DISSECTION OF INSECT-PLANT INTERACTION VIA MOLECULAR, BIOCHEMICAL AND GENETIC APPROACH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0193964
• Project Start Date: Sep 5, 2008
• Project End Date: Sep 4, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Entomology

Non Technical Summary
Genetic modification of crop plants to introduce insect resistance is a promising technology for insect management, but it is (i) threatened by the potential of insects to overcome the few available resistance genes and (ii) limited by the scarcity of useful resistance genes. Minimal attention has been given to understanding the molecular mechanisms by which insects circumvent proteinaceous antinutritional factors that may serve as plant defenses. The general goal of the proposed research is to understand how insects avoid the negative effects of plant toxins and anti-nutritional factors. Using the cowpea bruchid, Callosobruchus maculatus, as a model for other insects, we will profile midgut gene expression under challenge from individual anti-insect compounds using DNA microarray technology, and construct a MIAME-compliant gene expression matrix that highlights responses of insect genes to a series of dietary toxins; We will verify and quantify changes in the expression of specific messages that result from feeding the larvae the selected toxins. We will further study Transcriptional regulation of insect counter defense genes. The proposed work will (i) shed light on molecular mechanisms by which the cowpea bruchid responds to this range of plant defensive molecules or anti-nutritional factors and may (ii) reveal new vulnerabilities in an insect that may eventually be exploited for better insect control or better management of resistance genes to increase sustainability.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1040	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;

Keywords

adaptation

cowpea bruchid

microarray

plant defense

qrt-pcr

scn

transcriptome

transcription factor

Goals / Objectives
The long-term goal of my research program is to better understand insect-plant interactions in order to design more effective and durable plant defense to enhance U.S. agriculture. Specific research objectives are: 1. Use DNA microarray technology to profile gene expression in the alimentary tracts of cowpea bruchid larvae that have been fed selected anti-nutritional compounds, to better understand how insect pests overcome plant defense and human-imposed pest management. 2. Define cis-elements of selected insect counter-defense genes and interacting trans-acting factors to understand their roles in response to inhibitor challenge. 3. To dissect plant defense mechanisms in order to enhance natural responses of plants to insect pests and achieve more effective plant defense. This study helped uncover the mechanisms utilized by insect pests to minimize the effects of plant defensive protease inhibitors. Such understanding is critical to the success of biotechnology-based insect pest management.

Project Methods
In order to address how insects respond to the challenges of specific and general anti-nutrients, we have completed the setup of the platform for cDNA microarrays. The collection contains 20,352 cDNA clones from midguts of cowpea bruchid larvae. We are investigating cowpea bruchid midgut transcriptomic responses to anti-nutritional factors such as soybean cysteine protease inhibitor, alpha-amylase inhibitor, and cellulose using microarrays. Microarray slides were queried with probes from at least two biological replicates for each time point of each feeding treatment. Selected transcript response of cowpea bruchid larvae to dietary treatments will be further analyzed by quantitative RT-PCR. Essentially, we are providing the insects with suboptimal amounts of all nutrients, rather than a single specific nutrient. We hope to identify genes that are consistently differentially expressed when the insects are exposed to any one of these compounds. Such genes would allow us to further investigate, subsequent to the scope of this project, pathways that insects use to avoid starvation. We hypothesize that plant protease inhibitors trigger diverse signaling pathways to induce sets of coordinately regulated counter-defense genes. We intend to define cis-elements of selected insect counter-defense genes and interacting trans-acting factors to better understand their roles in response to plant protease inhibitor challenge. The identification of regulatory genes involved in the adaptive response may provide new targets for novel pest control strategies. For this, we will apply electrophoretic mobility shift assays, insect cell culture and other related techniques.

