Molecular mechanisms of insect adaptation to plant defense: Lessons learned from a Bruchid beetle

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 477-481

Insect adaptation to plant defense 477

477© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences

Insect Science (2008) 15, 477-481, DOI 10.1111/j.1744-7917.2008.00236.x

Keyan Zhu-Salzman1, 2 and Ren Sen Zeng3

1Department of Entomology, 2Vegetable & Fruit Improvement Center, Texas A&M University, College Station, Texas, USA; 3Institute of

Tropical and Subtropical Ecology, South China Agricultural University, Guangzhou, China

Molecular mechanisms of insect adaptation to plant defense:Lessons learned from a Bruchid beetle

Abstract Plants can accumulate, constitutively and/or after induction, a wide variety ofdefense compounds in their tissues that confer resistance to herbivorous insects. Thenaturally occurring plant resistance gene pool can serve as an arsenal in pest managementvia transgenic approaches. As insect-plant interaction research rapidly advances, it hasgradually become clear that the effects of plant defense compounds are determined not onlyby their toxicity toward target sites, but also by how insects respond to the challenge. Insectdigestive tracts are not passive targets of plant defense, but often can adapt to dietarychallenge and successfully deal with various plant toxins and anti-metabolites. This adaptiveresponse has posed an obstacle to biotechnology-based pest control approaches, whichunderscores the importance of understanding insect adaptive mechanisms. Molecularstudies on the impact of protease inhibitors on insect digestion have contributed significantlyto our understanding of insect adaptation to plant defense. This review will focus on exposinghow the insect responds to protease inhibitors by both qualitative and quantitative remod-eling of their digestive proteases using the cowpea bruchid-soybean cysteine proteaseinhibitor N system.

Key words adaptation, cowpea bruchid, cysteine protease, gene regulation, genomics,protease inhibitor, scN

Correspondence: Keyan Zhu-Salzman, Department ofEntomology, Texas A&M University, College Station, Texas77843, USA. Tel: +1 979 458 3357; fax: 979 862 4790; email:[email protected]

This paper was contributed to the International Symposium onInsect Midgut Biology, April 7-11, 2008, Guangzhou, China.

Cowpea bruchid: a model insect to studyadaptation

Since plants are constantly confronted by insect herbivory,they have evolved an array of defense mechanisms to battleagainst insect insults (Felton, 2005; Kessler & Baldwin,2002; Walling, 2000; Zhu-Salzman et al., 2008). In plantseeds and various tissues, there are typically proteaseinhibitors, α-amylase inhibitors, lectins and so on (Carlini

& Grossi-de-Sá, 2002; Koiwa et al., 1997; Murdock &Shade, 2002; Zhu-Salzman et al., 1998). Insect bioassaysas well as transgenic plants have shown that these com-pounds often retard insect growth and development. Pro-tease inhibitors inhibit insect digestive enzymes that nor-mally hydrolyze dietary proteins into amino acids, thebuilding blocks for biosyntheses of insect structural andfunctional proteins. Digestive enzymes are classified intoserine-, cysteine-, aspartic- and metallo-proteases (Terra &Ferreira, 1994), and protease inhibitors are named accord-ingly (Michaud, 2000). These interfere with the proteolyticprocess, causing reduction in amino acid assimilation byinsects. Nutrient starvation leads to delay in insect devel-opment and even cause insect death (Hilder et al., 1987;Koiwa et al., 1998; Mosolov et al., 2001; Schuler et al.,1998).

The cowpea bruchid, Callosobruchus maculatus is the


478 K. Zhu-Salzman & R.S. Zeng

activities. Also, proteases capable of degrading and thusdisarming scN were induced in the scN-adapted group.This shift to scN-insensitive and scN-degrading proteasesdid not occur in the unadapted control 4th instar larvae(Zhu-Salzman et al., 2003). These data show that insectsadapt to the presence of the inhibitor by both qualitativeand quantitative remodeling of their digestive proteases.Further, this adaptation is indeed induced by dietarychallenge, rather than being a natural developmentalprocess. The early developmental delay presumably re-sulted from reallocation of genomic resources needed forreconfiguration of gene expression to facilitate counter-defense (Moon et al., 2004). Then, once the adjustmentsare complete, the insects become insensitive to the inhibi-tor and develop normally even though the inhibitors arestill present.

