Friday, June 3, 2011

By Kerryn Dunse and Marilyn Anderson

This ongoing battle between plants and insects has led to the development of multiple defensive and offensive approaches by both insects and plants. Plants produce a multitude of constitutive and inducible defense barriers to reduce damage by insects. They include physical barriers such as waxy layers and trichomes as well as molecular barriers, ranging from secondary metabolites to proteins.

One exquisite example of this finely tuned plant-insect interaction is the defensive function of proteinase inhibitors (PIs) in solanaceous plants. Plants respond to insect attack by upregulating the expression of PIs in stems and leaves. The PIs interfere with protein digestion, which results in growth retardation or death of the insect pest. The struggle between plants and insects to survive and thrive has driven the diversification and expansion of gene families encoding both plant PIs and the target insect proteinases.

Regulation of PIs in plant defense

Clarence Ryan and colleagues were the first to characterize genes that encode potato type I and potato type II serine proteinase inhibitors and to discover an intriguing level of complexity that communities of plants use against insect attack. For example, when a caterpillar chews on tobacco or tomato plant leaves, a local signal derived from the insect or plant2 stimulates the expression of PIs and other defense related compounds. The plant then produces a small mobile peptide called systemin that moves systemically via phloem from wounded to unwounded leaves, prearming unwounded leaves before they are damaged by the insect. Furthermore, the plants then produce a volatile molecule, methyl jasmonate, that travels to neighboring unwounded plants and triggers the signaling pathway that activates PI genes and heightens the defenses of the whole plant population3.

Baldwin and colleagues have dissected various components of plant defense signaling pathways that lead to production of jasmonic acid (JA) and accumulation of proteinase inhibitors4-5. An antisense approach was used to knockdown expression of various upstream genes that blocked production of JA. In one example, transcription factors from the WRKY family, which mediate direct and indirect defenses in Nicotiana attenuate, were silenced, severely reducing the levels of JA in response to wounding by insects6. Accumulation of proteinase inhibitors and volatile alarm signals was significantly diminished in these plants, resulting in substantial herbivore damage when grown in their natural environment. When the defense response was reinstated by treatment with exogenous JA, PI production was restored and Manduca sexta larvae feeding on these plants exhibited the same slow growth as larvae feeding on native plants. Given that knocking out a signaling pathway in these plants may have caused changes other than PI production that might affect insect growth, Baldwin and coworkers went further and demonstrated that the endogenous proteinase inhibitors were directly responsible for reduced herbivore performance7.

Application of PIs for plant protection

Early observations linking the induction of PI gene expression to reduced insect fitness inspired researchers to investigate the potential ability of PIs to protect transgenic plants against herbivory from insect pests. As early as 1987, transgenic plants expressing heterologous proteinase inhibitors were reported to be more resistant to damage caused by chewing insects; there are now numerous examples of transgenic plants expressing PIs with improved protection against insects (reviewed by8-9). Although PIs have great potential, there are still no transgenic plants expressing PIs in commercial development.

A number of parameters have been identified that influence the effectiveness of PIs. These include insect gut pH, larval developmental stage, quantity and quality of dietary protein, and concentration of PI. Another important factor leading to the failure of PIs to provide adequate protection against insects has been the use of single PIs directed to a subset of digestive proteinases, which leaves other proteases unaffected and thus fails to provide sustainable plant protection. The application of PIs in crop protection depends on a better understanding of how insects respond and adapt to PIs.

PI mechanism of action

The antimetabolic effect of PIs on insects is not completely understood, but there are several responses that have been described. 1) Growth and development of insect larvae is severely retarded without significant changes in the gut proteolytic activity. This observation led to the theory that hyperproduction of proteases would in turn cause depletion of essential amino acids. 2) Growth and development of insect larvae is severely retarded and is associated with a severe reduction in proteolytic activity. In this case, amino acids for growth and development are limited, due to the lack of processing of dietary protein by digestive proteases.

On the other hand, insects can employ a large number of mechanisms to overcome the negative effects of proteinase inhibitors. There are several adaptive mechanisms described: 1) upregulation of proteinases to compensate for inhibition of another group of proteinases; 2) production of proteinases that degrade PI; and 3) production of PI-insensitive proteases. Insects can use a combination of these mechanisms to maintain sufficient proteolysis for growth and development.

It is often difficult to determine which of these adaptive processes is operating, because most research articles only describe changes in gut enzyme activity or gene transcription levels after PI ingestion. The changes in levels of enzyme activity could have resulted from PI binding to the enzyme, reduction in gene expression or enzyme secretion, or increased degradation of proteases. Understanding how insects respond to PIs on a case-by-case basis will provide valuable knowledge that may enable the development of PIs as a viable transgenic plant protection technology.