Progress 09/05/08 to 09/04/14

Outputs
Target Audience: Researchers, teachers, extension specialists and graduate/undergraduate students in the areas of entomology, plant protection, post-harvest products and pest management will benefit from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? through publications and conference presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The insect digestive system is the first line of defense protecting cells and tissues of the body from a broad spectrum of toxins and anti-nutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20,352 cDNAs were prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing anti-nutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative RT-PCR confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute, high dose of scN. The identified differentially-regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signaling, defense and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, while others exhibited developmental stage-specificity. Acute, high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN while structural, defense and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses. Arabidopsis vegetative storage protein (AtVSP) is an acid phosphatase that has anti-insect activity [Liu et al., 2005. Plant Physiology 139, 1545-1556]. To investigate the functionality of AtVSP in planta as an anti-insect defense protein, we produced AtVSP-overexpressing andAtVSP-silenced transgenic Arabidopsis lines, and evaluated impact on grasshoppers. No significant difference was detected in weight gain and growth rate when feeding on wild type, overexpressing, or silenced lines, respectively. Furthermore, AtVSP protein was undetectable in either the midgut or frass of grasshoppers reared on transgenic plants suggesting that AtVSP was unable to withstand proteolytic degradation. When midgut extracts from various nymphal stages were incubated with bacterially expressed AtVSP, AtVSP was hydrolyzed rapidly by grasshopper gut extract, in contrast with its fate when incubated with cowpea bruchid midgut extract. Multiple proteases detected in the midgut of grasshoppers may play important roles in determining the insect response to AtVSP. Results indicate that stability of an anti-insect protein in insect guts is a crucial property integral to the defense protein. BOTRYTIS-INDUCED KINASE1 (BIK1) plays important roles in induced defense against fungal and bacterial pathogens anda chewing insect herbivore. However, it remains unknown whether BIK1 functions in plant defense againstphloem-feedingaphids. We studied the potential function of BIK1 in Arabidopsis infested with the green peach aphid, Myzus persicae. In contrast to the previously reported positive role of intact BIK1 in defense response, loss of BIK1 function adversely impacted aphid settling, feeding and reproduction. Relative to wild-type plants, bik1 displayed higher aphid-induced H2O2 accumulation and more severe lesions, resembling a hypersensitive response (HR) against pathogens. The bik1 mutant showed elevated basal as well as induced salicylic acid and ethylene accumulation. Intriguingly, elevated salicylic acid levels did not contribute to the HR-like symptoms or to the heightened aphid resistance associated with the bik1 mutant. Elevated ethylene levels in bik1 accounted for an initial, short-term antixenotic activity. Introducing a loss of function mutation in the aphid resistance and senescence-promoting gene PHYTOALEXIN DEFICIENT4 (PAD4) into the bik1 background blocked both aphid resistance and HR-like symptoms, indicating bik1-mediated resistance to aphids is PAD4-dependent.Arabidopsis BIK1 thus confers susceptibility to aphid infestation through its suppression of PAD4 function. Results underscore the role of reactive oxygen species and cell death in plant defense against phloem-feeding insects.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Lei, J., S.A. Finlayson, R.A. Salzman, L. Shan and K. Zhu-Salzman (2014) BOTRYTIS-INDUCED KINASE1 modulates Arabidopsis resistance to green peach aphids via PHYTOALEXIN DEFICIENT4. Plant Physiol. 165: 1657-1670
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Ye, M., Y.Y. Song, J. Long, R.L. Wang, S.R. Baerson, Z. Pan, K. Zhu-Salzman, J.F. Xie, K.Z. Cai, S.M. Luo and R.S. Zeng (2013) Priming of jasmonate-mediated anti-herbivore defense responses in rice by silicon. Proc. Natl. Acad. Sci. USA. 110: E3631-E3639
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Ahn, J-E., X. Zhou, S.E. Dowd, R.S. Chapkin and K. Zhu-Salzman (2013) Insight into hypoxia tolerance in cowpea bruchid: metabolic repression and heat shock protein regulation via hypoxia-inducible factor 1. PLoS ONE 8: e57267
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Cheng, W., J. Lei, J-E. Ahn, T.-X. Liu and K. Zhu-Salzman (2012) Effects of decreased O2 and elevated CO2 on survival, development, and gene expression in cowpea bruchids. J. Insect Physiol. 58: 792-800
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Chi, Y.H., X. Jing, J. Lei, J-E. Ahn, Y.D. Koo, D-J. Yun, S.Y. Lee, S. Behmer, H. Koiwa and K. Zhu-Salzman (2011) Stability of AtVSP in the insect digestive canal determines its defensive capability. J. Insect Physiol. 57: 391-399
• Type: Journal Articles Status: Published Year Published: 2009 Citation: Ahn, J-E. and K. Zhu-Salzman (2009) CmCatD, a cathepsin D-like protease has a potential role in insect defense against a phytocystatin. J. Insect Physiol. 55: 678-685
• Type: Journal Articles Status: Published Year Published: 2009 Citation: Chi, Y.H., R.A. Salzman, S. Balfe, J-E. Ahn, W. Sun, J. Moon, D-J. Yun, S.Y. Lee, T.J.V. Higgins, B. Pittendrigh, L.L. Murdock and K. Zhu-Salzman (2009) Cowpea bruchid midgut transcriptome response to a soybean cystatincosts and benefits of counter-defense. Insect Mol. Biol. 18: 97-110

