Increasing Plant Pest Resistance with Bt + RNAi Pyramids

Lately we’ve written a few posts about how crop resistance may be increased in the future. Our last post covered the development of a material able to pass RNAi protection to crops, and the one prior to that covered the multitude of sustainable new methods that may play a role in disease management.

This month, the Plant Biotechnology Journal (which is open – thank you Wiley) published an article looking at a possible way of reducing the evolutionary pressure caused by transgenic crops with one specific method of protection (Bt cotton in this study) as well as improving protection against pests that have developed resistance.

Bt cotton, a transgenic crop which contains a gene for a toxin (Cry toxin) found in Bacillus thuringiensis, provides protection against a range of insects. Prior to this gene being inserted into cotton, corn and a number of other crops, the Baccilus thuringiensis bacteria would be sprayed onto the crops to provide the same protection.

As successful as Bt crops have been, the use of a singular method of pest control puts pressure on the pests to evolve a method of overcoming the effects of the control mechanism. In the case of Bt cotton crops, farmers have begun using lines of transgenic cotton containing a number of different Bt toxins designed to kill the same pest. However, these pyramids of multiple Bt toxin genes are not entirely effective due to the toxins interfering with each other or causing cross-resistance within the pests.

Pyramids of Bt toxins and RNAi

The research tested the possibility of constructing a cotton plant containing both a Bt toxin gene and one of two genes for a double stranded RNA aimed at interfering with two particular genes within the pest Helicoverpa armigera, a moth which develops by feeding on important crops such as cotton and corn.

To test whether the pyramids improved crop defence against H. armigera, cotton plants containing one of the two dsRNAs, the Bt toxin gene, a pyramid of Bt plus one of either of the RNAi constructs or a control plant were challenged with either a Bt susceptible or Bt resistant pest. Two essential genes which have previously been shown to effect pupation of H. armigera were the targets of the dsRNA constructs. The pest fed with an artificial diet containing either of the dsRNAs  was shown to result in an increase in pest mortality and those pests that did survive had a lower weight compared to those feeding on the control plant.

Cotton plants were then transformed via Agrobacterium tumefacnians-mediated transformation to create lines of cotton with either of the dsRNAs or a Green Fluorescence Protein as a control and then selfed the resulting lines to create lines homozygous for each of the genes. Resulting lines were shown through Southern blot analysis to contain only one of the inserted genes.

Susceptible pests fed either of the dsRNA transformed plants showed lower rates of transcription of the target genes.

Having created lines of cotton containing dsRNA able to effect pest growth, the researchers crossed the dsRNA lines with Bt lines in order create crops containing both traits and developed those lines into homozygous cotton plants with  the same number of copies of each of the genes.

Did the Pyramids make any difference?

Before testing the pyramided crops to compare their effect on Bt resistant pests to that of Bt-only crops, resistant H. armigera were fed Bt, transgenic GFP or non-transgenic cotton with no significant difference in their mortality or growth rates. Susceptible H. armigera pests fed the same diets showed the expected increased mortality when fed the Bt cotton diet.

When the resistant pests were fed the RNAi cotton crops, mortality rates were similar to that in susceptible pests, with both lines of pest having a mortality rate close to that of Bt susceptible pests when fed transgenic Bt plants. Of the two RNAi crops, there was no real difference in mortality rates and development between the two genes targeted by the RNAi constructs. Nor was there any significant difference between the effects of RNAi crops on resistant pest mortality and crops containing the Bt + RNAi construct, demonstrating that the pyramid did not have any effect on the pest save for the presence of the RNAi component of the pyramid.

Bt susceptible pests grown on the pyramid cotton crops did show an increase in mortality and days to development when compared to the RNAi crops alone. Using the index of multiplicative survival (“IMS” – comparing the mortality rates of the pyramid to the expected mortality rate of the pyramid which is calculated by multiplying the mortality rates of the pests when raised on crops containing only one or the other the pyramided genes) to determine whether the Bt and RNAi components were acting separately or in concert to cause the effect seen. Using this method of analysis it was thought that the two genes contained in the pyramid act independently against the susceptible pest.

Overall, the cotton crops containing the pyramids showed increased protection against both the susceptible and the resistant lines of pest.

bt and rnai pyramid figure 1

Figure 3b from article. Comparative mortality rates between susceptible and resistant H. armigera on wild type (W0), Green Fluorescence Protein transformed (GFP), Bt, two RNAi transformed crops (JHA and JHB) and Bt + RNAi pyramids. Astrix indicates statistically significant differences between the two lines of pest.

Just in case there is a possibility that Bt resistance resulted in a fitness cost to the H. armigera that may interfere with the analysis, resistant lines were fed on wild type cotton and the transgenic GFP cotton and their development rates monitored. Interestingly, resistance to Bt does come with a cost to development time, resistant pests having a 15 to 16% increase in development time compared to their susceptible cousins. Mortality between the two lines however did not show any difference.

What effect may use of the construct have in reducing resistance evolution?

Computer simulations were used to demonstrate what effect the use of pyramid cotton will have on resistance evolution in a number of scenarios with parameters taken from common growing conditions in northern China and varying levels of pest fitness cost of resistance and time for resistance development.

The amount of refuge land used in the scenarios had a significant impact on resistance development times. Using a refuge percentage of 50%, it was found that adding RNAi defence either in succession with Bt crops or in a pyramid crop increased the time to development of resistance against the defence when compared to Bt crops alone. Using pessimistic parameters for the development of resistance and fitness cost (faster evolution and little to no fitness cost associated with resistance development) and a refuge percentage of 50, it was demonstrated that the time to resistance increased by 5 years when RNAi was used in tandom with Bt crops while time resistance increased to 10 used when the two methods were used consecutively in a pyramid.

Bt plus RNAi Fig 2

Figure 5 from article. Simulations predicting years to resistance under a) realistic scenario, b) Optimistic scenario and c) Pessimistic Scenario with differing percentages of refuge area.

Conclusion

This may be the first time we have discussed a paper which experimented on something other than an important food crop. But Bt transformed food crops are in widespread use and the reliance on only one method of pest control results in the types of problems we are seeing today with the evolution of resistance. Therefore, developing the ability to provide more sustainable, longer term protection to crops could be fast-tracked using a technology like this where the gene targeted by the RNAi can be designed for a specific pest with minimal side effects on related species of insect.

The accuracy of the computer simulations is a little difficult to make out without a better knowledge of the underlying data but could be the basis of field tests and more sophisticated simulations.

The development of RNAi technology, from examples like this to the creation of crop protection technologies like BioClay, is impressive and seems likely to play significant role in the future protection of food production.

 

 

 

 

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