Team:NTHU Taiwan/Engineering

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For our project, we need to let our plasmid pass through E.coli, Agrobacterium, pine callus and put B. xylophilus on the callus to verify our sequence design. Therefore, we should make sure that each part is on the right track.

For B. xylophilus, because we need a large amount of the nematodes for the subsequent experiments, such as qPCR for target genes. We had to maintain their survival without a significant decline in number. So we made every effort to improve the growth environment of B. xylophilus.

The subculture of callus needs two weeks, and it is easy to be contaminated by bacteria and influenced by light. It’s better to guarantee both the medium and the process of taking an embryo are not interrupted.

Cloning is the most important part of engineering success. Because our design involves long hairpin RNA which needs to be expressed in pine callus. The choice of proper plasmid and sequence is easier to conduct Agrobacterium tumefaciens-mediated gene transfer and hence let the plasmid be able to express on the callus.

To make sure our design is able to kill the nematodes on pine and realize change for nematodes after digesting pine callus with our plasmid, the design for reasonable modeling is also necessary for our project.

To fulfill a scientific design is always not easy; however, we should never give up thinking of the idea to make our project better and complete. The following is the process we improve and reach our goal for each part of our experimental design.

Cloning success

Our experiments were based on previous papers. At the beginning, we wanted to make shRNA expressed in E.coli. Since these essays didn’t provide dsRNA sequence, we found some websites which helped us design shRNA. However, after going through Kyoto iGEM 2017 website, we realized that pine wilt nematodes don’t uptake E.coli. Therefore, we turned our focus to trees.

We were looking forward to creating transformed pine trees. In our concept, if pine trees could express dsRNA and RNAi mechanisms, this severe issue could be eradicated. Then, we found a handbook which explained the way to conduct hairpin structure cloning on plants. Besides, that book also gave us a novel idea --- long hairpin structure. As a result, we performed cloning experiments according to the handbook. We bought pHANNIBAL plasmids and designed sense and antisense strands. Moreover, we referred to paper to design long hairpin structures --- with whole or 350bp(short version) of exon. Unfortunately, with some unknown contamination, long hairpin construction failed. The sense strand was successfully cloned into pHANNIBAL, but it degraded a few days after the experiment. Thus, we altered our design, using binary vector and shRNA to achieve our goal (Fig. 1-1).

Fig. 1-1
Transformation success. We used pBI121 digestion product as negative control. Different amounts of ligation products were added to ensure that transformation was successful.

We used a website to build optimal shRNA. The experiment started from two primers being annealed. Then, they were inserted into a binary vector, pBI121. With the CaMV 35S promoter, we anticipated that shRNA could be expressed in pine trees (Fig. 1-2).

Fig. 1-2
Transformation success. We used pBI121 digestion product as negative control. Different amounts of ligation products were added to ensure that transformation was successful.

To certify that cloning was complete, we conducted a digestion check rather than a PCR check. Since hairpin structure may cause self-annealing which meant primers couldn’t bind to target genes. Additionally, our target genes were too small to be seen on agar gel while the original plasmid contained around 1800 bp within restriction sites. Therefore, we could know whether our target genes were cloned into the plasmid by bands appearing on the gel (Fig. 1-3).

Fig. 1-3
(A-F) samples, A and C were successfully cloned into plasmid while B, D, E, F were not. (G) Digestion of original plasmid indicated 1800 bp band. (H) Original plasmid contained 1800 bp sequence making it larger than other digested plasmids.

Culturing Bursaphelenchus xylophilus

The reason we decided to subculture B. xylophilus is because we want to make sure that the callus with our designed vector can successfully decrease the bad effect of nematodes. But due to COVID-19, our experiment process got delayed. So we only subcultured them.

In the beginning, we contacted Prof. Jiue-in Yang to know whether she had B. xylophilus in her lab or not. Fortunately, she had B. xylophilus and she was interested in our project, so we had a meeting on Google to make sure we needed to culture B. xylophilus and the opinion or resource that she can give us.

On June 17, 2021, we received a set of tubes including one tube of B. xylophilus and two tubes of A. citri, the food for B. xylophilus. As soon as we received these tubes, we started to subculture them. But sadly, after two weeks, tubes of A. citri we subcultured didn’t grow well(Fig. 2-1). To be honest, it seems very few A. citri were alive in tubes. To solve this problem, we sent an email to Prof. Jiue-in Yang’s student--Mrs.Lu. She said that the problem led to failure remaining unclear and she would send a set of tubes and a video link to us. With her help, we got a more successful result in the subculture of this time. But the A. citri still seems fewer and more gray than those in the tube that Mrs.Lu gave us(Fig. 2-2). So we decided to visit Prof. Jiue-in Yang’s lab in NTU to understand which part of our experiment went wrong.