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Researchers, teachers, extension specialists and graduate/undergraduate students in the areas of entomology, plant protection, post-harvest products and pest management will benefit from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Through quality publications and scientific meeting presentations What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Knowledge on mechanisms of resistance is essential for breeding and selecting varieties resistant to insects. We conductedbiochemical and molecular analyses to understand the basic resistance in crop plants and uncover the mechanisms utilized by insects to minimize the effects of plant defense and environmental stresses.Our study also suggested that use of modified atmospheres with depleted oxygenis an environmentally friendly alternative for control of stored grain insect pests. Southwestern corn borer (SWCB, Diatraea grandiosella) and fall armyworm (FAW, Spodoptera frugiperda) are major pests of sorghum in the southern United States. Host plant resistance is a desirable means for reducing plant damage and yield losses from both insects. We evaluated 12 sorghum lines for whorl-stage resistance to leaf-feeding SWCB and FAW in greenhouse and laboratory bioassays. Differential plant responses were detected against the two insects. Among 12 lines tested, CM1821, Della and PI196583 were resistant to both insects, while BTx2752 was largely susceptible. Line R.09110 was resistant to SWCB, but susceptible to FAW, whereas Redbine-60 was susceptible to SWCB, but not to FAW. In addition, we quantified various chemical components in the plants and determined their association with insect resistance. Tannin and chlorophyll in leaves did not show any significant correlation with resistance to either insects, but contents of soluble protein in general were negatively correlated with resistance to both insects. Endogenous soluble sugar and dhurrin were only positively correlated with resistance to SWCB, but not with FAW resistance. To gain some molecular insight into resistance mechanism of sorghum to SWCB, we performed qPCR reactions for key genes encoding enzymes involved in dhurrin and JA biosynthesis on selected resistant or susceptible lines. Although these genes were rapidly and strongly induced by insect feeding in all lines, the observed resistance is likely explained by higher constitutive dhurrin contents in some resistant lines and higher basal JA biosynthesis in others. Our results suggest that sorghum utilizes multiple strategies to defend itself against SWCB. Oxygen is of fundamental importance for most living organisms including insects. Hermetic storage uses airtight containment facilities to withhold oxygen required for development, thus preventing damage by insect pests in stored grain. Cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth when exposed to 2% oxygen. However, although population expansion is temporarily arrested, the bruchids can survive extended periods of hypoxia and recover development if normoxic conditions resume. To understand fundamental mechanisms that enable insects to cope with oxygen deprivation, we constructed a 3'-anchored cDNA library from 4th instar larvae subjected to normoxic and hypoxic treatments, and performed 454-pyrosequencing. Quality filtering and contig assembly resulted in 20,846 unique sequences. Of these, 5,335 sequences had hits in BlastX searches, constituting a 2,979 unigene set. Further analysis based on gene ontology terms indicated that 1,036 genes were involved in a diverse range of cellular functions. Genes encoding putative glycolytic and TCA cycle enzymes as well as components of respiratory chain complexes were selected and assessed for transcript responses to low oxygen. The majority of these genes were down-regulated, suggesting that hypoxia repressed metabolic activity. However, a group of genes encoding heat shock proteins (HSPs) was induced. Promoter analyses of representative HSP genes suggested the involvement of hypoxia-inducible transcription factor 1 (HIF1) in regulating these hypoxia-induced genes. Its activator function has been confirmed by transient co-transfection into S2 cells of constructs of HIF1 subunits and the HSP promoter-driven reporter.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Cheng, W., J. Lei, W.L. Rooney, T.-X. Liu and K. Zhu-Salzman (2013) High basal defense gene expression determines sorghum resistance to the whorl-feeding insect southwestern corn borer. Insect Sci. 20: 307-317
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Ahn, J-E., X. Zhou, S.E. Dowd, R.S. Chapkin and K. Zhu-Salzman (2013) Insight into hypoxia tolerance in cowpea bruchid: metabolic repression and heat shock protein regulation via hypoxia-inducible factor 1. PLoS ONE 8: e57267
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Sun, Y., H. Guo, K. Zhu-Salzman and F. Ge (2013) Elevated CO2 increases the abundance of the peach aphid on Arabidopsis by reducing jasmonic acid defenses. Plant Sci. 210: 128 140
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Cheng, W., J. Lei, J-E. Ahn, Y. Wang, C.L. Lei and K. Zhu-Salzman (2013) CO2 enhances effects of hypoxia on mortality, development, and gene expression in cowpea bruchid, Callosobruchus maculatus. J. Insect Physiol. 59: 1160-1168
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Guo, H., Y. Sun, B. Tong, M. Harris, K. Zhu-Salzman, and F. Ge (2013) Pea aphid promotes amino acid metabolism both in Medicago truncatula and bacteriocytes to favor aphid population growth under elevated CO2. Global Change Biol. 19: 32103223

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The furanocoumarin compound bergapten is a plant secondary metabolite that has anti-insect function. When incorporated into artificial diet, it retarded cowpea bruchid development, decreased fecundity, and caused mortality at a sufficient dose. cDNA microarray analysis indicated that cowpea bruchid altered expression of 543 midgut genes in response to dietary bergapten. Among these bergapten-regulated genes are those encoding proteins related to nutrient transport and metabolism, development, detoxification, defense and various cellular functions. Such differential gene regulation presumably facilitates the bruchids' countering the negative effect of dietary bergapten. Many genes did not have homology with known genes in a BLASTX search, or had homology only with genes of unknown function. Interestingly, when compared with the transcriptomic profile of cowpea bruchids treated with dietary soybean cysteine protease inhibitor N (scN), 195 out of 200 coregulated midgut genes are oppositely regulated by the two compounds. Simultaneous administration of bergapten and scN attenuated magnitude of change in selected oppositely-regulated genes, as well as led to synergistic delay in insect development. In addition, we examined the impact of hypoxia and hypercapnia on cowpea bruchids. Two O2/CO2 combinations were used; (i) 10% O2 + 10% CO2 (ii) 2% O2 + 18% CO2 ( N2 was maintained at 80%). In ambient atmosphere, the rate of O2 consumption and CO2 output at different stages was: eggs ≈ 1st instar＜2nd instar ≈ pupae ≈ adults＜3rd instar＜4th instar. When exposed to 10% O2 + 10% CO2, eggs, larvae and pupae were able to complete development and successfully enter the next developmental stage, although developmental time and mortality varied at different stages. In contrast, more severe hypoxic/hypercapnic treatment, i.e. 2% O2 + 18% CO2, led to cessation of development of all stages. Effects on eggs and adults were most dramatic while the 3rd and 4th instar larvae were least sensitive. Patterns of gene expression and proteolysis suggest that cowpea bruchids suppress their metabolic activity and increase stress tolerance when challenged by O2 deprivation. Transcript abundance as well as proteolytic activity recovered once normoxic conditions resumed. Taken together, cowpea bruchids were able to cope with hypoxic and hypercapnic stress. This ability was particularly strong in the late larval stage. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We found that targeting insect vulnerable sites that may compromise each other's counter-defensive response has the potential to increase the efficacy of the anti-insect molecules. Simultaneous expression of defense proteins like these presumably enhances anti-insect efficacy. Our study also suggested that use of modified atmospheres with depleted O2 is an environmentally friendly alternative for control of stored grain insect pests.