Controlling Lepidopteran pests

In a recent paper10, we described which of these adaptive mechanisms operate in the highly destructive insect pests Helicoverpa armigera and H. punctigera when challenged with a series of serine proteinase inhibitors from the flowers of the ornamental tobacco Nicotiana alata (NaPI). These insects belong to the infamous insect order Lepidoptera, which are major pests of more than 100 species of crop plant worldwide. In 2001 the global cotton market alone was worth 20 billion dollars, and yield loss combined with the cost of controlling lepidopteran pests was estimated at $3 billion dollars annually11.

H. armigera species have been difficult to control due to development of resistance to several major insecticides. This problem was addressed by the commercialization of transgenic crop plants expressing Bt toxin, which also reduced use of pesticides to control lepidopteran species. Insecticide use fell by half in Indian cotton production and by 85% in Australian cotton production with introduction of Bt cotton. Although use of Bt toxin genes has been a highly successful technological advance to control lepidopteran pests, and more than 58 million hectares of Bt crops were grown globally in 2010, there is evidence that field populations of insects are beginning to develop resistance. Until now, this resistance has not caused any major crop losses, but the proliferation of resistance to Bt remains a real threat to global food production. A continued effort to discover insecticidal molecules with different targets to Bt is critical for sustainable protection of crop plants against insect pests.

The Na-proPI multidomain potato type II inhibitor and its application in crop protection

The potato inhibitor II (PinII) family of serine proteinase inhibitors is widely distributed in Solanaceae. This family arose from gene duplication of an ancestral gene encoding a single proteinase inhibitor domain. The most well known members of this family are two domain inhibitors from potato and tomato, although members with one, three, four, six, and eight proteinase inhibitor domains have been described. NaPI is a 40.3 kDa protein composed of an N-terminal endoplasmic reticulum signal peptide that precedes six 6 kDa proteinase inhibitor domains followed by a C-terminal vacuolar-targeting signal (VTS). Unusual folding of PI domains results in a circular bracelet-like structure clasped by three disulfide bonds between N- and C-terminal fragments. Processing NaPI in the secretory pathway removes the signal peptide, the VTS, and the conserved linker that joins PI domains, releasing the six PIs, two of which are chymotrypsin inhibitors and four are trypsin specific. These peptides are potent inhibitors of trypsin and chymotrypsin, the major digestive proteinases in lepidopteran larvae.

The NaPI gene differs from most other PI genes for insect control tested in transgenic plants because six individual proteinase inhibitors are produced from a single NaPI transcript, in contrast to the typical single or double domain PIs produced from other genes. We considered that this would allow us to produce higher levels of PIs in transgenic plants and also introduce the opportunity to swap one or more of the PI domains in the multidomain precursor with PIs tailored to the suite of proteinases in the gut of a target insect.

We began by studying how H. punctigera larvae respond to ingestion of the six 6 kDa PIs produced by NaPI. Larvae exposed to both artificial diets and transgenic tobacco plants containing the PIs were much smaller and exhibited more mortality than insects on control diets12. Although we achieved up to 80% mortality, we focused on the 20% of larvae that appeared unaffected by PI and asked which of the adaptive procedures we described earlier had been used by these insects to bypass the toxic effects of the PIs. We discovered that more than 95% of trypsin activity had been eliminated in surviving insects, but a chymotrypsin had been upregulated that was not inhibited by the 6 kDa NaPI chymotrypsin inhibitors10. Thus the insects had responded by producing a PI resistant protease.

We thought if chymotrypsin activity were essential for continued protein digestion and hence survival in the presence of NaPIs, then we needed to find a new inhibitor that was effective against the PI-resistant chymotrypsin. We discovered that an inhibitor from potato inhibitor I class (StPin1A) was an excellent inhibitor of NaPI-resistant chymotrypsin. Even though Pin I inhibitors are encoded by a completely different family of genes, these genes are expressed in the same tissues as Pin II inhibitors in Solanaceous species and are also regulated by wounding and methyl jasmonate. This makes biological sense, since it is likely that the two families of genes have co-evolved in response to development of PI-resistant proteases in insects. Thus it appears that proteinase inhibitors from at least two distinct classes are needed to provide sustainable insect protection in a transgenic plant, which explains why expression of a transgene encoding a single proteinase inhibitor has failed to provide protection in the past.

We tested this hypothesis by feeding a combination of NaPI and StPin1A to the closely related and more damaging species H. armigera. Larval growth was much more inhibited by the combination of the six 6 kDa NaPI inhibitors and StPin1A compared to the NaPI or the StPin1A inhibitors alone. Using this knowledge we created transgenic cotton plants expressing both genes and tested them under insect pressure in the field. Plants expressing both inhibitors produced 20% more lint than control plants or plants expressing just the NaPI or the StPin1A gene10.