On August 18, 2021, we visited Prof. Jiue-in Yang’s lab in NTU and made the whole experiment we did in our lab. After watching the process, Mrs.Lu said that our experiment method didn’t have any problems. It might be other factors, like the environment of the lab or the humidity in the Incubator. When we returned to NTHU, she sent a set of tubes to us again. This time we subcultured B. xylophilus and A. citri by using an alcohol lamp and alcohol to make sure that our experiment wouldn’t be polluted during the aseptic process. We are glad to say that we got a better result after doing our experiment in the sterilized area(Fig. 2-3).

Fig. 2-1

Fig. 2-2

Fig. 2-3

Callus of Pinus armandii

Since we couldn't use the whole plant to conduct experiments, we aimed to gene-modify pine callus to fight the infection of B. xylophilus. First, we had to choose which kind of pine species we should use.

There are three reasons that we chose Pinus armandii as our experimental subject:

First, P. armandii is easy to get pine wilt disease, which means it is vulnerable to B. xylophilus.

Second, nematodes can not be cultured on all of the calli of pine trees that can be infected. There is a paper which states that the nematodes can be cultured on the callus of P. armandii.

Third, P. armandii can grow well under the weather conditions in Taiwan and its seed is easy to get.

Due to the pandemic, COVID-19, we couldn’t buy the suggested medium -- LM (Lollably medium). After discussing with Professor Wen-Liang Chen from NCTU, we replaced the LM medium with the MS medium, which is the most common medium for pine tree callus but less nutritious compared to LM.

We used MS that contain 2,4-D, 6-BA, inositol, and sucrose to culture our callus. In the start, we also added kinetin into the medium, but the callus didn’t grow well, it seemed not growing. We guess the problem might be the proportion of the auxin and the cytokine. The ratio of cytokinin to auxin is usually 1:1 to induce the formation of plant callus. Since we already had 6-BA, we decided not to use kinetin, and make auxin(2,4-D) and cytokine(6-BA) more balanced. Fig. 3-1 and Fig. 3-2 were the cultivation of callus with and without kinetin.

Inositol is important to plant growth and development, also it is believed to play an important role in cell division. We tried to add extra inositol in our medium and got a satisfactory result (Fig. 3-3~3-4).

Fig. 3-1 Culturing callus with a medium containing kinetin.

Fig. 3-2 Culturing callus with medium without kinetin.

Fig. 3-3 Culturing callus with extra inositol.

Fig. 3-4 Culturing callus without inositol.

Dry Lab

In our project, we try to plant a modified pine tree to stop the spreading of B. xylophilus. Due to Taiwan’s laws, we can’t modify a complete pine tree in the laboratory, therefore we use the callus of P. armandii in replacement. To examine whether our modified pine tree can resist nematode or not, we build a model to compute it. The model of B. xylophilus is devised and constructed by us, we referred to papers or report to make it close to the real situation.

Design of our model

From past studies, we know that the cell consumption by nematodes is the main reason why pine trees withered. We calculate the consumption and the population size of nematodes in both unmodified and modified pine tree. If the population size of nematodes in the pine trees can be reduced to zero before the consumption exceeds the max consumption that would kill the pine tree, we could say that the pine tree is immune to nematodes.

To calculate the consumption, we read the previous paper to find the length of nematodes versus age. We found that the length of nematodes is an exponential function of age when they are growing, which means that the growth rate of nematode is proportional to its length. To explain this, we came up with some hypotheses.

The nematode can acquire nutrients from consumption, and most of these nutrients is used on growing before the nematode is mature. The digestion rate is proportional to the surface area. From the conclusion shown above and the property, the intestinal tract must be full of nutrients the whole time. Therefore, in the intestinal tract, the rate nutrients appear (ingestion rate) must be faster than the rate nutrients disappear (digestion rate). Because nematodes can control when to excrete, we thought nematodes would only ingest after it finishes the digestion. Therefore, the consumption rate depends on which is slower between the ingestion rate and the digestion rate.

After the nematode is mature, it stops growing. The consumption rate would be a constant too. We found that nematode can’t eat before hatching. So, the energy they acquire from consumption is used to reproduce and growing for the larva of nematode.