Publications

• 49. Guo, F., J. Lei, Y. Sun, Y. H. Chi, F. Ge, B. Patil, H. Koiwa, R.S. Zeng and K. Zhu-Salzman (2012) Antagonistic regulation, yet synergistic defense: Effect of bergapten and protease inhibitor on development of cowpea bruchid, Callosobruchus maculatus. PLoS ONE 7: e41877
• 48. Jing, X., H. Vogel, R. Grebenok, K. Zhu-Salzman and S. Behmer (2012) Dietary sterols/steroids and the generalist caterpillar Helicoverpa zea: physiology, biochemistry and midgut gene expression. Insect Biochem. Mol. Biol. 42: 835-45
• 47. Ren, S., S. Weeda, K. Zhu-Salzman and T. Ferrell (2012) Novel Arabidopsis jasmonate-responsive mutants have variations in bacterial disease resistance. J. Biotech Res. 4: 80-91
• 46. Guo, H., Y. Sun, Q. Ren, K. Zhu-Salzman, L. Kang, C. Wang, C. Li and F. Ge (2012) Elevated CO2 reduces the resistance and tolerance of tomato plants to Helicoverpa armigera by suppressing the JA signaling pathway. PLoS ONE 7: e41426
• 45. Wu, G.Z, L. Hu, M. Ye, R.L. Wang, K. Zhu-Salzman and R.S. Zeng (2012) Effects of soybean trypsin inhibitor and defense signaling compounds on detoxification enzymes in Spodoptera litura (F.) larvae. Chinese J. Appl. Ecol. 23: 1952-8
• 44. Ye, M., S.M. Luo, J.F. Xie, Y.F. Li, T. Xu, Y. Liu, Y.Y. Song, K. Zhu-Salzman and R.S. Zeng (2012) Silencing COI1 in rice increases susceptibility to chewing insects and impairs inducible defense. PLoS ONE 7: e36214
• 43. Cheng, W., J. Lei, J-E. Ahn, T.-X. Liu and K. Zhu-Salzman (2012) Effects of decreased O2 and elevated CO2 on survival, development, and gene expression in cowpea bruchids. J. Insect Physiol. 58: 792-800
• 42. Wu, G.Z, N.W. Yin, L. Hu, M. Ye, Y.Y. Song, K. Zhu-Salzman and R.S. Zeng (2012) Effects of soybean trypsin inhibitor and defense signaling compounds on the protective enzymes in Spodoptera litura larvae. Chinese J. Ecol. 31: 652-658

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We have previously demonstrated that Arabidopsis vegetative storage protein (AtVSP) is an acid phosphatase that has anti-insect activity in in vitro feeding assays [Liu et al., 2005. Plant Physiology 139, 1545-1556]. To investigate the functionality of AtVSP in planta as an anti-insect defense protein, we produced AtVSP-overexpressing as well as AtVSP-silenced transgenic Arabidopsis lines, and evaluated impact on the polyphagous American grasshopper Schistocerca americana. Grasshoppers showed no significant difference in weight gain and growth rate when feeding on wild type, overexpressing, or silenced lines, respectively. In addition, AtVSP protein was undetectable in either the midgut or frass of grasshoppers reared on transgenic plants suggesting that AtVSP was unable to withstand proteolytic degradation. To determine the stability of the AtVSP protein in grasshopper digestive canal, midgut extracts from various nymphal stages were incubated with bacterially expressed AtVSP for different periods of time. AtVSP was hydrolyzed rapidly by grasshopper gut extract, in stark contrast with its fate when incubated with cowpea bruchid midgut extract. Multiple proteases have been detected in the midgut of grasshoppers, which may play important roles in determining the insect response to AtVSP. Results indicate that stability of an anti-insect protein in insect guts is a crucial property integral to the defense protein. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Overexpression of defense proteins against herbivorous insect attack presumably enhances anti-insect efficacy. Our study raised an important issue; that is, only when the transgenic protein is able to survive the passage through the insect digestive canal, could its defense functionality be realized. Proteolysis-susceptible, but otherwise anti-insect proteins have little practical use.

Publications

• Chi, Y.H., X. Jing, J. Lei, J-E. Ahn, Y.D. Koo, D-J. Yun, S.Y. Lee, S. Bhemer, H. Koiwa and K. Zhu-Salzman (2011) Stability of AtVSP in the insect digestive canal determines its defensive capability. J. Insect Physiol. 57: 391-399
• Chi, Y.H. , J-E. Ahn, D-J. Yun, S.Y. Lee, T.-X. Liu and K. Zhu-Salzman (2011) Changes in oxygen and carbon dioxide environment alter gene expression of cowpea bruchids. J. Insect Physiol. 57: 220-230