Insect adaptation to plant protease inhibitors has beenobserved in a number of insects (Bown et al., 1997;Broadway, 1997; Brunelle et al., 2004; Brunelle et al.,1999; Cloutier et al., 2000; De Leo et al., 1998; Jongsmaet al., 1995; Jongsma & Bolter, 1997; Mazumdar-Leighton& Broadway, 2001). They not only were able to resist theinhibitor effects, some could even ingest more plant mate-rials and gain more weight on transgenic plants expressingprotease inhibitors than on control plants. Biochemicalanalyses indicated that insects possess effective and dy-namic digestive systems that allow them to feed anddevelop on plants with suboptimal amino acids. Generally,they deploy several strategies to counter the proteaseinhibitor effects: (i) overproduction of existing, inhibitor-sensitive digestive proteases to out-titer the inhibitors (Ahnet al., 2004; Broadway, 1997; De Leo et al., 1998); (ii)increased expression of inhibitor-insensitive proteaseisoforms (Bolter & Jongsma, 1995; Bown et al., 1997;Broadway, 1997; Cloutier et al., 2000; Jongsma et al.,1995; Koo et al., 2008; Liu et al., 2004; Mazumdar-Leighton & Broadway, 2001; Zhu-Salzman et al., 2003);and (iii) activation of proteases that hydrolyze and thusdetoxify plant inhibitors (Ahn et al., 2004; Giri et al., 1998;Ishimoto & Chrispeels, 1996; Michaud et al., 1995; Zhu-Salzman et al., 2003).

Differential regulation of insect digestiveproteases

The plasticity and wide diversity of insect digestive pro-teases has gradually gained appreciation. Cowpea bruchidsmodulate transcripts and protein products of major diges-tive cysteine protease CmCP isoforms (Zhu-Salzman etal., 2003). These proteases play dual roles: breaking downdietary proteins to meet nutritional requirements, as well as

most serious post-harvest pest of cowpea and other le-gumes worldwide (Taylor, 1981). It has a high reproduc-tive capacity (40-60 eggs per female) and short develop-mental cycle (3-4 weeks per generation). Populations canexpand rapidly such that 90% or higher of harvested grainmay be destroyed within a few months. Developing cow-pea cultivars resistant to biotic stresses via transgenictechnology or plant breeding represent pest managementstrategies that are environmentally friendly. Such cultivarsare highly desirable because they require little or no addi-tional inputs.

These naturally occurring plant defense genes werethought to be a good gene reservoir for transgenic pestcontrol. However, the reality contradicts what we hadexpected. Insects readily develop resistance when fedthese proteins. Like many other coleopteran insects, thecowpea bruchid uses cysteine proteases as their majordigestive enzymes (Murdock et al., 1987; Terra & Ferreira,1994), although aspartic proteases have also been detectedin this species (Amirhusin et al., 2007; Silva & Xavier-Filho, 1991; Zhu-Salzman et al., 2003). Feeding the insectswith artificial seeds containing 0.2% of soybean cysteineprotease inhibitor N (scN), a concentration reasonablyachievable in transgenic plants, resulted in significantfeeding suppression and developmental delay (Jongsma &Bolter, 1997). However, using a feeding monitor that candetect ultrasonic feeding sounds generated by larval feed-ing inside the artificial seeds (Shade et al., 1990), it wasapparent that the negative impact caused by dietary scNonly occurred during the 1st, 2nd and 3rd instars, but by thetime the insects reached the 4th instar, rates of feeding anddevelopment were comparable to those reared on an inhibi-tor-free diet (Zhu-Salzman et al., 2003). This clear-cutadaptive feeding behavior, indicative of recovery from theinitial growth suppression by scN, thus makes the cowpeabruchid an attractive model system to study how insects areable to circumvent the inhibitory effect.

Biochemical studies on insect counter-defensemechanisms

To determine whether the recovery of normal feeding andgrowth of the cowpea bruchid is part of a programmeddevelopmental process, that is, whether the 4th instarlarvae are naturally more tolerant to scN, gut extracts from4th instar larvae reared on scN-containing diet (scN-adapted) and from those reared on scN-free control diet(scN-unadapted) were prepared. Total gut protease activ-ity of the adapted cohort was significantly higher than thatof the unadapted group. Of this increase, 50% was attrib-uted to scN-insensitive cysteine and aspartic protease



functioning in insect counter-defense. At least 30 CmCPsexist in the midgut of cowpea bruchids, which can befurther clustered into CmCPAs and CmCPBs based onsequence similarity. The bruchids selectively expressedCmCPB over CmCPA when fed on diet containing scN.Studies using recombinant proteins revealed a number ofadvantages in selectively accumulating CmCPB transcripts:(i) CmCPBs have higher intrinsic proteolytic activity thanCmCPAs; (ii) CmCPBs are more efficient in autoprocessing,a process that removes the propeptide and converts thelatent proenzyme to its active mature protease form; and(iii) CmCPBs possess exclusive scN-degrading activitywhich did not exist in CmCPA (Ahn et al., 2004). Thesuperiority of CmCPBs is dependent on efficient propeptideautoprocessing and degradation (Ahn et al., 2007b). Abil-ity of modulating differential proteolytic activity at bothtranscriptional as well as post-translational levels no doubthas helped insects cope with suboptimal dietary conditions.