Now, we consider the environment of the modified pine trees. In our project, we used a kind of RNAi that could inhibit the production of pectin lyase. When nematodes eat RNAi particles, they would become RISC in nematodes’ cells and they would degrade the mRNA of pectin lyase. It is proportional to the amount of consumption because the amount of RISC is proportional to the amount of RNAi. We assume the degradation rate of mRNA is proportional to the amount of RISC.

When the system reaches the equilibrium state, the degradation rate would equal to the production rate, so we could write down the equation of [mRNA] with given consumption x:

Where α is the degradation rate per concentration induced by consumption, and s is the natural degradation rate per concentration. Compares to αx, s is neglectable.

From the paper, we found that the production rate of mRNA would be decreased by the repressor when the concentration of mRNA is too high. We assumed that the relation between the production rate of mRNA and the concentration of mRNA has two modes: linear and highly nonlinear.

For linear mode, we assume the relation between the production rate of mRNA and the concentration of mRNA is an oblique line:

P_0 is the production rate when [mRNA] is zero, and [mRNA]_max is the max concentration of mRNA.

As for the highly nonlinear mode, we assume the relation between the production rate of mRNA and the concentration of mRNA is a step function:

Substitute P_r into equation, we have:


We set [mRNA]_max as 1, and α/P_0 can be seen as the toxicity of our system.

Now we have the relation between [mRNA] and the consumption rate. Then, we need to figure out the relation between mRNA and ingestion rate. We consider a cell its cell wall thickness is d and nematode can acquire n nutrient after eating it. Times for a nematode to break down its cell wall is T. Breaking rate for a nematode is proportional to the concentration of pectin lyase p(t), so we could write down:

Concentration of pectin lyase is dependent on the concentration of mRNA.

Because the ingestion rate of nematode can be written as I=n/T , substitute T from equation (?) into it, we have:

The integral rate is proportional to the root of the concentration of mRNA.

Parameters in this model

With these conclusions shown above, we could build a birth-death model and calculate the consumption if we have these parameters: the ingestion rate, the digestion rate, the birth rate, the death rate, and the toxicity.
We could write down the equation of growth for nematodes:

The product of A and B is the digestion rate per unit length of nematode. The solution for this equation is:

Where t_0 is the hatching time of nematodes, and l_0 is the length right after the nematodes hatched (initial length).
Fitting the data we found from papers, we have:

Figure 1: Length of nematode versus time, we choose the hatching point as the zero point of time. Fitting it with l(t)=187.23e^(0.03232(t-23.57))

Fitting result: l(t)=187.23e^(0.03232(t-23.57)). We get the digestion rate.

From the paper, we found the average ages for nematode is roughly 600 hours. To simplify computation, we assume the nematode will die when it aged for 600 hours. We also found that a female nematode can reproduce roughly 80 eggs in 600-74=526 hours. So, we could calculate out the reproduction rate in an hour: 0.076.

As for the initial ingestion rate and the toxicity, we saw them as the control variables. We could compute how much toxicity our modified pine tree needs to prevent withering of the pine trees at a given initial ingestion rate.

Symbols Meaning Description
l(t) Length of nematode Function of times, we can get it by iteration
l0 Length of nematode right after it hatched 187.23 (mm), we get it from fitting length data
D Digestion rate of nematode 0.032 (1/hrs), we get it from fitting length data
I(t) Ingestion rate of nematode Function of times, we can get it by iteration
I0 Initial ingestion rate Controllable variables
B0 Birth rate of healthy nematodes 0.076 (1/hrs), we get it from paper
[mRNA](t) Concentration of mRNA Function of times, we can get it by iteration
[mRNA]_max Max concentration of mRNA We set it as 1 because we only need to know the ratio of [mRNA]
C(t) Consumption Function of time, we can get it by iteration.
C_tot Total consumption Accumulation of C(t)
P_rna (C_tot) Production rate of mRNA Function of total consumption, it was discussed in design of model
P_0 Initial production rate of mRNA
α Degradation rate of mRNA induced by consumption
P Toxicity of modified pine tree We defined the ratio of α and P_0 as the toxicity of the modified pine tree

Table: Symbols of our model


In our model, we use iteration to compute the curve of consumption, birthrate, amount of mRNA, and size. To simplify the computation, we ignore all dimensions except time.

We separate nematodes into 2 kinds to iterate: parent and progeny. Parent nematodes were brought in by the Monochamus alternatus at the beginning of the computation, they were all mature. We thought that the chance for long-horned beetles will be stopped at the same position at the same tree is very small, so the parent nematodes wouldn’t increase in this model. And we set the initial population size of nematodes as 1. The progeny nematodes could be borne by both parent and progeny nematodes.