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: CmCatB, a cathepsin B-type cysteine protease, is insensitive to inhibition by the soybean cysteine protease inhibitor scN. Cowpea bruchids dramatically induce CmCatB expression when major digestive proteases are inactivated by dietary scN, which is presumably an adaptive strategy that insects use to minimize effects of nutrient deficiency. In this study, we cloned the cowpea bruchid hepatocyte nuclear factor 4 (CmHNF-4) and demonstrated its involvement in transcriptional activation of CmCatB in the digestive tract of scN-adapted bruchids. Electrophoretic mobility shift assays demonstrated that CmHNF-4 binds to a CmCatB promoter region containing two tandem chicken ovalbumin upstream promoter (COUP) sites, which is also the cis-element for Seven-up (CmSvp), a previously identified transcriptional repressor of CmCatB. While CmSvp is predominantly expressed in unadapted insect midgut, CmHNF-4 is more abundant in adapted bruchids. When transiently expressed in Drosophila S2 cells, CmHNF-4 substantially increased CmCatB expression through COUP-binding. CmSvp inhibited CmHNF-4-mediated transcriptional activation even in the absence of its DNA-binding domain. Thus antagonism resulted, at least in part, from protein-protein interactions between CmSvp and CmHNF-4. Association of the two transcription factors was subsequently confirmed by GST pull-down assays. Interestingly, anti-CmHNF-4 serum caused a supershift not only with nuclear extracts of scN-adapted insect midgut, but with that of unadapted control insects as well. Presence of CmHNF-4 in unadapted insects further supported the idea that interplay between CmSvp and CmHNF-4 controls CmCatB transcription activation. Together, these results suggest that coordination between CmHNF-4 and CmSvp is important in counter-defense gene regulation in insects. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
How insect pests regulate the expression of counter-defense genes to cope with plant defense is an important issue, not only from a basic science perspective, but also because it is crucial for the success of biotechnology-based pest control measures. Our study revealed a new function of HNF-4 and Svp; they contribute to reconfiguration of insect counter-defense genes in the midgut under dietary challenge.

Publications

• Ahn, J-E., L.A. Guarino and K. Zhu-Salzman (2010) Coordination of hepatocyte nuclear factor 4 and seven-up controls insect counter-defense cathepsin B expression. J. Biol. Chem. 285: 6573-6584
• Chi, Y.H., Y.D. Koo, S.Y. Dai, J-E. Ahn, D-J. Yun, S.Y. Lee and K. Zhu-Salzman (2010) N-Glycosylation at non-canonical Asn-X-Cys sequence of recombinant cowpea bruchid cathepsin B-like counter-defense protein. Comp. Biochem. Physiol. 156B: 40-47
• Peters, L., K. Zhu-Salzman and T. Pankiw (2010) Effect of primer pheromones and pollen diet on the food producing glands of worker honey bees (Apis mellifera L.). J. Insect Physiol. 56: 132-137

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: When fed on a diet containing a proteinaceous cysteine protease inhibitor from soybean (scN), cowpea bruchid larvae enhance their overall digestive capacity to counter the inhibitory effect. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20,352 cDNAs were prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing anti-nutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative RT-PCR confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 hrs. A total of 1,756 scN- responsive cDNAs were sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a BLAST search. The identified differentially-regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signaling, defense and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, while others exhibited developmental stage-specificity. Acute (24 hr), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN while structural, defense and stress-related genes were largely down-regulated. In addition, we found that elevated proteolytic activity is attributed not only to the major digestive cysteine proteases (CmCPs), but to aspartic proteases, a minor midgut protease component. We isolated a CmCatD cDNA from cowpea bruchid midgut that shares substantial sequence similarity with cathepsin D-like aspartic proteases of other organisms. Its transcript profile was developmentally regulated and subject to alteration by dietary scN. CmCatD transcripts were more abundant in scN-fed 3rd and 4th instar midguts than in control. The bacterially expressed recombinant CmCatD proprotein was capable of autoprocessing under acidic conditions, and mature CmCatD also exhibited pH-dependent proteolytic activity. CmCatD trans-activated CmCPs and vice versa, suggesting a cooperation between the minor midgut CmCatD and major digestive CmCPs. Further, CmCatD was able to degrade scN after extensive incubation. This activity partially restored CmCP proteolytic activity otherwise inhibited by scN. Thus CmCatD could facilitate insects' coping with the challenge of dietary scN by exerting its scN-insensitive and scN-degrading activity, freeing cysteine proteases for food degradation. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The insect digestive system is the first line of defense protecting cells and tissues of the body from a broad spectrum of toxins and anti-nutritional factors in its food. Our results show that insects actively mobilize genomic resources in the alimentary tract, including coordination of the functionality of the two classes of digestive proteases to fend off the negative effect of scN, and fulfill their nutrient requirements. The enhanced or restored digestibility, crucial for insect survival, may be bought at the cost of weakened response to other stresses. Such understanding helps the success of biotechnology-based insect pest management.

Publications

• Peters, L., K. Zhu-Salzman and T. Pankiw (2009) Effect of primer pheromones and pollen diet on the food producing glands of worker honey bees (Apis mellifera L.). J. Insect Physiol. (in press)
• Ahn, J-E. and K. Zhu-Salzman (2009) CmCatD, a cathepsin D-like protease has a potential role in insect defense against a phytocystatin. J. Insect Physiol. 55: 678-685
• Chi, Y.H., R.A. Salzman, S. Balfe, J-E. Ahn, W. Sun, J. Moon, D-J. Yun, S.Y. Lee, T.J.V. Higgins, B. Pittendrigh, L.L. Murdock and K. Zhu-Salzman (2009) Cowpea bruchid midgut transcriptome response to a soybean cystatin, costs and benefits of counter-defense. Insect Mol. Biol. 18: 97-110

