PCB6528 Spring 2016 Organelle genomes, gene expression and signaling - Discussion paper 1
Zhang, J, S A Khan, C Hasse, S Ruf, DG Heckel and R Bock (2015) Full crop protection from an
insect pest by expression of long double-stranded RNAs in plastids. Science 347:991
You don’t need to prepare these to turn in, but they will be used for a starting point in our class
discussion and some may re-appear at exam time.
1 – What two hypotheses were tested in this work?
2 – What known features of insect biology were exploited in developing transgenic control
strategies?
3 – What target plant issues were the authors attempting to circumvent in developing
transgenic plants resistant to insect pests?
4 – Why is Colorado potato beetle (CPB) an excellent test case?
5 – Why were the CPB actin (ACT) and shrub (SHR) genes selected for this work? How was the
efficacy of these double stranded RNAs (dsRNAs) pre-tested for CPB control?
6 – Why did the in vivo work for CPB control start with tobacco?
7 – Where were the constructs placed in the tobacco genome? (Fig 1A)
8 – What was the selectable marker for plastid transformation and how does it work? (Fig 1A)
9 – What promoter and terminator sequences were used to drive the selectable marker? What
was there biological source and why do you think this source was selected? (Fig 1A)
10 – What were the different strategies used to produce dsRNAs? What promoters and
terminators were selected here and why? (Fig 1A)
11 – What is homoplasmy? What strategies were used to make sure homoplasmic plants were
recovered? Why is it important to achieve homoplasmy? Which assay for homoplasmy do you
think was the most rigorous and why?
12 – How was dsRNA accumulation assayed? (Fig 1B) What would be alternative approaches for
this and what is the advantage of the assay chosen? (Hint – Which of the approaches produced
the highest accumulation of dsRNA? Which approach was eventually chosen for further work
and why?)
13 – Which target gene produced the most dsRNA when expressed in potato plastids? Why do
you think there was a difference in dsRNA accumulation between leaf and tuber? (Fig 1B,C)
14 – For comparison, a nuclear transgene was also designed for production of dsRNA in potato.
Besides genome location, how did this construct differ from the plastid construct? Discuss the
challenges of developing comparable plastid and nuclear constructs. Despite construct
differences, what did the authors conclude about the efficacy of plastid vs nuclear constructs
for dsRNA production? To what did they attribute these differences (Fig 1D,E)
15 – How was protection against the CPB assayed and who was the big winner in terms of
insecticidal activity? (Fig 2)
16 – What is the evidenced that insecticidal activity is due to RNAi effects in the insect? (Figs
S11, S14) Why do the authors believe that target plants must produce large quantities of dsRNA
to trigger RNAi in the insect pest?
17 – Based on all results, what do you conclude about the original hypotheses? What are the
practical advantages and disadvantages of plastid dsRNA production as a pest control strategy?