Mechanism of resistance to NaPI

Even though Lepidopteran larvae have a huge impact on global food and fiber production, relatively little is known about the biochemistry of their major digestive enzymes, the trypsins and chymotrypsins. The features that enable lepidopteran enzymes to operate at the highest biological pH ever recorded (pH ~10 – 11) have not been described, and it is not known why the activation peptide of insect chymotrypsins is 3 - 4 times longer than chymotrypsins from other species. No structures are available, and until now the changes that render some enzymes resistant to inhibition by plant proteinase inhibitors have not been defined. The nature of these changes is intriguing because any alterations in the enzyme that prevent binding of the PIs must not interfere with normal hydrolysis of proteins, or the enzyme would be rendered inactive.

To address this question, we looked for the enzyme responsible for resistance to NaPI13. We exploited the interaction between NaPI and StPin1A to develop affinity columns that enabled us to purify the putative NaPI-resistant chymotrypsin and simultaneously purify an NaPI-susceptible chymotrypsin. The unique amino acid sequence from these two proteins allowed the design of specific primers for cDNA cloning and sequencing to confirm that the encoded proteins were indeed chymotrypsins that shared 72% sequence identity at the amino acid level. The encoded zymogens were expressed using baculovirus and insect cell cultures and, after purification from the medium, were activated with immobilized trypsin.

The putative NaPI-resistant chymotrypsin was not inhibited by NaPI but was strongly inhibited by StPin1A, and the NaPI-susceptible chymotrypsin was strongly inhibited by both PIs, confirming that we had cloned genes encoding a PI resistant and a PI susceptible chymotrypsin. We identified the residues that prevented NaPI-binding to the PI resistant chymotrypsin by substituting residues in the NaPI-susceptible chymotrypsin with residues from the NaPI-resistant chymotrypsin. Chymotrypsins with the alterations were once again expressed using baculovirus and tested for susceptibility to NaPI and StPin1A inhibitors. We discovered that substituting the four consecutive amino acids LANF in the S1’ binding pocket of NaPI-susceptible chymotrypsin with amino acids VIDL from the NaPI-resistant chymotrypsin converted the NaPI-susceptible chymotrypsin to a NaPI-resistant chymotrypsin. Molecular modeling predicted that the F>L sub-stitution had likely resulted in the loss of several beneficial contacts between inhibitor and enzyme.

Summary

Our work with two proteinase inhibitors from the structurally distinct potato type I and II families demonstrates the application of PIs for crop protection. Understanding how insects develop resistance to the potato type II inhibitor NaPI is not only important from a resistance management perspective but may enable the design and combinations of PIs that avoid this type of resistance. Engineering potent new PIs is currently hampered by the lack of structures of insect proteases, particularly from Lepidoptera.

The goal of insect control in agricultural biotechnology is to develop a durable, multimechanistic approach, which could include genes that target different biochemical or physiological process of which proteinase inhibitors will be an essential component.

References

1. Ryan CA & Pearce G. Systemin: a polypeptide signal for plant defensive genes. Annu Rev Cell Dev Biol 14, 1-17 (1998).
2. Schmelz EA, et al. Fragments of ATP synthase mediate plant perception of insect attack. Proceedings of the National Academy of Sciences 103, 8894-8899 (2006).
3. Farmer EE & Ryan CA. Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proc Natl Acad Sci U S A 87, 7713-7716 (1990).
4. Halitschke R & Baldwin I. Jasmonates and related compounds in plant-insect interactions. J. Plant Growth Regul. 23, 238-245 (2004).
5. Halitschke R & Baldwin IT. Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36, 794-807 (2003).
6. Skibbe M, Qu N, Galis I, & Baldwin IT. Induced plant defenses in the natural environment: Nicotiana attenuata WRKY3 and WRKY6 coordinate responses to herbivory. Plant Cell 20, 1984-2000 (2008).
7. Zavala JA, Patankar AG, Gase K, Hui D & Baldwin IT. Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 134, 1181-1190 (2004).
8. Carlini CR & Grossi-de-Sa MF. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. Toxicon 40, 1515-1539 (2002).
9. Schluter U, et al. Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective. J Exp Bot 61, 4169-4183 (2010).
10. Dunse KM, et al. Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field. Proc Natl Acad Sci U S A 107, 15011-15015 (2010).
11. James C. Global review of commercialized transgenic crops. International Service for the Acquisition of Agri-biotech Applications Ithaca NY. ISAAA briefs no. 26(2002).
12. Heath RL, et al. Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests. J. Insect Physiol. 43, 833-842 (1997).
13. Dunse KM, et al. Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor. Proc Natl Acad Sci U S A 107, 15016-15021 (2010).