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Insects are capable of readjusting their digestive regimes in response to dietary challenge. The soybean cysteine protease inhibitor scN inhibits digestive proteolytic activity in cowpea bruchids (Callosobruchus maculatus), and causes larval growth retardation. However, in response to scN, insects become less susceptible to scN by producing scN-insensitive proteases, or by proteolytic fragmentation of scN. Cowpea bruchids strongly induce CmCatB1 transcripts when fed diet containing scN. CmCatB1 shares significant sequence similarity with cathepsin B-like cysteine proteases. In this study, we isolated another cDNA, namely CmCatB2 that encodes a protein sequence otherwise identical to CmCatB1, but lacking a 70 amino acid internal section. CmCatB1 and CmCatB2 likely resulted from alternate splicing events. Only the CmCatB1 transcript, however, exhibited differential expression in response to dietary scN. This transcript activation was only detectible in larvae, the developmental stage associated with food ingestion. The scN-activated and developmentally regulated CmCatB1 expression pattern suggests it may have a unique function in insect counter defense against anti-nutritional factors. Heterologously expressed recombinant CmCatB1 protein exhibited enzymatic activity in a pH-dependent manner. Activity of the protein was inhibited by both the cysteine protease inhibitor E-64 and the cathepsin B-specific inhibitor CA-074, verifying its cathepsin B-like cysteine protease nature. Interestingly, the enzymatic activity was unaffected by the presence of scN. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We have provided functional evidence suggesting that CmCatB1 confers inhibitor-insensitive enzymatic activity to cowpea bruchids, which is crucial for insect survival when challenged by dietary protease inhibitors. This study helped uncover the mechanisms utilized by insect pests to minimize the effects of plant defensive protease inhibitors. Such understanding is critical to the success of biotechnology-based insect pest management.

Publications

• Zhu-Salzman, K. and R.S. Zeng (2008) Molecular mechanisms of insect adaptation to plant defense: lessons learned from a bruchid beetle. Insect Sci. 15: 477-481
• Koo, Y.D., J-E. Ahn, R.A. Salzman, J. Moon, Y.H. Chi, D-J. Yun, S.Y. Lee, H. Koiwa and K. Zhu-Salzman (2008) Functional expression of an insect cathepsin B-like counter-defense protein. Insect Mol. Biol. 17:235-45
• Zhu-Salzman, K., D.S. Luthe and G.W. Felton (2008) Arthropod-inducible proteins: Broad spectrum defenses against multiple herbivores. Plant Physiol. 146: 852-858

Progress 01/01/07 to 12/31/07

Outputs
Targeting multiple digestive proteases may be more effective in insect pest control than inhibition of a single enzyme class. We therefore explored possible interactions of three antimetabolic protease inhibitors fed to cowpea bruchids in artificial diets, using a recombinant soybean cysteine protease inhibitor scN, an aspartic protease inhibitor pepstatin A, and soybean Kunitz trypsin inhibitor KI. scN and pepstatin, inhibiting major digestive cysteine and aspartic proteases respectively, significantly prolonged the developmental time of cowpea bruchids individually. When combined, the anti-insect effect was synergistic, i.e., the toxicity of the mixture was markedly greater than that of scN or pepstatin alone. KI alone did not impact insect development even at relatively high concentrations, but its anti-insect properties became apparent when acting jointly with scN or scN plus pepstatin. Incubating KI with bruchid midgut extract showed that it was partially degraded. This instability may explain its lack of anti-insect activity. However, this proteolytic degradation was inhibited by scN and/or pepstatin. Protection of KI from proteolysis in the insect digestive tract thus could be the basis for the synergistic effect. These observations support the concept that cowpea bruchid gut proteases play a dual role; digesting protein for nutrient needs and protecting insects by inactivating dietary proteins that may otherwise be toxic. Our results also suggest that transgenic resistance strategies that involve multigene products are likely to have enhanced efficacy and durability.

Impacts
Having multiple digestive enzymes could be a functional overlap to ensure protein degradation. Transgene products are at risk of losing their effectiveness due to insect ability to overcome defense mechanisms. This research provides additional evidence supporting that the effect of multiple inhibitors in the diet could be much greater than individual inhibitors on insect development. Such understanding is critical to the success of biotechnology-based insect pest management.

Publications

• Ahn, J-E. , L.A. Guarino and K. Zhu-Salzman (2007) Seven-up facilitates insect counter-defense by suppressing cathepsin B expression. FEBS J. 274: 2800-2814
• Amirhusin, B., R.E. Shade, H. Koiwa, P.M. Hasegawa, R.A. Bressan, L.L. Murdock and K. Zhu-Salzman (2007) Protease inhibitors from several classes work synergistically against Callosobruchus maculatus. J. Insect Physiol. 53: 734-740

Progress 01/01/06 to 12/31/06

Outputs
The soybean cysteine protease inhibitor scN inhibits digestive proteolytic activity in cowpea bruchids, and causes larval growth retardation. However, in response to scN, insects become less susceptible to scN by producing scN-insensitive proteases, or by proteolytic fragmentation of scN. Evidence indicated that bruchids selectively induced CmCPs (the major digestive enzymes) from subfamily B. Enzymes from this subfamily were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally upregulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B in these regards. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pairwise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

Impacts
A biotechnology-based approach to pest management offers an alternative to reliance on chemical pesticides. However, transgene products are at risk of losing their effectiveness due to the ability of insects to overcome defense mechanisms. This study helped uncover the mechanisms utilized by insect pests to minimize the effects of plant defensive protease inhibitors. Such understanding is critical to the success of biotechnology-based insect pest management.