Discovery of inhibitor-insensitive proteasesusing a genomic approach

One tactic cowpea bruchids use to overcome the effects ofscN is to increase expression of scN-insensitive proteaseisoforms (Zhu-Salzman et al., 2003). Inhibitory assaysclearly showed that this inhibitor-insensitive activity wasnot conditioned by major digestive enzyme CmCPs (Ahnet al., 2004). The advent of functional genomics andproteomics has shed new light on the processes in insectsthat enable them to circumvent dietary challenges. As thefirst line of counter-defense that protects the vulnerableinsect cells and tissues from a broad spectrum of toxins andanti-nutritional factors in the food, the alimentary tractactively responds when the insect faces dietary challenges.The cells lining the digestive tract go through globalchanges in gene expression and in the protein repertoire.Microarray analyses on a cowpea bruchid midgut cDNAlibrary enriched in scN-responsive genes indicated thatscN-responding genes include more than just major diges-tive enzymes targeted by the scN inhibitor. Also regulatedare genes encoding detoxification proteins, antimicrobialpeptides and various metabolic proteins (Moon et al.,2004).

Among up-regulated genes were also cathepsin B-likecysteine protease genes (CmCatB). This finding isintriguing, because its human ortholog cathepsin B pos-sesses an “occluding loop” that has been shown to partiallyblock the access of substrates and inhibitors (Illy et al.,1997; Musil et al., 1991). Further, accumulation of CmCatBtranscript peaked in the 4th instar under scN challenge,concordant with the time of adaptation (Moon et al., 2004;

Zhu-Salzman et al., 2003). Full-length sequence informa-tion on the cowpea bruchid CmCatB from gut tissueindicated that it has an occluding loop sequence. Based onthis information, it is likely that CmCatB enzymes play arole in cowpea bruchid adaptation by rendering cowpeabruchids less susceptible to scN inhibition.

To further test this hypothesis, CmCatB was expressed inthe Pichia pastoris system (Koo et al., 2008). The heterolo-gously expressed recombinant protein exhibited enzy-matic activity that could be inhibited by both E-64 (a broadspectrum cysteine protease inhibitor) and CA-074 (a cathe-psin B-specific cysteine protease inhibitor), confirming itscathepsin B-like nature. Most interestingly, scN was un-able to inhibit CmCatB activity, possibly due to the loopstructure blocking the access of the inhibitor to the catalyticsite of the enzyme. This finding provides molecular andbiochemical evidence that may explain the scN-inducedand scN-insensitive activity in adapted insect guts.

Much less is known regarding how insects sense dietarychallenge and activate counter-defense-related genes.Analyses of the CmCatB promoter concluded that CmCatBtranscriptional regulation is partly mediated by a COUPregulatory cis-element in the promoter and a nuclear-localized trans-acting factor seven-up (CmSvp), the COUP-TF homolog (Ahn et al., 2007a). Electrophoretic mobilityshift assays detected differential DNA binding activitybetween nuclear extracts of scN-adapted and scN-unadaptedmidguts. CmSvp appeared to be involved in the negativeregulation of CmCatB, repressing its expression undernormal growth conditions, but helping insects cope withplant defense compounds by releasing the repression wheninsects are challenged by dietary protease inhibitors.

Conclusions

Billions of dollars are lost each year in agriculture due toinsect pests. Traditional reliance on synthetic chemicalinsecticides has become more and more unacceptable dueto environmental and human health consequences. Alter-native crop protection strategies must be developed(Christou et al., 2006; Halpin, 2005). A safer way tomanage insect pests is to develop crops that carry genesimparting insect resistance. Plants have evolved naturaldefense mechanisms, and current methods of biotechnol-ogy allow for the transfer of these genes from one speciesto another.

While it remains a viable approach to use plant naturaldefense genes for insect pest management (Bent & Yu,1999; Carlini & Grossi-de-Sá, 2002; Hilder & Boulter,1999; Schuler et al., 1998), a detailed understanding ofinsect counteracting mechanisms becomes essential. The


480 K. Zhu-Salzman & R.S. Zeng

lack of information on the molecular and genetic bases ofinsect adaptation to plant defense is a barrier to biotechnol-ogy-based approaches to insect pest management. Underheavy selection pressure, insect populations emerge thatcan overcome any given resistance gene. Strategies basedon a single plant defense gene are insufficient and likelywill reveal another layer of insect counter-defense alreadyprepared. It is imperative to have a global and comprehen-sive understanding of insect-plant interactions. Techno-logical advances allow researchers to ask questions such ashow many genes are involved in insect adaptation to plantdefense, and how their expression is coordinated to facili-tate this process. Exposing transcription activation andsignaling networks of insect counter-defense will lead theway to more intelligently designed strategies for insectcontrol, as neutralizing regulators of the insect adaptivecascade should be more effective than blocking eachindividual downstream effector gene.

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Accepted June 23, 2008

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Molecular mechanisms of insect adaptation to plant defense: Lessons learned from a Bruchid beetle