We mentioned that the consumption rate depends on which is smaller between ingestion rate and digestion rate. The former is the product of the initial ingestion rate and √([mRNA]) , and the latter is the product of digestion rate per length and the length of nematode. If the latter is larger, the consumption rate would decrease, and the birthrate would decrease too. That is, we could write down:



Bn would be zero for immature nematodes, ln would be constant for mature nematodes.

Here, we only plot the linear mode of the production rate of mRNA, the initial ingestion rate is set to 1. We used red lines to represent parent nematodes and blue lines to represent progeny nematodes. For unmodified pine trees, toxicity is 0. As for modified pine trees, toxicity is 1.








Figure 2: Iteration result for the unmodified pine trees. (a), (b): birthrate versus time. (c), (d): consumption versus time. (e), (f): [mRNA] versus time. (g): length versus time.








Figure 3: Iteration result for the modified trees. (a), (b): birthrate versus time. (c), (d): consumption versus time. (e), (f): [mRNA] versus time. (g): length versus time.

By iteration result, we could use them to calculate the evolution of population size and the total consumption.

Max consumption before pine tree withered

As mentioned before, the consumption is the main reason why pine trees withered. Therefore, we need to calculate the max consumption in an unmodified pine tree before it withered. According to previous reports, after being infected by nematodes, pine trees would stop secreting resin in 6-20 days. In 20-30 days, the transpiration of pine trees would decline, and leaves would start withering. At the same time, lots of funguses and bacteria invade the sapwood of pine trees. Therefore, nematodes receive lots of nutrients and the population size increases rapidly in the whole tree. We choose 6 days as the point that the consumption is too large for pine trees to cure themselves. We choose 6 days because we want to make sure that all modified pine trees are immune to nematodes. Therefore, we choose the day that pine trees start having symptoms.



Figure 4: Evolution of population size of nematodes in unmodified pine trees (a) and the accumulation of total consumption (b).

The result is shown above, it shows the evolution of population size and the accumulation of total consumption versus times. And the total consumption in 6 days after being infected is 40.86.

In the same way, we could calculate the evolution of population size and the accumulation of total consumption versus times with any given toxicity and initial ingestion rate. If the population size of nematodes drops to 0.01, we claim that all nematodes are dead in this one tree. By comparing the consumption with the max consumption, we can determine whether this pine tree can survive under the attack of nematodes.

The result of the model is shown in dry lab: model.


[1] Xiu-Wen Qiu, Xiao-Qin Wu, Lin Huang, and Jian-Ren Ye. (2016) Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus. Int J Mol Sci.17(1): 125.

[2] Han-Yu Fu, Jia-Hong Ren, Lin Huang, Hao Li, and Jian-Ren Ye. (2014) Screening and Functional Analysis of the Peroxiredoxin Specifically Expressed in Bursaphelenchus xylophilus—The Causative Agent of Pine Wilt Disease.Int J Mol Sci,15(6): 10215–10232.

[3] Bowen Wang, Xin Hao, Jiayao Xu, Yan Ma, and Ling Ma1. (2019) Transcriptome-Based Analysis Reveals a Crucial Role of BxGPCR17454 in Low Temperature Response of Pine Wood Nematode. Int J Mol Sci, 20(12): 2898.

[4] The Handbook of Plant Functional Genomics: Concepts and Protocols. Chapter 10 RNA Interference. Chris A. Brosnan,Emily J. McCallum,José R. Botella,Bernard J. Carroll. Book Editors:Prof. Dr. Günter Kahl,Prof. Dr. Khalid Meksem.

[5] Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu and Ralph Scorza. (2007) Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in Nicotiana benthamiana and Prunus domestica. American Society for Horticultural Science Volume 132 issue 6.

[6] Thermo Fisher Scientific BLOCK-iT™ RNAi Designer

[7] Somatic Embryogenesis of Immature Embryos in Pinus armandii (accessed Oct 20, 2021).

[8] Liu, X. Z.; Liu, Z.; Yang, Y. M.; Zhang, H. Y. Production of Transgenic Pinus armandii Plants Harbouring BtCryIII(A) Gene. Biol. Plant. 2010, 54 (4), 711–714.

[9] Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode, Bursaphelenchus xylophilus, PLoS ONE 4(11):e7593 DOI:10.1371/journal.pone.0007593

[10] 金門國家公園松材線蟲萎凋病防治計畫



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