Publications

• Ahn, J-E., M.R. Lovingshimer, R.A. Salzman, J.K. Presnail, A.L. Lu, H. Koiwa and K. Zhu-Salzman (2006) Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors through modulating propeptides of major digestive enzymes. Insect Mol. Biol. (in press)
• Zhu-Salzman, K. and L.L. Murdock (2006) Cowpea: insects, ecology and control. An invited article for the Encyclopedia of Pest Management, published by Marcel Dekker, pp1-3.

Progress 01/01/05 to 12/31/05

Outputs
We have made significant progress with characterization of the Arabidopsis vegetative storage protein (AtVSP). Vegetative storage proteins are proteinaceous reserves that temporarily store unneeded amino acids and therefore buffer the availability of nitrogen and other nutrients. Although indirect evidence previously suggested that AtVSP could play a role in defense against herbivorous insects, direct evidence for biochemical and anti-insect activities of the Arabidopsis protein were previously lacking. To test the hypothesis, we selected AtVSP2, a wound-, mJA-, insect feeding- and phosphate deprivation-induced gene, for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects which have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic Asp119 residue at the conserved DXDXT signature motif was substituted by Glu via site-directed mutagenesis. This single amino acid alteration did not compromise the protein secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Consequently, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.

Impacts
Understanding the molecular and genetic bases of plant defense and insect counter-defense mechanisms is not only important from a basic science perspective, it is also crucial for the development of novel biotechnology-based pest control practice. Advances in genomics and bioinformatics provide the keys to this new understanding. We believe our approach will have the potential to reveal new vulnerabilities in an insect that may eventually be exploited for better insect control.

Publications

• Liu, Y., J-E. Ahn, S. Datta, R.A. Salzman, J. Moon, B. Huyghues-Despointes, B. Pittendrigh, L.L. Murdock, H. Koiwa and K. Zhu-Salzman (2005) Arabidopsis vegetative storage protein is an anti-insect acid phosphatase. Plant Physiol. 139: 1545-1556
• Sagili, R.R., T. Pankiw and K. Zhu-Salzman (2005) Effects of soybean trypsin inhibitor on hypopharyngeal gland protein content, total midgut protease activity and survival of the honey bee (Apis mellifera L.). J. Insect Physiol. 51: 953-957
• Zhu-Salzman, K., J.L. Bi and T.-X. Liu (2005) Molecular strategies of plant defense and insect counter-defense. Insect Science 12: 3-15

Progress 01/01/04 to 12/31/04

Outputs
We have used cowpea bruchid (Callosobruchus maculatus), southern corn rootworm (Diabrotica undecimpunctata howardi) and sorghum-greenbug (Schizaphis graminum) as our systems to study insect-plant interactions. Using high-throughput DNA microarrays, we identified greenbug-regulated genes in sorghum, and compared greenbug-responsive transcript profiles with those after treatments by methyl jasmonate and salicylic acid. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding. On the insect side, we assembled EST collections from cowpea bruchid guts, as well as from southern corn rootworm. Subsequent microarrays and northern blotting allowed us to identify insect genes that are responsive to a plant defense protease inhibitor scN in insect diets. scN-adapted insects induced genes encoding protein and carbohydrate digestive enzymes, probably to help meet its carbon and nitrogen requirements. Up-regulation of antimicrobial and detoxification protein genes may represent a generalized defense response. Induction of a peritrophin gene suggested that strengthening the peritrophic membrane plays a role in coping with protease inhibitors. Genes down-regulated by scN reflected physiological adjustments of the insects to scN challenge. A large portion of the responsive genes, presumably involved in carrying out the counter-defense response, were of unknown function. Multiple regulatory mechanisms of counter defense-related genes, i.e. developmental regulation (in southern corn rootworm) and in response to dietary toxin (such as cowpea bruchid and southern corn rootworm) may allow insects to evade the effect of plant defense proteins, and impose an obstacle to biotechnology-based insect control.

Impacts
Understanding the molecular and genetic bases of plant defense and insect counter-defense mechanisms is not only important from a basic science perspective, it is also crucial for the development of novel biotechnology-based pest control practice. Advances in genomics and bioinformatics provide the keys to this new understanding. We believe our approach will have the potential to reveal new vulnerabilities in an insect that may eventually be exploited for better insect control.

Publications

• Liu, Y., R.A. Salzman, T. Pankiw and K. Zhu-Salzman (2004) Transcriptional regulation in southern corn rootworm larvae challenged by soyacystatin N. Insect Biochem. Mol. Biol. 34: 1069-1077
• Pankiw, T., R. Roman, R.R. Sagili, and K. Zhu-Salzman (2004) Pheromone-modulated behavioral suites influence colony growth in the honey bee (Apis mellifera). Naturwissenschaften 91: 575-578
• Ahn, J-E., R.A. Salzman, S.C. Braunagel, H. Koiwa and K. Zhu-Salzman (2004) Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor. Insect Mol. Biol. 13: 649-657
• Moon, J., R.A. Salzman, J-E, Ahn, H. Koiwa and K. Zhu-Salzman (2004) Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defense protease inhibitor. Insect Mol. Biol. 13: 283-291
• Amirhusin, B., R.E. Shade, H. Koiwa, P.M. Hasegawa, R.A. Bressan, L.L. Murdock and K. Zhu-Salzman (2004) Soyacystatin N inhibits proteolysis of wheat a-amylase inhibitor and potentiates toxicity against cowpea bruchid, Callosobruchus maculates. J. Econ. Entomol. (in press)
• Zhu-Salzman, K., R.A. Salzman, J-E. Ahn and H. Koiwa (2004) Transcriptional regulation of sorghum defense determinants against a phloem-feeding aphid. Plant Physiol. 134: 420-431

