

In a word, plant-parasitic nematodes cause serious losses to agricultural crops worldwide. The traditional methods based on the use of nematocides and antihelminthic drugs are associated with major environmental and health concerns, so the development of biocontrol agents to control nematodes is of major importance [5]. In this case, our project is designed to control the losses caused by nematodes effectively with biocontrol agents; and after the bait-kill system successfully established, we are looking forward to further expansion to other agricultural pests for the sake of achieving our ultimate goal – establishing a database containing attractant base and toxic protein base in allusion to all sorts of agricultural pests.

Our BioDesign

In our project, we designed three modules separately to attract and poison nematodes and then control the biocontrol agents. Module 1: We made E. coli synthesize limonene to lure plant-parasitic nematodes. Module 2: E. coli would kill them with two kinds of toxic proteins Bace 16 and MpL. Module 3: we introduced a photo-regulated bi-direction transcription system to the whole system, by which means we would be able to regulate the expression of attractant and toxic proteins through the control of light. That’s to say, the E. coli would express attractant to lure nematodes when exposed to light whereas express toxic proteins to kill them as a result of the promoter’s reverse in the dark.

Module 1：Bait more ›

Limonene（Fig. 3） is a monoterpenoid with two conformations in the nature, d-configuration and l-configuration. Limonene can attract root knot nematodes of citrus (Tylenchulus semipenetrans) or other plant parasitic nematodes. Our design based on the following two limonene synthase sequences which are named Citrus unshiu CitMTSE1 (GenBank number: AB110636.1) for d-limonene synthase[6] and Mentha spicata 4S-limonene synthase (GenBank number: L13459) for l-limonene[7]. At the same time, we also designed another synthase sequence which can synthesize the isoprene GPP, the precusor substance of synthesizing the limonene. And the sequence (GenBank number: AF513112) is from Abies grandis[8]. We transformed these parts into E. coli to realize the expression of limonene in prokaryotic cell.

Fig. 3 Ball-and-stick model of the d-isomer

Module 2：Killer more ›

Bace16 and MpL(Fig. 4) both have a good toxicity toward the nematodes. Bace16 is a kind of serine protease from nematode-parasitic bacteria named Bacillus nematocida. After the expression of Bace16 has completed, the protein can be secreted out of the cell. This proteases could degrade the intestine protein of the nematodes thereby the nematodes will be killed[9]. MpL is a novel lectin isolated from a kind of parasol mushroom (Macrolepiota procera). It is a kind of intracellular expression protein. MpL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults[10].

Fig. 4 Three-dimensional structures of rMpL in complex with carbohydrates

Module3：Suicide more ›

Our engineering E. coli could not only carry out a series of functions by the bait and killer system, but also suicide after a certain time. The suicide system is controlled by quorum sensing, bacteria can accumulate an organic named AHL. AHL can move across the cytomembrane freely; when it accumulates to a certain degree, it will combine with the intracellular proteins to induce the activation of the promoter of the suicide gene mazF. Later the suicide system starts and the E. coli will be killed.

Photoinduced bidirectional transcriptional system more ›

In order to attract and kill the nematodes, we also built a light regulated bidirectional transcription system. In this system, the expression of the attractant organics and the toxin are under different conditions. We have already known the promoter PompR is regulated by red light; when there is no light, the promoter combines with the ompR protein to function. gp35 is a serine integrase, this intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, and results in the reverse of the sequence（there it means the promoter Pcon） between attB and attP, changing the two sites to attL and attR at the meantime. As a consequence, the promotor moves on to express the toxic protein instead of the attractant.

Modelingmore ›

Our modeling consists of the following three parts:

  1. To make the project applicable in real life, we designed a device with engineering bacteria inside, which can be placed in soil to attract and kill nematodes.
  2. Assuming there is a farmland, we took advantage of gas diffusion and nematodes' movement analogue simulation to find the best position for the device to be placed.
  3. We established a database to broaden the scope of our applications, combined with our methods, to kill more different pests. We hope this new environment-friendly method, based on principles of synthetic biology, could be shared with and improved by the researchers all over the world.
Fig. 5 Design of Device 2.0

Human Practice more ›

During our project, we constantly did the communication and practice. We improved our plan by doing human practice. At the same time, we also helped people understand the meaning of synthetic biology and taste the charm of synthetic biology. Moreover, according to our survey, the farmer did not realize the damage of nematodes and we provided some suggestions to them. We interviewed related experts, dropped in the countryside, analyzed the soil sample, communicated with other teams, did assistance and so on. The goal we would like to achieve is to help more people with the development of ourselves.

  1. Chen Lijie, Duan Yuxi. Classification and Identification of Plant Nematodes [C]. Chin J Parasitol Parasit Dis Dec. 2006, Vol. 24, Suppl.
  2. Chitwood, David J. Research on plant‐parasitic nematode biology conducted by the United States Department of Agriculture–Agricultural Research Service[J]. Pest Management Science, 2003, 59(6-7):748-753.
  3. 付令国. 植物病原线虫的发生与防治技术初探[J]. 农业与技术 2015, Vol.35, No.09
  4. 董娜, 张路平, 康云晖. 植物线虫寄生策略及致病机理[J]. 河北师范大学学报：自然科学版, 2003, 27:298-301. DOI:doi:10.3969/j.issn.1000-5854.2003.03.024.
  5. Duncon LW (1991) Current options for nematode management. Annu Rev Phytopathol 29:469–490CrossRef
  6. Ali J G, Alborn H T, Stelinski L L. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes[J]. Journal of Ecology, 2011, 99(1):26–35
  7. Rodríguez A, San A V, Cervera M, et al. The monoterpene limonene in orange peels attracts pests and microorganisms.[J]. Plant Signal Behav, 2011, 6(11):1820-1823.
  8. Du F L, Yu H L, Xu J H, et al. Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli[J]. Bioresources & Bioprocessing, 2014, 1.
  9. Niu Qiuhong , Huang Xiaowei , Tian Baoyu , et al. Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor[J]. Applied Microbiology & Biotechnology, 2006, 69(6):722-730.
  10. Žurga S, Pohleven J, Renko M, et al. A novel β‐trefoil lectin from the parasol mushroom (Macrolepiota procera) is nematotoxic[J]. FEBS Journal, 2014, 281(15): 3489-3506.