Progress 01/01/03 to 12/31/03

Outputs
We have shown that fusion protein that covalently joined scN with the Griffonia lectin GSII had much higher toxicity than was expected for the additive activities of the two components alone. Several scenarios can explain the markedly higher anti-insect activity of the fusion protein than the mixture at the same dosage level; (i) interaction of the rGSII domain with glycoligands in the insect midgut epithelium, or peritrophic matrix, brought the scN domain into close proximity to the epithelial cells. Thus newly synthesized digestive proteases could be sequestered before their secretion to the gut lumen where bulk dietary proteins are located, (ii) tethering scN to the rGSII domain may have prevented the inhibitor from moving along with ingested food, consequently increasing its effective concentration in the insect gut, (iii) adaptive strategies under single toxin challenge may not be effective when insects are challenged by more than one toxin, and (iv) covalently attaching rGSII to scN may have blocked the access of scN-insensitive proteases to the fusion molecule, therefore preventing proteolytic destruction of the scN moiety. We have also started using subtractive hybridization and DNA microarray technology to investigate insect gut gene regulation upon dietary challenge. This effort has proven to be fruitful. Simultaneously, we have applied these technologies in studying response of monocot sorghum to a sucking insect, greenbug aphid. Results help us understand how plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, meanwhile gene expression patterns reveal that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery. We found aphids only weakly induced genes in the wounding/jasmonate-regulated defense pathway, but strongly and more rapidly induced genes conferring pathogen defense. We also demonstrated that the jasmonate-induced defense response deterred aphid infestation but the pathogen defense pathway did not. Thus it appears aphids have developed strategies to misguide plants to activate an ineffective pathway and to avoid stimulating effective plant defense.

Impacts
Understanding the molecular and genetic bases of plant defense and insect counter-defense mechanisms is not only important from a basic science perspective, it is also crucial for the development of novel biotechnology-based pest control practice. Advances in genomics and bioinformatics provide the keys to this new understanding. We believe our approach will have the potential to reveal new vulnerabilities in an insect that may eventually be exploited for better insect control.

Publications

• Zhu-Salzman, K., H. Li, P.E. Klein and R.L. Gorena (2003) Using high-throughput amplified fragment length polymorphism to distinguish sorghum greenbug (Homoptera: Aphididae) biotypes. Agr. Forest Entomol. 5: 311-315
• Zhu-Salzman, K., R.A. Salzman, J-E. Ahn and H. Koiwa (2003) Transcriptional regulation of sorghum defense determinants against a phloem-feeding aphid. in press
• Zhu-Salzman, K., J-E. Ahn, R.A. Salzman, H. Koiwa, R.E. Shade and S. Balfe (2003) Fusion of a soybean cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically. Agr. Forest Entomol. 5: 317-323

Progress 01/01/02 to 12/31/02

Outputs
We have used cowpea bruchid (Callosobruchus maculatus) and sorghum greenbug (Schizaphis graminum) as our systems to study insect genetic diversity and adaptation to plant defense mechanisms. Despite the lack of apparent morphological differences, variation in genetic information among greenbug populations can be detected using genetic techniques. We found 1,775 markers among eight greenbug biotypes using amplified fragment length polymorphism (AFLP), a powerful method to generate large number of DNA polymorphic markers. Interestingly, cluster analysis clearly showed that sorghum biotypes share higher polymorphisms among themselves than with non-sorghum biotypes, suggesting that common genetic factor(s) are shared by sorghum greenbug biotypes, contributing to their high fitness on this crop. Detection of genetic divergence is the first step toward mapping insect resistance to plant defense. Using the defensive soybean cysteine protease inhibitor scN and the cowpea bruchid beetle (that utilizes cysteine proteases for digestion) as our research system, we have characterized the involvement of cysteine proteases in cowpea bruchid protein digestion, and conducted in-depth studies to uncover insect counter defense mechanisms. We found that over 30 cysteine protease genes have provided insects great flexibility to differentially express different isoforms as needed. Qualitative and quantitative remodeling of digestive proteases also involves another enzyme group, the aspartic proteases. Further, the bruchid can adapt to the challenge of plant protease inhibitors by activating scN-insensitive and scN-degrading protease activity.

Impacts
Insect-resistant hybrids and genetically engineered plants that express insect-resistance genes have been successful, widely used control measures for insect pest management. But the useful life of resistant lines may be as short as a few years, because of the appearance of new insect biotypes that are unaffected by the resistance factors. The proposed study will help us gain insight into the extent of genetic diversity among insect pests and mechanisms insects use to adapt to plant natural defense and to overcome human-imposed management practices. Such an understanding is not only important from a basic science perspective, but also crucial for breeding and biotechnology-based pest control practices.

Publications

• Zhu-Salzman, K., H. Koiwa, R.A. Salzman, R.E. Shade and J-E Ahn (2002) Cowpea Bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor. Insect Mol. Biol. (in press)
• Zhu-Salzman, K., P.K. Hammen, R.A. Salzman, H. Koiwa, R.A. Bressan, L.L. Murdock and P.M. Hasegawa (2002) Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII). Comp. Biochem. Physiol. 132:327-334
• Zhu-Salzman, K. (2002) Book review "Recombinant Protease Inhibitors in Plants". Ann. Entomol. Soc. Am. 95: 651